Levulinic acid (LA) is a biomass-derived platform chemical which has attracted increasing interest in recent years due to the possibility to be converted into added-value derivatives, such as biofuels, fragrances, solvents, pharmaceuticals, and plasticizers [1], thus justifying the increasing worldwide market demand for LA production [2]. LA is traditionally produced in water medium via dehydration of C6 sugars through the formation of 5-hydroxymethylfurfural as the main reaction intermediate, the overall reaction occurring in the presence of a suitable acid catalyst [3–7]. Among the LA-derived platforms chemicals, alkyl levulinates appear significantly attractive due to their potential applications developed in recent years for the global market scenario, such as fuel blending additives for diesel/gasoline, and as intermediates for the synthesis of valuable polymers, perfumes, and flavoring formulations [8,9]. Levulinates can be synthesized by the esterification of pure LA with a simple equilibrium reaction, requiring a mild acid catalysis/reaction conditions, and generally affording very high yields towards the desired ester products. Both the reduced number of process units and the enhanced performances of new technological solutions, such as the reactive distillation, should allow significant improvements in the economics of the esterification process [10–12]. However, despite these ascertained potentials, the catalysis issue can be further improved, taking into account both the synthetic strategy and the adopted feedstock. Up to now, much work has been done on the synthesis of methyl and ethyl levulinates, which were recognized as effective additives for diesel and biodiesel transportation fuels, showing excellent performances, including non-toxicity, high lubricity and good flashpoint stability and flow properties under cold conditions [13–15]. In addition, the conversion of these short-chain alkyl levulinates into more added-value bio-products, such as γ-valerolactone, is preferred respect to that of LA due to the improved selectivity. Moreover, the hydrogenation of alkyl levulinates with short alkyl chains facilitates down-stream processing as separation of alcohol in the final step is easier and cheaper, compared to water [16]. The synthesis of alkyl levulinates was carried out in the presence of homogeneous or, more advantageously, heterogeneous catalysts due to their easy recovery from the reaction mixture, starting from pure LA or expensive pure model precursors, such as C6 carbohydrates (glucose, fructose, and clean cellulose), C5 derivatives (furfuryl alcohol), and even real lignocellulosic biomasses [17,18]. The one-pot synthesis of these levulinates directly from monosaccharides, polysaccharides, and, above all, starting from lignocellulosic biomass, has gained more interest due to the low cost of these feedstocks, and the feasibility of this approach was demonstrated, in particular for the biomass alcoholysis to ethyl levulinate [19]. A key advantage of the direct alcoholysis is represented by the limited formation of undesired furanic products, named *humins*, when using alcohol (instead of water) as the solvent for biomass conversion [20,21]. On the other hand, the yields of levulinate esters from real biomass are generally lower than those obtained from pure model compounds due to the usually higher recalcitrance of the former [18], and to the increased formation of reaction by-products, such as formates, HMF ethers and, above all, dialkyl ethers, originating from the alcohol dehydration [20,21]. Differently, *n*-butyl levulinate (BL) was less studied, but its use as an efficient fuel additive was already demonstrated [22], resulting in a more promising diesel additive than EL [18,23]. In addition, *n*-butanol (*n*-BuOH) is a green reagent/solvent, being obtainable by fermentation and also by catalytic conversion of bio-ethanol [24], thus further justifying the interest towards the sustainable production of BL. Regarding the possible pathways for BL production, as previously stated for methyl and ethyl levulinates, it can also be obtained with a two-steps process from C5 or C6 carbohydrates or their conversion products (Pathway A or Pathway B1, respectively, in Figure 1) or, more advantageously, with a one-pot approach from C6 carbohydrates (Pathway B2, Figure 1). In the first case, furfuryl alcohol or LA (from hemicellulose and cellulose fractions, respectively) must be synthesized in the first step, recovered, and properly purified before the subsequent stage, consisting of acid alcoholysis or esterification, respectively. The C5 route (Pathway A, Figure 1) is a three-step process consisting of: (1) acid-catalyzed hydrolysis of the hemicellulose fraction to simpler C5 sugars and their dehydration to furfural; (2) hydrogenation of furfural over a suitable catalyst to furfuryl alcohol; (3) acid alcoholysis of furfuryl alcohol to BL, occurring in the presence of strong acid catalysts [25]. The C5 route was investigated in the literature adopting furfuryl alcohol as starting feedstock, in the presence of heterogeneous catalysts, due to their easier separation from the liquid reaction mixture [26–29]. On the other hand, BL synthesis through the C6 route (Pathway B1, Figure 1) provides the hydrolysis of the C6 carbohydrates to LA, followed by its esterification in *n*-BuOH, and both steps occur in the presence of a suitable acid catalyst.

**Figure 1.** C5 and C6 sugar-based routes to *n*-butyl levulinate.

Regarding this C6 route, in the literature, it is possible to find many BL synthesis from pure LA, the intermediate compound, usually preferring the use of heterogeneous catalysts, achieving excellent yields (>90 mol%) under sustainable reaction conditions [9,30,31]. On the contrary, BL synthesis from C6 carbohydrates was not exploited with the same emphasis, although this approach should result very attractive from the industrial perspective if realized in a single step without any intermediate purification procedures (Pathway B2, Figure 1), thus decreasing the BL production cost. In this context, some authors have reported the one-pot butanolysis of microcrystalline cellulose to BL [32–41], which is already very difficult to achieve due to its recalcitrance to the solubilization/conversion, while the butanolysis of the real biomass, which includes lignin as a further recalcitrant component, is even unexplored. In this regard, a simplified scheme of the C6 fraction butanolysis pathway is shown in Figure 2.

**Figure 2.** C6 fraction butanolysis pathway starting from cellulose feedstock (adapted from [40]).

Butanolysis of the C6 fraction is a complex pathway, which involves the formation of many reactive species, in particular butyl glucosides and furanic derivatives as the main reaction intermediates, in addition to butyl formate (BF) as the main reaction co-product. Furanic intermediates are very reactive species, which could condense to solid insoluble polyfurans, the humins [40]. The first step of the butanolysis process consists of the depolymerization of cellulose chains to form glucosides, followed by the subsequent formation of furan derivatives, whereas the final step involves the conversion of the furanic intermediates to BL, and all these steps occur in the presence of an acid catalyst [40]. When the above reaction is performed adopting a real solid lignocellulosic biomass, the use of homogeneous catalysts is the best choice. Some typical drawbacks, such as the possible corrosion of the equipment, and the recovery of the acid catalyst, need to be further improved by adopting very low acid concentrations and more technological work-up solutions. Moreover, the use of a very low acid concentration in the alcoholysis reaction, which helps to minimize the corrosion of the equipment, should also control the formation extent of by-products, in particular the dialkyl ether [8,14]. In this context, it is noteworthy the work of Démolis et al. [37], who achieved the highest BL molar yield of about 50 mol%, working in an autoclave at 200 ◦C for 30 min, adopting pure cellulose as starting feedstock (2.4 wt%), with a very low concentration of H2SO4 (0.6 wt%). However, this good BL yield, although academically interesting, was obtained with a low starting cellulose loading, which should represent significant limitations for the development on the intensified industrial scale.

Definitely, at this state-of-the-art, the main bottlenecks of the published works are related to the adoption of (1) model compounds as starting substrates instead of the cheaper and largely available real biomasses, and (2) low substrate loading, which is not a limit for an academic investigation, in a preliminary phase, but it is certainly for the next industrial scale-up. Therefore, the resolution of both these aspects is fundamental for the BL development towards the biofuel market, and this work contributes to filling this gap. In this context, wood is the most abundant type of lignocellulosic biomass and, more in detail, *Eucalyptus* is a widespread, fast-growing, and widely distributed species, which shows a good adaptation to grow in zones with a high probability of freezing and affording decreased susceptibility to diseases [42–44]. It already shows an interesting potential in many industrial fields, as in the paper-making production, where it is already used as a valuable and cheap fiber source. Moreover, it is an ideal energy crop, thanks to its high yield, low energy input for production, low cost, minimal contents of contaminants, and low nutrient requirements. From a different perspective, it may represent a promising feedstock for many biochemical conversion processes, given its high content of C5 and C6 carbohydrates (about 60 wt%) [42–44]. The use of this feedstock is particularly advantageous if the complete fractionation and the successive valorization of each component are achieved, according to the perspective of an integrated biorefinery [45]. In this context, the aim of the present work is the complete exploitation of *Eucalyptus nitens* biomass. For this purpose, pre-treated autohydrolyzed-delignified wood (ADW) *Eucalyptus nitens* was obtained from a first autohydrolysis treatment of the starting raw biomass in order to remove and exploit hemicellulose and water-soluble extractives, followed by a second step of delignification on the resulting solid through the HCl-catalyzed acetic acid treatment (Acetosolv method). The recovered cellulose-rich feedstock was employed for the one-pot production of BL in *n*-BuOH, adopting microwave (MW) and/or traditional (TR) heating, in the presence of very dilute sulfuric acid as a homogeneous catalyst. MW heating represents an important tool because it can reduce reaction time and energy consumption, thus improving the efficiency of the process [46,47]. In the specific case of LA esterification, remarkable thermal (kinetic) advantages of MW towards this reaction were already reported by Ahmad et al. [48]. The choice of H2SO4 as the acid catalyst was done taking into account its promising catalytic performances in the alcoholysis reaction to methyl and ethyl levulinates [49,50], while other acid catalysts, such as HCl or H3PO4, resulted less active, for example in the case of the one-pot reaction from cellulose to ethyl levulinate [49]. The effects of the main reaction parameters, temperature, reaction time, and acid concentration were investigated by a traditional One-Factor-at-a-Time (OFAT) approach and further optimized by Response Surface Methodology (RSM), developing a Face-Centered Central Composite Design (FCCD), from the perspective of developing the BL process intensification. Finally, a preliminary study of diesel engine performances and emissions for a model mixture with analogous

composition to that produced from the alcoholysis reaction was performed in order to evaluate its potential application as an additive for diesel fuel, without separation of each component.

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

#### *2.1. Characterization of the Eucalyptus nitens Samples*

The chemical composition of the starting untreated *Eucalyptus nitens* was as follows: 42.0 wt% of cellulose, 14.5 wt% of hemicellulose, 21.4 wt% of Klason lignin, and 22.1 wt% of unidentified compounds (including acid-soluble lignin, ash, extractives, waxes). After carrying out the autohydrolysis and Acetosolv pretreatments, the mass yield of the ADW *Eucalyptus nitens* sample was 45.0 wt% of the starting raw biomass, and its chemical composition resulted to be as follows: 85.0 wt% cellulose, 2.0 wt% hemicellulose, 4.1 wt% Klason lignin, 4.8 wt% acetyl groups, and 4.1 wt% of unidentified other compounds. The compositional analysis of untreated and ADW *Eucalyptus nitens* samples confirms the effective enrichment in cellulose and the depletion in hemicellulose and lignin as a consequence of the chemical pre-treatments [51].

XRD analysis of the untreated and ADW *Eucalyptus nitens* samples was attained in order to estimate the crystallinity index (CI) of the cellulose fraction, a paramount parameter for understanding the behavior of biomass to the subsequent butanolysis reaction, achievable under an appropriate severity degree [52]. The XRD spectra of the starting untreated and ADW *Eucalyptus nitens* biomasses are reported in Figure 3. Here, deconvoluted curves were reported, including that due to amorphous cellulose (at about 2θ = 21.5◦) and those related to the crystalline planes, with Miller indices of 101, 10¯ı, 002 e 040.

**Figure 3.** XRD spectra of (**a**) untreated and (**b**) autohydrolyzed-delignified wood (ADW) *Eucalyptus nitens* samples.

A higher crystallinity degree was obtained for the ADW sample rather than for the untreated one (46.8 versus 43.3%, respectively), ascribed to the partial removal of both lignin and hemicellulose fractions for the ADW sample as a consequence of the pre-treatment, leading to greater exposure of the crystalline cellulose fraction [53]. Besides, autohydrolysis pre-treatment allowed the preferential removal of the amorphous component of the cellulose, leaving almost unchanged the crystalline portion [54].

FT–IR characterization of the untreated and ADW *Eucalyptus nitens* samples was also carried out, and the acquired spectra are reported in Figure 4.

**Figure 4.** FT–IR spectra of (**a**) untreated and (**b**) ADW *Eucalyptus nitens* samples.

In the IR spectrum of the untreated *Eucalyptus nitens* sample, typical bands of biomass macro-components, cellulose, hemicellulose, and lignin derivatives, are detected, such as that at about 3400 cm−1, assigned to the O-H stretching, and that at about 2900 cm−1, due to the C-H stretching. Moreover, the absorption band at about 1730 cm−1 is assigned to the C=O stretching of ester bonds, such as acetyl derivatives, while those at 1600 cm−1 and 1510 cm−1 indicate the presence of C=C ring vibrations, which are typical of lignin units [55,56]. In the region between 1500 and 1300 cm−1, absorption bands ascribed to the bending of the O-H bonds and the vibrations of the methyl and methylene groups of both lignin and cellulose are present. The absorption bands between 1300 and 1200 cm−1 are due to the stretching of the C-O bonds of the alcoholic, phenolic, and carboxyl groups. The shoulder at about 1160 cm−1 can be assigned to the stretching of the C-O-C bond of the hemicellulose and cellulose, while the absorption bands at about 1030 cm−1 and that at about 900 cm−1 are due to the stretching of the C-O-C β-glycosidic bonds of the cellulose [55,56]. Regarding the IR spectrum of ADW *Eucalyptus nitens* biomass, the absorption bands of lignin rings at 1600 cm−1 and 1510 cm−1 are absent, thus confirming the efficacy of the Organosolv treatment. In addition, a new absorption band is present at about 1050 cm−1, which is uniquely assigned to the C–O stretching of the cellulose [57], thus indirectly confirming the occurred cellulose enrichment for the ADW *Eucalyptus nitens* sample. The other absorption bands are similar to those discussed for the untreated *Eucalyptus nitens* biomass.

#### *2.2. Univariate Optimization: OFAT Approach*

After having demonstrated the occurred cellulose enrichment of the ADW *Eucalyptus nitens*, this biomass was our preferred choice for performing the next one-pot butanolysis to BL, thus further developing the biorefinery concept of this biomass. For this purpose, alcoholysis of ADW *Eucalyptus nitens* (range of biomass loading: 7–20 wt%) in *n*-BuOH was preliminarily investigated by a traditional One-Factor-at-a-Time (OFAT) approach, employing both MW and TR heating, in the presence of 1.2 wt% H2SO4 (Table 1). Starting from the published results [8,36], in the beginning, the biomass loading of 7 wt% and the temperature of 190 ◦C, under MW heating, were selected for studying the behavior of the reaction (Runs 1–3, Table 1). At the increase of the reaction time, the BL molar yield raised to 42 mol% after 15 min. The extension of the reaction time did not affect the BL molar yield, which was stable at 42 mol%. Under the optimized reaction conditions (MW, 190 ◦C, 15 min), the comparison between ADW and untreated *Eucalyptus nitens* biomass was investigated (Runs 2 and 4, Table 1): the employment of the starting crude *Eucalyptus nitens* without any pre-treatment allowed us to obtain the same BL molar yield of 42 mol% as the corresponding ADW sample, but the BL concentrations in the final reaction mixtures were 23 g/L for the ADW sample against 12 g/L for the crude *Eucalyptus nitens* due to the higher cellulose content in the ADW biomass. Finally, a further test employing TR heating was carried out (Run 5, Table 1) employing the ADW *Eucalyptus nitens* wood as substrate: after 120 min, the BL molar yield of 49 mol% was obtained together with the BL concentration of 27 g/L, demonstrating that analogous promising results can be also achieved with TR heating, even if a longer reaction time was necessary.

**Table 1.** One-pot butanolysis of the untreated or ADW *Eucalyptus nitens* to *n*-butyl levulinate (BL), adopting microwave (MW), or traditional (TR) heating. Reaction conditions: biomass loading 7 wt%, 190 ◦C, H2SO4 1.2 wt%.

Taking into account the low cost of the starting biomass, it is more important to achieve high BL concentrations in the final mixture rather than to maximize the BL molar yield respect to cellulose fraction present in the starting biomass, making the entire process economically convenient due to the significant reduction of the purification cost, from the perspective of the high gravity approach [58]. On this basis, the biomass loading was increased to 14 and 20 wt% and the obtained results adopting MW heating, working at 190 ◦C, for 15 min, in the presence of H2SO4 1.2 wt%, are shown in Figure 5.

**Figure 5.** One-pot MW-assisted butanolysis of the ADW *Eucalyptus nitens* sample to BL, adopting different biomass loadings (7, 14, and 20 wt%). Reaction conditions: 190 ◦C, 15 min, H2SO4 1.2 wt%, MW heating.

The increase of the initial biomass loading caused the decrease of BL molar yield as expected considering that, when a higher initial biomass loading is employed, adopting the same amount of catalyst, not only the catalyst/biomass weight ratio decreases, but also the mixture mixing can become more difficult, working in slurry phase. However, at the increase of initial biomass loading, the decrease of BL molar yield was not significant and it was associated with a huge increase of BL concentrations, highlighting the effectiveness of the high gravity approach. The same reactions were also carried out under TR heating to confirm the feasibility of this reaction on a larger scale, and the comparison between the two systems is shown in Table 2.

**Table 2.** One-pot butanolysis of the ADW *Eucalyptus nitens* to BL, with different biomass loadings (7, 14, and 20 wt%), adopting MW and TR heating systems. Reaction conditions: 190 ◦C, H2SO4 1.2 wt%.

The shift from the MW to the TR heating system was demonstrated, in the latter case requiring much longer reaction times to get comparable BL molar yields. As already achieved for the MW heating, also for TR, the systematic decrease of the BL molar yield, occurring with the increase of the initial biomass loading, may be due to the increase of the substrate/catalyst ratio, leading to an insufficient amount of catalyst, and to the inefficient agitation of the reaction slurry.

At this level of investigation, the above tests with TR heating confirm the MW data, further justifying and claiming our high gravity approach. Good results were achieved with the biomass loading of 20 wt% (Runs 10 and 11, Table 2), and therefore, 25 wt% of biomass loading was also tested, always under TR heating. Unfortunately, in this last case, although the decrease of BL molar yield (35 mol%) was not significant with a related very high BL concentration (87 g/L), considerable practical difficulties were encountered in filtering and recovering the liquid phase. For this reason, even in the case of TR heating, the best result, in the application perspective, is obtained with the biomass loading of 20 wt%. The achieved results are very interesting because, up to now, BL molar yield higher than 30 mol%, corresponding to the best BL concentration of about 70 g/L, with the initial biomass loading of 20 wt%, has never been ascertained under TR heating, opening the way towards the industrial adoption of this approach for BL synthesis.

On the basis of the promising results obtained with the biomass loading of 20 wt%, the best alcoholysis test under TR heating (Run 10, Table 2) was further investigated, adopting lower reaction times (30 and 60 min), to gather more information about the kinetics. These new data are reported in Figure 6. This figure shows that the most significant improvement, in terms of BL molar yield, was achieved already after 60 min. Lower reaction times (30 min) are not sufficient for the complete conversion of the reaction intermediates to BL, while higher ones (120 min) are not advantageous, not leading to further raise of BL molar yield. In terms of BL concentration, the increase is remarkably moving from 30 to 60 min (52 and 72 g/L, respectively), while it remains almost constant at longer reaction times.

**Figure 6.** Kinetics of the one-pot butanolysis of the ADW *Eucalyptus nitens* sample (20 wt%) to BL. Reaction conditions: biomass loading 20 wt%, 190 ◦C, H2SO4 1.2 wt%, TR heating.

The increase of the final BL concentration is certainly a key parameter for the industrial scale-up of the reaction. In our case, only by acting on the biomass loading (up to the maximum of 20 wt%), it was possible to significantly increase the final BL concentration from the perspective of the high gravity approach. To further boost the concentration of the desired BL, it is possible to reprocess the mother liquor with fresh biomass, without adding further solvent and catalyst, according to the cross-flow approach [42]. This approach is certainly advantageous and smart, especially starting from cheap biomasses, as in our case. In this regard, the mother liquor deriving from the best alcoholysis run carried out under TR heating (run at 60 min, Figure 6) was used for a subsequent analogous alcoholysis reaction. In this additional step, a lower biomass loading (10 wt%) was adopted due to the very high viscosity of the liquor, and the results are reported in Table 3.

The final high BL concentration achieved with an additional alcoholysis step (85 g/L) justifies the validity of our approach. Regarding the BL molar yield, the small decrease occurred in the second step, despite the lower biomass loading, is probably due to the presence of reaction by-products obtained at the end of the first alcoholysis step, such as dibutyl ether (DBE), in other words to the lower amount of

*n*-BuOH available for the second alcoholysis step. However, these differences, in terms of BL molar yield, are not significant, being largely rewarded by the increase in BL concentration, the latter being a much more important process output, especially from an industrial perspective, allowing significant cost reduction of purification and separation treatments.

**Table 3.** Cross-flow butanolysis of ADW *Eucalyptus nitens* to BL. Reaction conditions: 190 ◦C, 60 min, H2SO4 1.2 wt% (added only at the 1st step), TR heating.

#### *2.3. Design of Experiments and Optimization by RSM*

The above promising preliminary results, prompted us to study the combined effect of three main factors, including temperature, reaction time and catalyst loading, on the butanolysis reaction, adopting an FCCD (α = 1). BL molar yield was chosen as the response of interest, but the other main components of the reaction mixture were also determined. The ranges of the independent variables for planning the DOE were selected on the previous OFAT screening: temperature, x1 (160–200 ◦C); reaction time, x2 (30–180 min), catalyst loading, x3 (0.2–3 wt%). These actual parameters were coded in three levels according to Equation (1):

$$\mathbf{X}\_{i} = (\mathbf{x}\_{i} - \mathbf{x}\_{0}) / \Delta \mathbf{x} \tag{1}$$

where Xi is the coded value of the independent variable, xi is the real value of the independent variable, x0 is the real value of the independent variable at the center point, and Δx is the step change value. The complete case studies of 18 experiments, realized at the constant biomass loading of 20 wt%, are shown in Table 4, together with the respective experimental responses.

**Table 4.** Experimental design and BL molar yield (%) response of the Face-Centered Central Composite Design (FCCD) for different combinations of temperature, reaction time, and catalyst loading, all realized at the constant biomass loading of 20 wt%.

The experimental data were analyzed by Design-Expert software and a second-order polynomial model was developed to correlate the process parameters with the response, thus obtaining Equation (2):

$$\begin{array}{c} \text{Y} = 40.54 + 6.2 \text{T} \mathbf{X}\_1 + 3.97 \mathbf{X}\_2 + 9.63 \mathbf{X}\_3 - 1.41 \mathbf{X}\_1 \mathbf{X}\_2 - 5.59 \mathbf{X}\_1 \mathbf{X}\_3 - 1.84 \mathbf{X}\_2 \mathbf{X}\_3 - 3.95 \mathbf{X}\_1^2 \\ \qquad - 1.45 \mathbf{X}\_2^2 - 9.45 \mathbf{X}\_3^2 \end{array} \tag{2}$$

According to the monomial coefficient value of the regression model equation, the order of priority among the main effects of impact factors is the following: catalyst loading > temperature > reaction time. Linear parameters have a significant synergistic effect on the response, since they have a positive coefficient, whereas the remaining combined and quadratic terms show significant antagonistic effects, thus highlighting the importance of the DOE optimization.

In Table 5, the results of the analysis of variance (ANOVA) are summarized to test the soundness and suitability of the model [59]. The mean squares values were calculated by dividing the sum of the squares of each variation source by their degrees of freedom, and a 95% confidence level was used to determine the statistical significance in all analyses. Results were assessed with *p*-value and *F*-value as the main statistical parameters of interest. The R2 value for the quadratic model is 0.9102, which demonstrates a close agreement between experimental and predicted values of the BL molar yield. The R2 adjusted is 0.8091 (>0.6), expressing that the model is significant. An adequate precision value of 11.2385 (>4) highlights a good signal, therefore this model can be used to describe the design space. Regarding the other parameters, both high *F-* and low *p*-values indicate the high significance of the corresponding coefficients of the model [58]. In our case, the model has an *F*-value of 9.01 (much greater than unity) and a *p*-value of 0.0025 (<0.05), which also implies that the model is significant. There is only a 0.25% chance that such a large *F*-value could occur due to noise. *F*-values of C, A, AC, and C2 in Table 5 show that these are significant model terms, firstly C (catalyst loading) and secondly A (temperature), thus confirming the key roles of both these reaction variables on the butanolysis reaction, while reaction time has a modest effect. This latter aspect is certainly assessed to the chemistry of the butanolysis reaction, but also to the very efficient MW heating, which narrows the range of reaction time for reaching the optimal BL molar yield. Moreover, the same order of importance of the independent variables is deduced taking into account the *p*-values, whose significances are ascertained because of *p* < 0.05.

**Table 5.** ANOVA for the response surface quadratic model.

*p* < 0.05 is considered significant.

Diagnostic plots (predicted vs. actual plot and normal plot of residues) were checked for the adequacy and accuracy of the proposed model equation. The predicted vs. actual plot indicates that the points should be aligned with a straight line, and the normal plot of residues shows whether the residuals are in normal distribution [60]. A predicted vs. an actual plot of BL molar yield is shown in Figure 7a, which shows as the predicted values are close to the observed ones, in agreement with the above discussion. Additionally, the residuals showed a good fit to a normal distribution, indicating a high significance (Figure 7b).

**Figure 7.** (**a**) Predicted versus actual plot, and (**b**) normal plot of residues.

Design-Expert software was used to produce three-dimensional (3D) response surfaces and two-dimensional (2D) contour plots. The 3D surfaces and 2D contour plots are graphical representations of the regression equation for the optimization of reaction conditions, which are very useful to visualize the relationship between the response variables and experimental levels of each factor. In such plots, the response functions of two factors are presented, while the remaining factor is kept constant at the central values. These graphs are shown in Figure 8.

**Figure 8.** Three-dimensional (3D) response surfaces and two-dimensional (2D) contour plots: effect of temperature, reaction time, and acid concentration on BL molar yield. (**a**,**b**) catalyst loading was kept constant at 1.6 wt%; (**c**,**d**) reaction time was kept constant at 105 min; (**e**,**f**) temperature was kept constant at 180 ◦C.

Figure 8a,b confirms that the positive effect of the temperature on BL molar yield is more significant than that of the reaction time, at constant catalyst loading. In detail, a temperature higher than 180 ◦C is necessary to ensure the highest BL molar yield (about 40 mol%), together with relatively short times (up to about 120 min). On the other hand, a temperature lower than 180 ◦C is insufficient to achieve high BL molar yield, regardless of the adopted reaction times. Figure 8c,d elucidates that, firstly the catalyst loading, and secondly the temperature, strongly affect the BL yield, at constant time. This trend provides that a catalyst loading higher than 1.6 wt% is necessary to accomplish a very high BL molar yield, and that a lower catalyst loading should be associated with a corresponding higher reaction temperature (190–200 ◦C). Lastly, Figure 8e,f further confirms that, at a constant temperature, the catalyst loading has a strong non-linear influence on BL molar yield, and it should be higher than 1.6 wt%, while reaction time has a weaker effect, showing a feeble and presumably not significant curvature, as previously stated.

Starting from the above discussion, it is evident that the optimal solution for the BL optimization is not univocal, but involves rather a spatial region of the 3D response surfaces and 2D contour plots, depending on the combined choice of independent variables. The final stage of the design is the determination of the criteria for optimization and model validation. The optimization criterion was the maximum BL molar yield within the space design, with the independent variables kept within the range. For this purpose, starting from the acquired response surfaces and contour plots (Figure 8), the ranges of catalyst loading, temperature and reaction time, were further narrowed to those of greatest and practical interest for maximizing BL molar yield, avoiding the highest levels, in agreement with a more sustainable optimization approach. The identified ranges of interest were selected as follows: 180–190 ◦C for the temperature, 1.6–2.3 wt% for the catalyst loading, and 90–150 min for the reaction time. One of the possible solutions at the optimum levels (183 ◦C, 146 min, H2SO4 1.9 wt%) was experimentally carried out, and the experimental BL molar yield was compared with that predicted, as shown in Table 6. The results confirm the good agreement between the predictive and experimental results, at the optimum levels for BL synthesis, thus demonstrating the validity of our proposed model.

**Table 6.** Predicted and experimental BL yield: model validation.

#### *2.4. Identification of the Reaction By-Products and Application Perspectives of the Final Reaction Mixture*

Before developing the possible engine applications of the final alcoholic mixture, it is necessary to analyze more in-depth its chemical composition, to better define its final use as biofuel. In this context, some authors have identified some reaction intermediates/by-products, but their quantification has not been reported [37], which is very useful for dealing with an in-depth discussion about the possible applications of this alcoholic mixture. Some possible intermediates/by-products were already defined in the Section 1 (Figure 2), in particular, furanic derivatives and glucosides as main reaction intermediates, and BF as the main reaction co-product. Furanic intermediates are very reactive species, which could condense to give solid polyfurans, or *humins* [40] and, in our case, their partial solubilization in the alcoholic mixture is more favored, if compared to the traditional hydrothermal path due to the presence of the alcoholic solvent, which acts as a polymerization inhibitor for *humins* growth [21,61]. Taking into account the chemical composition of ADW *Eucalyptus nitens*, which has a significant content of acetyl groups (4.8 wt%), deriving from the upstream Acetosolv treatment, these groups can be released during the alcoholysis, thus enabling the acid-catalyzed formation of butyl acetate (BA). Lastly, *n*-BuOH can be etherified to give dibutyl ether (DBE) and water, the latter in equimolar amount respect to DBE, and also this reaction favorably occurs in the presence of the adopted sulfuric acid catalyst [37].

To confirm the presence of the above by-products, the reaction mixtures recovered from the experiments planned for the FCCD (Table 4) and the model validation (Table 6), were qualitatively analyzed by GC–MS, identifying BF, DBE, and BA as main reaction by-products, together with the unconverted *n*-BuOH. These compounds were subsequently quantified by GC–FID, and the corresponding mass yields in the organic phase are reported in Table 7, together with that of the product of interest (BL).

**Table 7.** Composition of the organic reaction mixtures recovered from the experiments planned for the FCCD and for the model validation, working at the constant biomass loading of 20 wt%.

The above data show that the variation of BA and BL yield within the investigated ranges of the independent variables is modest, if compared with that of DBE (and consequently that of *n*-BuOH), which represents the main reaction by-product, even in the case of the optimum experiment for BL synthesis (Run 19, Table 7). DBE represents a high cetane component (CN = 100) and it was already tested in blend with diesel fuel, leading to very short ignition delays, so its possible application in compression ignition engines is favorable and attractive [22]. However, different experimental conditions should allow a significant modulation of the DBE to *n*-BuOH weight ratio, etherification being significantly favored by the acidity increase (compare Runs 1 and 8). On this basis, the best experimental choice for performing the biomass butanolysis should lead to a good production of both BL and DBE, while the unconverted *n*-BuOH could be eventually recovered and reused within the same process [62]. In principle, the organic ternary mixture BL/DBE/*n*-BuOH could be immediately exploited, without separation of its components as an innovative diesel fuel additive, thus making the alcoholysis reaction a viable route to the direct production of a blending component. In addition, the amount of the adopted mineral acid for the butanolysis reaction should be as low as possible, to avoid costly work-up procedures and, on this basis, the reaction mixture deriving from Run 1 represents the best compromise for developing the next application of this mixture as a diesel additive. In order to explore this never reported perspective, a preliminary study was carried out employing a model mixture BL/DBE/*n*-BuOH as an additive for diesel fuel. At this preliminary level of investigation, the addition of BA, which is a minor product closely related to the adopted biomass, was not considered.

#### *2.5. Engine Experimental Activity*

A preliminary engine experimental activity was carried out to verify the influence of these oxygenated fuel additives on diesel engine performance. As aforementioned, the ternary mixture available from one-pot butanolysis of raw and ADW biomass is mainly composed of BL, DBE, and unreacted *n*-BuOH. These compounds represent valuable oxygenated fuels and their properties

were already investigated, singularly, in blend with diesel [22,63–66]. The properties of all fuel components are shown in Table 8 and compared with those of commercial diesel fuel.

**Table 8.** Overview of biomass resources available from the literature [13,21,61].

a Density at 25 ◦C. b Density at 20 ◦C. c Cetane Number. d Lower Heating Value. e Kinematic viscosity at 40 ◦C.

The above properties show that both *n*-BuOH and BL have a lower Cetane Number than diesel. This is a known behavior reported by Koivisto et al. [66,67] for alcohols and levulinates, including *n*-BuOH and BL, respectively. These compounds are characterized by higher ignition delays (i.e., lower Cetane Number) than alkanes of the same carbon atom chain length. However, ethers, such as DBE, have an opposite behavior and show lower ignition delays in comparison with alkanes [67]. Taking into account the components of the ternary mixture obtained from alcoholysis reaction of biomass, DBE can play a fundamental role as a cetane enhancer, making its controlled coproduction in the alcoholysis highly valuable in this applicative perspective. Moreover, this characteristic enables us to test diesel blended with a high-volume percentage of the ternary mixture. In addition, the use of the *n*-BuOH/DBE/BL mixture leads to an increase in the fuel oxygen content. Generally, oxygenated diesel blends ensure, especially in the areas of the cylinder with a low air-to-fuel ratio, the presence of oxygen directly from the fuel and, consequently, soot precursors reduction [22,68]. In this context, *n*-BuOH and BL represent the two components of the mixture which mostly influence the oxygen content in the final blend with diesel.

By considering that the final composition of the reaction can be easily tuned, a model mixture with a composition similar to that of Run 1 in Table 7 was prepared and tested on a small diesel engine. The mixture, whose composition is reported in Table 9, was blended in three different volume percentages with diesel: 10, 20, and 30 vol % (named MIX1 10%, MIX1 20%, MIX1 30%).

**Table 9.** Composition of the prepared ternary mixture.

This mixture was utilized in the experimental engine at different rpm values (1500, 2000, and 2500 rpm) at full power (Tmax). Data of the engine performance were compared with those obtained with diesel fuel alone, and all the data of the engine performances are reported in Figure 9. Fuel injection timing was maintained constant along with the experimentation.

**Figure 9.** Engine performances obtained with diesel fuel, alone and in blends with *n*-BuOH/dibutyl ether (DBE)/BL.

The use of different blending (from 10 to 30 vol %) with diesel fuel does not significantly affect the power of the employed engine, meaning that the calorific values and the reactivity of these mixtures do not significantly differ from those of commercial diesel fuel. Moreover, no considerable variation of HC and NOx emissions occurs with the use of the adopted mixtures. Literature reports different nitrogen oxides behavior as a function of the fuel type, which indicates that its emission strongly depends on several other factors besides the employed fuel, and these are not easily detachable within the performed analysis [69,70].

On the other hand, a strong reduction of both CO and soot emission was obtained as the blend of both the ternary mixtures was increased. This can be addressed to the increased combustion oxygen availability which plays a key role in the formation process of the carbon-based pollutants, such as soot or CO. Particularly, the oxygen provided by the fuel reduces the low air excess zones which are the main cause of the soot formation. Furthermore, more oxygen is available from fuel, and less carbon is present for CO or soot formation. Svensson et al. [71] found that the soot emissions could be reduced to 0 when fuel oxygen content reaches 27–35 wt%. Another aspect that contributes to decreased CO and soot when applying the two mixtures is the lower boiling point of the oxygenated components in comparison with diesel fuel. This leads to a kind of "droplet explosion" once the fuel mixture is introduced in a hot ambient, such as the cylinder at the end of the compression stroke, increasing the spray fragmentation and mixing [72,73], so increasing the combustion completion of carbon-based molecules is necessary.

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

#### *3.1. Materials*

*Eucalyptus nitens* was collected locally in the Galicia region (Spain). The starting untreated and ADW *Eucalyptus nitens* biomasses were milled with a knife mill, using a 0.5 mm metal mesh, air-dried, and further processed according to the following pre-treatments. Regarding the ADW treatment, the raw *Eucalyptus nitens* was first subjected to autohydrolysis. For this purpose, the sample was suspended in water and treated in a stainless steel reactor (Parr Instruments Company, Moline, IL, USA) under non-isothermal conditions, adopting a water/biomass weight ratio of 10/1, up to the final temperature of 193 ◦C [51]. Then, the solid residue was recovered and subsequently underwent an Acetosolv treatment, with a mixture of 90.00% acetic acid, 9.78% water, and 0.22% hydrochloric acid. For this purpose, the temperature was maintained at 134 ◦C for 30 min, adopting the solid/liquid

weight ratio of 1/10 [51]. Solid residue obtained from this treatment was recovered by filtration, washed with water, and finally air-dried.

#### *3.2. Characterization of the Starting Eucalyptus nitens Samples*

The compositional analysis of the starting untreated and ADW *Eucalyptus nitens* samples was carried out based on the standard NREL procedures [74,75]. XRD analysis was carried out using a vertical goniometer diffractometer D2-PHASER (Bruker, Billerica, MA, USA). The analyses were performed using the CuKα radiation at 1.54 Å as the X-ray source. The interval used was 5◦ < 2θ < 40◦, with a resolution of 0.016◦. DIFFRAC software (Bruker AXS, Karlsruhe, Germany) was used for spectra processing. The crystallinity index of the *Eucalyptus nitens* samples was calculated after deconvolution of the curves, which was carried out by the PeakFit software (Systat Software Inc., San Jose, CA, USA), taking into account the contribution of the amorphous component (at about 2θ = 21.5◦) and the peaks related to the crystalline plans with Miller indices 101, 10¯ı, 002 e 040, as reported in the literature [52]. The integration of the areas of these peaks allowed the estimation of the crystallinity index (CI) of the cellulose, based on Equation (3):

$$\text{CI}(\%) = \left[1 - (\text{A}\_{\text{AM}}/\text{A}\_{\text{Total}})\right] \times 100\tag{3}$$

where AAM is the area of the peak corresponding to the amorphous cellulose, and ATOT is the total area of all peaks.

Fourier Transform–Infrared (FT–IR) characterization of the biomass samples was performed with a Perkin-Elmer Spectrum-Two spectrophotometer (Perkin–Elmer, Waltham, MA, USA), equipped with an Attenuated Total Reflectance (ATR) apparatus. The acquisition of each spectrum has provided 12 scans, with a resolution of 8 cm−1, in the wavenumber range between 4000 and 450 cm−1.

#### *3.3. Alcoholysis Experiments*

MW-assisted alcoholysis of the untreated and ADW *Eucalyptus nitens* samples to BL was performed in the single-mode MW reactor (CEM Discover S-class System), employing the 35 mL vessel with a Teflon stir bar. Once the starting biomass, *n*-BuOH, *n*-dodecane (internal standard) and sulfuric acid (catalyst) were weighed in the vessel, the reactor was closed and the sealed system was irradiated up to the set-point temperature. The maximum pulsed-power of 300 W was used to heat the samples. During the reaction, pressure and temperature values were continuously acquired with the software and controlled with a feedback algorithm to maintain the constant temperature. At the end of each hydrolysis reaction, the reactor was rapidly cooled at room temperature by blown air and the solid-liquid slurry was recovered, filtered under vacuum, properly diluted with acetone, and analyzed by Gas Chromatography.

Alcoholysis experiments with conventional heating were carried out in the 60 mL glass reactor. Once the starting biomass, *n*-BuOH, *n*-dodecane (internal standard) and sulfuric acid (catalyst) were weighed in the reactor, it was closed and the sealed system was placed in an oil bath, previously heated to the set-point temperature. At the end of each alcoholysis reaction, the reactor was rapidly cooled at room temperature by blown air and the solid-liquid slurry was recovered, filtered under vacuum, properly diluted with acetone, and analyzed by Gas Chromatography. For this purpose, the reaction products were qualitatively identified by Gas Chromatography coupled with Mass Spectrometry (GC–MS), and subsequently quantified by Gas Chromatography coupled with Flame Ionization Detector (GC–FID). Regarding GC–MS analysis, a gas chromatograph Hewlett-Packard (Hewlett-Packard HP, Palo Alto, CA, USA) HP 6890 equipped with an MSDHP 5973 detector and with a G.C. column Phenomenex Zebron with a 100% methyl polysiloxane stationary phase (30 m × 0.25 mm × 0.25 μm), was used. The transport gas was helium 5.5 and the flow was 1 mL/min. The temperatures of the injection port and detector were set at 250 ◦C and 290 ◦C, respectively. The carrier pressure at 100 kPa and the split flow at 3.40 ms−1 were adopted. The oven was heated at 60 ◦C for 3 min, and then the

temperature was raised at 10 ◦C/min up to 260 ◦C for 5 min, and lastly, 10 ◦C/min up to 280 ◦C for 3 min. GC–FID analysis was carried out by a DANI GC 1000 DPC (Dani Instruments S.P.A., Cologno Monzese, Italy) gas chromatograph, equipped with a fused silica capillary column—HP-PONA cross-linked methyl silicone gum (20 m × 0.2 mm × 0.5 μm). The FID ports were set at 250 ◦C. The oven temperature program was set at 90 ◦C for 3 min and then increased at the rate of 10 ◦C/min up to 260 ◦C, where it was maintained for 5 min, then up to 280 ◦C with the rate of 10 ◦C/min and maintained for 3 min. Nitrogen was used as the carrier gas, at the flow rate of 0.2 mL/min. Quantitative determination of BL, DBE, BA, and unconverted *n*-BuOH was carried out with the internal standard method, using *n*-dodecane as the internal standard. Each analysis was carried out in duplicate and the reproducibility of the technique was within 5%.

The yield to BL was calculated as follows:

$$\text{Yield to BL (mol\%)} = \text{(mol BL/mol C}\_6\text{H}\_{10}\text{O}\_5 \text{ units in the starting biomass)} \times 100 \tag{4}$$

Besides, in the case of the Cross-Flow experiments, BL yield was calculated as follows:

$$\begin{aligned} \text{Yield to BL (mol\%)} &= \text{(mol BL obtained in the 2nd step/mol C}\_6\text{H}\_{10}\text{O}\_5 \text{ units} \\ &\quad \text{in the biomass added in the 2nd step} \times 100 \end{aligned} \tag{5}$$

#### *3.4. Experimental Design*

Response Surface Methodology (RSM) and Face-Centered Central Design (FCCD) were employed for the reaction optimization by maximizing the response, that is BL molar yield, investigating appropriate ranges of the independent variables. The chosen independent variables are temperature, reaction time, and catalyst loading, as reported in Table 4. Their levels were selected starting from preliminary One-Factor-at-a-Time (OFAT) experiments. The experimental design in this study required 18 experimental runs, which included 4 replicates. The software Design-Expert 12 (<IP_ADDRESS>) Trial Version (Stat-Ease, Inc., Minneapolis, MN, USA) was adopted to process and analyze the results. The data were fitted to the polynomial model and presented in the analysis of variance (ANOVA). The quadratic equation that represents the correlation between independent variables and the response can be expressed by the quadratic polynomial Equation (6):

$$Y = b\_0 + b\_1 X\_1 + b\_2 X\_2 + b\_3 X\_3 + b\_{11} X\_1^2 + b\_{22} X\_2^2 + b\_{33} X\_3^2 + b\_{12} X\_1 X\_2 + b\_{13} X\_1 X\_3 + b\_{23} X\_2 X\_3 \tag{6}$$

where *Y* is the predicted response, *b*0 the constant, *b*1, *b*2, and *b*3 the linear coefficients, *b*12, *b*13, and *b*23 the cross-product coefficients, and *b*11, *b*22, and *b*33 are the quadratic coefficients.

#### *3.5. Engine Experimental Setup*

A small diesel engine, whose specifications are reported in Table 10, was chosen and coupled with a Borghi and Saveri eddy current brake with rpm/torque controller. An AVL gravimetric fuel balance was used to online measure fuel consumption. An Environnement SA test bench, equipped with a Non-Dispersive Infra-Red (NDIR) Sensor, a paramagnetic sensor, a Heated Chemiluminescence Detector (HCLD), and a Heated Flame Ionization Detector (HFID) was employed to measure, respectively, CO and CO2, O2, NOx, and THC (Total Hydro-Carbons). The particulate matter was determined using a dedicated sample line and an AVL smoke meter. An exhaust gas K-type thermocouple was employed to verify the occurrence of the steady-state conditions, for each different test condition. Once the engine was stabilized in a particular operating condition, data were collected and analyzed to provide average values.

**Table 10.** Experimental engine characteristics.

#### **4. Conclusions**

In this work, autohydrolyzed-delignified *Eucalyptus nitens* wood was employed as cheap cellulose-rich feedstock for the one-pot alcoholysis to *n*-butyl levulinate, adopting *n*-butanol as green reagent/reaction medium, very dilute sulfuric acid as a homogeneous catalyst, and microwave as an efficient heating system. The effect of the main reaction parameters to *n*-butyl levulinate was investigated firstly by a traditional One-Factor-at-a-Time approach, to verify the feasibility of this reaction and identify the coarse ranges of the operating variables. Under the best reaction conditions (microwave heating, 190 ◦C, 15 min, biomass loading 20 wt%, 1.2 wt% H2SO4), the maximum *n*-butyl levulinate molar yield of about 30 mol% was achieved, using a very high biomass loading (20 wt%), an eligible aspect from the perspective of an intensified *high gravity* approach. However, even higher molar yields (up to about 40 mol%) were obtained adopting traditional heating (190 ◦C, 120 min, biomass loading 20 wt%, 1.2 wt% H2SO4), demonstrating the good feasibility of the reaction also with traditional heating systems, aspect of paramount industrial interest. The possibility of reprocessing the reaction mixture deriving from the optimized experiment by addition of fresh biomass, was evaluated, achieving the maximum *n*-butyl levulinate concentration of about 85 g/L after only one reprocessing of the mother liquor, and this is the highest *n*-butyl levulinate concentration hitherto reported in the literature starting from real biomass.

The butanolysis reaction was further optimized by Response Surface Methodology, utilizing a Face-Centered Central Composite Design. The chosen design appropriately describes the studied real system, which requires mild acidity and high temperature, for maximizing *n*-butyl levulinate production, while the effect of the reaction time is softened due to the efficient microwave heating. The significance of the independent variables and their possible interactions was tested using ANOVA with a 95% confidence level, and the model was experimentally validated at the optimal operating conditions for *n*-butyl levulinate production.

Finally, a preliminary study of diesel engine performances and emissions for a model mixture with a composition analogous to that of the main components of the reaction mixture was performed, to draw an indication of its potential application as an additive for diesel fuel, without performing the separation of each component.

**Author Contributions:** S.G., A.M.R.G., and S.F. conceived the experiments; S.G., A.M.R.G, C.A., S.F., M.L., D.L., and G.P. designed the experiments; S.G., M.L., and G.P. performed the experiments and analysis; all the authors analyzed the data; D.L. and S.G. wrote the paper; J.C.P., S.F., A.M.R.G., and C.A. revised and supervised the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Spanish Ministry of Economy and Competitivity supported this study in the framework of the research project "Modified aqueous media for wood Biorefineries" (reference CTQ2017-82962-R), partially funded by the FEDER program of the European Union. Mar López thanks the European Social Fund (ESF) for economic support and the "Xunta de Galicia" for her predoctoral grant (reference ED481A-2017/316). MIUR supported this study in the framework of the research project VISION PRIN 2017 FWC3WC\_002.

**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* **Sustainable Exploitation of Residual** *Cynara cardunculus* **L. to Levulinic Acid and** *n***-Butyl Levulinate**

**Anna Maria Raspolli Galletti 1,\*, Domenico Licursi 1, Serena Ciorba 1, Nicola Di Fidio 1, Valentina Coccia 2, Franco Cotana 2 and Claudia Antonetti 1,3**

**Abstract:** Hydrolysis and butanolysis of lignocellulosic biomass are efficient routes to produce two valuable bio-based platform chemicals, levulinic acid and *n*-butyl levulinate, which find increasing applications in the field of biofuels and for the synthesis of intermediates for chemical and pharmaceutical industries, food additives, surfactants, solvents and polymers. In this research, the acid-catalyzed hydrolysis of the waste residue of *Cynara cardunculus* L. (cardoon), remaining after seed removal for oil exploitation, was investigated. The cardoon residue was employed as-received and after a steam-explosion treatment which causes an enrichment in cellulose. The effects of the main reaction parameters, such as catalyst type and loading, reaction time, temperature and heating methodology, on the hydrolysis process were assessed. Levulinic acid molar yields up to about 50 mol% with levulinic acid concentrations of 62.1 g/L were reached. Moreover, the one-pot butanolysis of the steam-exploded cardoon with the bio-alcohol *n*-butanol was investigated, demonstrating the direct production of *n*-butyl levulinate with good yield, up to 42.5 mol%. These results demonstrate that such residual biomass represent a promising feedstock for the sustainable production of levulinic acid and *n*-butyl levulinate, opening the way to the complete exploitation of this crop.

**Keywords:** cardoon; waste biomass; hydrolysis; levulinic acid; alcoholysis; *n*-butyl levulinate; biofuels; microwaves

#### **1. Introduction**

Renewable resources have garnered increasing interest due to the shortage of petroleum, recurring rise in its price and environmental deterioration associated with its consumption, including pollutant by-products and greenhouse gases emission. The serious need to explore alternative resources respect to traditional ones for the production of chemicals and fuels has encouraged a new, more aware international policy of energy and economy. The European Union has recently incremented the renewable component of fuels and Italian legislation requires that 10% of fuels on sale in Italy are made of biofuel by 2020 [1,2]. The use of the waste biomass for energy purposes, instead of fossil fuels, should reduce the greenhouse effect, since biomass releases the same amount of carbon dioxide which has been previously trapped from atmosphere during photosynthesis. Additionally, it is worthy of notice that methane, a greenhouse gas 25 times more powerful than carbon dioxide, is emitted during the decomposition of organic material in landfills [3,4]. In this scenario, not only the simple thermal-valorization but also the chemical conversion of nonedible biomass to exploitable biomolecules should be developed and implemented, possibly avoiding any conflict with the food chain. In this regard, the ability to employ residual or waste biomasses as starting materials using water or a bioalcohol as reactants/reaction media

**Citation:** Raspolli Galletti, A.M.; Licursi, D.; Ciorba, S.; Di Fidio, N.; Coccia, V.; Cotana, F.; Antonetti, C. Sustainable Exploitation of Residual *Cynara cardunculus* L. to Levulinic Acid and *n*-Butyl Levulinate. *Catalysts* **2021**, *11*, 1082. https:// doi.org/10.3390/catal11091082

Academic Editor: Tomás García

Received: 1 August 2021 Accepted: 6 September 2021 Published: 8 September 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/).

represents an added value that fits well with the concept of green chemistry. In this context a growing interest has recently been directed to third-generation infesting plant species, such as the *Cynara cardunculus* (cardoon). It represents a promising resource to produce biomaterials and biochemicals and is a very common variety in the center of Italy and in the Mediterranean region [5]. *Cynara cardunculus* L. offers a wide spectrum of potential applications, being a rich source of fibers, oils and bioactive compounds [6,7]. Interestingly, the cultivation of such perennial herbaceous biomass shows significant advantages, such as good adaptability to climate change and growth on marginal or uncultivated lands with modest inputs, including little irrigation, care and minimal need of nutrients [8]. The seeds of the flower are exploited for oil production in food and bio-diesel supply chains. On the other hand, the nonedible lignocellulosic residues of this crop are reduced to a size of 20–40 mm by chipping and/or can undergo pretreatments that favor further exploitation of this biomass [9,10]. Steam-explosion is the most common and cost-effective method for pretreatment of lignocellulosic materials [11,12]. According to this process, chipped biomass is treated with high-pressure saturated steam for a few minutes at initial temperatures of 160–260 ◦C, corresponding to a pressure of 0.69–4.83 MPa, then the pressure is rapidly reduced, thus leading to explosive decompression of the biomass and fiber damage. The process causes hemicellulose degradation and lignin transformation, due to high employed temperatures, and, in addition, it reduces the cellulose crystallinity, thus increasing the effectiveness of an eventual subsequent cellulose valorization.

In the present research, two types of cardoon waste residues, remaining after seeds removal, were studied: the first one is the untreated defatted biomass, whereas the second one is the waste residue recovered after a steam-explosion pretreatment.

The hydrolysis of the cellulosic fraction of lignocellulosic biomass represents an efficient way to produce valuable platform chemicals, such as levulinic acid (LA) [13]. This last has been highlighted by the United States Department of Energy in 2010 as one of the 10 most promising building blocks in chemistry [14]. Due to its carboxyl and carbonyl functionalities, LA can be converted into various products for large-volume chemical markets, as biofuels, intermediates for chemical and pharmaceutical industries, food additives, surfactants, solvents and polymers [15]. In particular, the most promising LA derivatives for biofuel production are its alkyl esters, i.e., alkyl levulinates, γ-valerolactone, 2-methyltetrahydrofuran [16–19]. The LA yield is strongly affected by the cellulose content of the adopted feedstock [6,13] which can be changed by applying pretreatments [20]. Taking into consideration that LA is produced via hydrolysis of hexose sugars (glucose, fructose, mannose and galactose), cellulose-rich biomass proves the most suitable feedstock for its production [21].

The acid-catalyzed hydrolysis, assisted by microwave (MW) irradiation, was successfully performed for the two different samples of residual *Cynara cardunculus* L. and the effect of the main reaction parameters was investigated. Taking into account the high LA concentration reached, the hydrolysis of steam-exploded cardoon was also performed in a batch autoclave, in the perspective of a larger scaling-up. Moreover, in the same perspective of industrial applications, H2SO4 and HCl were selected as acid catalysts for LA synthesis due to their generally recognized advantages. They are characterized by low cost, abundant availability and high efficiency. Furthermore, they are usually employed in high TRL chemical processes, especially for the production of levulinic acid, such as the Biofine process and GF Biochemicals plants [22,23].

In addition, the direct production of *n*-butyl levulinate (BL) by alcoholysis with *n*butanol of the cellulose-rich steam-exploded biomass was also studied and discussed, being this strategy more promising than the generally adopted procedure of esterification of neat levulinic acid [24]. Indeed, the one-pot synthesis does not require intermediate operations concerning LA concentration and purification and reduces also the waste water treatment [25–27]. BL finds interesting applications not only as solvent and intermediate, but also as valuable bioblendstock for diesel fuel, being able to reduce the emissions of

particulates without increasing NOx emissions or worsening engine performance compared to neat conventional diesel fuel [27].

In Scheme 1 the two described approaches of valorization of cardoon by hydrolysis and alcoholysis are shown.

**Scheme 1.** Different hydrolysis and alcoholysis approaches of valorization of defatted *Cynara cardunculus* L. employed in the present research.

In the perspective of complete exploitation of the starting biomass, the solid residues recovered at the end of the hydrolysis and alcoholysis reactions were characterized by FT-IR spectroscopy, thermogravimetric and elemental analysis.

The obtained results of both hydrolysis and butanolysis runs highlight cardoon as a promising feedstock for multiproduct biorefineries. A notable example of a third-generation biorefinery fed by cardoon is the industrial site in Porto Torres (Italy). Novamont S.p.A. and ENI Versalis S.p.A. converted a petrochemical refinery into an integrated green chemicals plant, which involves local agriculture, including cultivation of cardoon and, currently, the production site is dedicated to bioplasticizers and biolubricants [28]. However, a new challenge for this innovative biorefinery is to integrate the manufacturing processes also with the synthesis of LA and ALs from the residual defatted biomasses, due to the excellent applicative perspectives of these bioproducts.

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

#### *2.1. Compositional Analysis of Crude Cardoon Samples*

The chemical compositions of the two investigated biomasses, the un-treated defatted cardoon residue (C) and the same biomass recovered after a steam-explosion pretreatment (E), are reported in Figure 1.

The steam-explosion pretreatment is a high-pressure and high-temperature physical process that has been widely used for biomasses in the last decades [29], since it allows increased availability of cellulose of the raw lignocellulosic matrix for further processing. The possible use of steam-exploded pretreated biomass is on one hand the production of bioethanol or more in general energy carriers [30] and on the other hand the possibility to obtain high value biochemical, such as the nanocrystalline cellulose [5] and other products useful for several chemical or pharmaceutical applications. The input matrices to the steam-explosion pretreatment section can be lignocellulosic softwoods, hardwoods and spruces but also residues obtained from agroindustrial activities, such as tomatoes, vineyard pruning or residues of infesting species, such as the *Cynara cardunculus* that can be considered third-generation biomass of growing interest for the production of sustainable biomaterials [31]. The process "intensity" is generally described by the severity factor (R0) that considers the process parameters, such as the temperature and the pressure. In the case of lignocellulosic materials, the process yields can be significantly increased using a double-step process of both solid and liquid fraction after the pretreatment [30]. The steam-explosion facility used for this research is available at the University of Perugia (CRB/CIRIAF) and it is composed of: (i) vapor generator; (ii) charging section for raw biomass; (iii) expansion valves; (iv) high-pressure reactor; (v) postexplosion tank and (vi) exploded liquid recovery section [30].

Both C and E samples show very different content of cellulose, about 38 and 65 wt%, respectively. In fact, after the steam-explosion pretreatment, the hemicellulose amount decreased from about 17 to 4 wt%, whereas extractives and ash were removed, as expected. On the other hand, lignin increased from about 17 up to 30 wt%, due to the reduction of the content of other components. The above-reported composition was determined on the dry biomasses and the enrichment of cellulose content in the steam-exploded sample proved very important from an industrial point of view because at equal biomass loading, it enables the processing of a higher amount of cellulose, which results in marked increases of target product concentrations in the reaction mixture with subsequent ease for the successive work-up, separation and purification processes. The available defatted raw sample C had a humidity amount of 5.9 wt%, while, as expected, the steam-exploded sample E had a humidity level of 73.6 wt%. Both wet as-received samples and dried ones were tested in the catalytic runs.

#### *2.2. MW-Assisted Hydrolysis of C and E Cardoon Samples*

A preliminary study on the effect of the catalyst types, their amount and humidity on LA formation was performed. The hydrolysis of dry cardoon, both C and E samples, was carried out using two acid catalysts, HCl and H2SO4. The amount of these mineral acids was calculated to have the same concentration of hydronium ions in the starting mixtures. Table 1 reports the results for the experiments performed employing a biomass loading of 10 wt% (on dry basis), working at 190 ◦C for 20 min in the MW reactor.

a Substrate to catalyst molar ratio: mol of anhydrous glucose unit in the starting biomass/mol of catalyst; b Glu = glucose; AA = acetic acid; FA = formic acid; LA = levulinic acid; c the sample was used after drying step; d the sample was employed as received.

In the presence of HCl, both dry samples (runs C1 and E1, Table 1) achieve comparable LA yields, 49.3 and 48.4 mol% respectively. Adopting the same dry samples, when H2SO4 was used (runs C2 and E2), lower LA molar yields were obtained, 41.5 and 40.7 mol% for C and E cardoon samples, respectively. As reported in previous studies, HCl enhances LA hydrolysis via a one-pot mechanism, since Cl− ions catalyze the intermediate 5-hydroxymethyl-2-furaldehyde (HMF) rehydration reaction, while SO4 2− ions are responsible for an inhibitor effect [32]. In all these runs, low amounts of unconverted glucose were detected, while no intermediates, such as furfural and HMF, were observed. On the other hand, appreciable amounts of formic acid (FA), which is another commodity chemical [8], were coformed by the hydrolysis of both C and E cardoon samples. Indeed, FA is coproduced during biomass hydrolysis [33] and its use in several fields has further encouraged the interest in LA synthesis. In our experiments, FA formation seems to be slightly facilitated when HCl is employed (compare runs C1 with C2 and runs E1 with E2) and for this reason, only HCl was selected for the subsequent investigation regarding the humidity grade. To investigate this parameter, both the cardoon samples C and E were adopted as-received with the humidity grade of 5.9 and 73.6 wt% respectively (runs C3 and E3), using the same experimental conditions of runs C1 and E1. As expected, dry and wet cardoon samples show similar LA molar yields, which are 49.3 and 50.7 mol% for dry and wet C (runs C1 and C3), respectively, and 48.4 and 49.7 mol% for dry and wet E (runs E1 and E3), respectively. Based on these results, since wet biomass can be directly used, only as-received wet cardoon samples were employed for a more detailed investigation. In the perspective of industrial application, the adoption of a high biomass loading is to be preferred, thus applying the high-gravity approach to achieve the highest products concentration. In other words, this is the same concept already applied regarding the cellulose content (on dry biomass) with the steam-explosion pretreatment. The high-gravity method presents several advantages for an industrial perspective: it increases the concentration of crude products, reduces the costs for their purification and waste-water treatment. On the other hand, in an over-loaded reactor, a more difficult physical agitation of the reaction slurry could lead to a significant decrease in the reaction rate. Table 2 reports the results of hydrolysis experiments carried out by increasing the biomass loading from 10 up to 20 wt% on dry basis, keeping constant the other reaction parameters (190 ◦C, 20 min, HCl as catalyst). In the same Table 2, the effect of the substrate to catalyst ratio was investigated at 20 wt% of biomass loading, for both wet C and E cardoon samples.

**Table 2.** Hydrolysis experiments on wet C and E cardoon samples with different biomass loadings and substrate/catalyst molar ratios. Experimental conditions: 190 ◦C, 20 min, HCl as catalyst, MW heating.

a determined on dry basis; b substrate to catalyst molar ratio: mol of anhydrous glucose unit in the starting biomass/mol of catalyst; c Glu = glucose; AA = acetic acid; FA = formic acid; LA = levulinic acid.

> For both C and E samples, no significant decrease of the yields was observed increasing the biomass loading. On the other hand, higher LA concentrations, from 15.9 up to 34.6 g/L for C samples, and from 25.5 to 58.9 g/L for E ones, were reached. Considering that lower concentrations of HCl allow minimizing environmental impact and process costs, Table 2

reports the results of experiments performed with lower HCl amounts, increasing the substrate/catalyst ratios from 0.9 up to 2.0 mol/mol, run C5, C6, C7 and runs E5, E6 and E7, for C and E samples respectively. These runs evidenced that LA molar yield is not affected by the reduction of the catalyst content, at least in the range of the investigated substrate to catalyst ratios. For both C and E samples, promising values of LA molar yields were obtained, as well as no significant formation of side-products. In the case of E biomass hydrolysis, even acetic acid was not observed, due to the low content of acetyl groups in this feedstock. As expected, higher LA concentrations were achieved from the cellulose-rich steam-exploded cardoon (E sample) rather than from that untreated (C sample), justifying the adopted pretreatment in the industrial perspective: the maximum reached value was 59.0 g/L for E and 34.6 g/L for C. On the other hand, it is interesting that the reactivity of the cellulosic fraction of the exploded biomass E is very similar to that of the untreated sample C, thus suggesting that pretreatment does not significantly modify the accessibility of the cellulose fibers. Regarding FA, its formation is almost equimolar with respect to LA for runs C3–E3 of Table 2, as expected from the overall hydrolysis reaction mechanism, whilst this correspondence does not fully fit for the remaining runs E4–E7 of Table 2 (lower FA molar concentration than the theoretical one), probably due to a combined effect of the type of feedstock, which has been previously steam-exploded (resulting more reactive than the crude sample to the hydrolysis), and of the high loading used for these runs, both leading to its more appreciable thermal degradation to CO2 and H2 in the liquid phase.

At the end of every run, a solid residue was recovered, accounting for about 30 wt% respect to the starting dry biomass. Due to the ponderal relevance of these residues, their characterization and applicative perspectives will be discussed later.

Regarding the presence of by-products, the reaction mixture certainly includes also low amounts of soluble impurities, which have not been considered up to now and which should be otherwise better characterized, to get information about the proper work-up procedures, although the concentration of any single by-product in the hydrolyzates is under the limit of detection of the HPLC analysis by routine refractive index detector (0.1 g/L) and, for this reason, not inserted in Tables 1 and 2. To detect trace amounts of impurities, higher sensitivity mass and/or UV detectors should be used. For this purpose, first of all, the crude hydrolysate obtained from run E7 of Table 2 was extracted by diethyl ether and the recovered extract was analyzed by the GC-MS technique. The analysis revealed the presence of oxygenated C5 compounds and of aromatics of lignin sources, such as guaiacol (2-methoxy phenol) and syringol (2,6-dimethoxy phenol) (see Figure S1, Supplementary Section,). The dehydration of C5 and C6 carbohydrates causes the formation of furanic monomers and soluble precursors of solid humins [34]. On the other hand, the above phenolic derivatives show characteristic absorptions in the ultraviolet region, in particular at 284 nm, due to carbonyl n π\* transitions [35], while stronger carbonyl π π\* transitions occur at a lower wavelength, at about 205 nm. Based on these statements, the HPLC-UV analysis of the crude hydrolysate was carried out at 284 and 205 nm, thus better differentiating furanic impurities from other carbonyl species, such as aliphatic carboxylic acids. Regarding the UV-HPLC chromatogram at 284 nm (Figure S2, Supplementary Section), it shows the presence of many furanic/aromatic species, which elute after 20 min, whereas at 205 nm aliphatic carbonyl species prevail (Figure S3, Supplementary Section). To obtain more in-depth information, the crude liquor was analyzed by HPLC-MS and total ion current (TIC) chromatogram related to all ions of all detected masses and UV chromatograms at 280, 250 nm and 205 nm are reported in the Supplementary Section, together with the chromatographic data of the main detected compounds including, where possible, the best matched chemical formula (Figure S4 and Table S1, respectively). At this level of investigation, given the high sensibility of this technique, the TIC signal due to the crude hydrolysate is very complex, including many impurities, besides LA (compound n◦ 17 of Table S1, Supplementary Section), which by far represents the main product of interest, as previously stated,. The cross-comparison with the GC-MS data confirms the presence of methoxyphenol and 2,6-dimethoxyphenol (compounds n◦ 36 and n◦ 38 of Table

S1 of Supplementary Section, respectively), as aromatics of lignin source. To obtain further useful information for the following discussion, some typical compounds deriving from biomass hydrolysis have been sought [36], paying particular attention to some selected carboxylic acids of interest [37]. On this basis, after having excluded the contribution of the LA as the main component, extracted ion chromatogram (XIC) of some typical organic acids deriving from biomass hydrolysis treatment were acquired (Figure S5/Table S2 and Figure S6/Table S3 of Supplementary Section), thus ascertaining the presence, in low abundance, of many of these, such as tartaric, malic, succinic, lactic, butyric, itaconic, maleic, pyruvic, glutaric, adipic, 2-hydroxy-2-methylbutyric, gluconic and citric acid. In addition, 5-HMF is still present, as well as its dimeric/trimeric derivatives, as confirmed by the corresponding XIC processing (Figure S7, Supplementary Section). Instead, furfural, the furanic compound deriving from the acid-catalyzed conversion of the hemicellulose fraction, has not been found, thus indirectly confirming the effectiveness of the steam-explosion treatment, which has allowed the preliminary removal of this biomass component. Lastly, glucose has been identified only in traces (Figure S5/Table S2 of Supplementary Section ), confirming the effectiveness of the acid-catalyzed conversion of the C6 fraction, as highly desired by our approach. Lastly, in order to demonstrate the cost-effectiveness of the hydrolysis process, the purification of the crude hydrolysate deriving from run E7 of Table 2 was carried out on laboratory scale adopting 2-methyltetrahydrofuran as extraction solvent, followed by subsequent fractional distillation of the corresponding extract, according to the experimental procedure reported in the Materials and Methods section. By this way, the final isolated LA yield of 20.3 wt% was ascertained, similar to that obtained from the HPLC analysis of the corresponding crude hydrolysate (run E7 of Table 2). The ascertained LA purity grade was 93%, estimated by both the GC and HPLC techniques, including FA, AA and angelica lactone as residual impurities. The latter compound originates from the acid-catalyzed LA dehydration, followed by ring closure, which typically occurs upon LA heating, during the purification procedure.

Steam-exploded cardoon E was selected for further studies regarding the effect of temperature on the hydrolysis reaction and Table 3 reports the results achieved at the lower temperature of 180 ◦C and the upper one of 200 ◦C, in comparison with the run at the temperature of 190 ◦C previously used. The obtained results confirm that 190 ◦C is the best temperature for the production of LA, as highlighted in the literature for the hydrothermal treatment of many lignocellulosic biomasses [13].

**Table 3.** Hydrolysis experiments on wet E cardoon sample, with different temperatures. Experimental conditions: reaction time = 20 min, sub/cat = 2.0 mol/mol, biomass loading = 20 wt% (on dry basis), HCl as catalyst (1.4 wt%), MW heating.

a Glu = glucose; AA = acetic acid; FA = formic acid; LA = levulinic acid.

Finally, the effect of the reaction time on LA formation was investigated (runs E7, E10 and E11, Figure 2). As reported in Figure 2, experiments were carried out on E sample at 20, 40 and 60 min of MW irradiation, under the same reaction conditions of temperature (190 ◦C), catalyst (HCl, 1.4 wt%), substrate-to-catalyst molar ratio (2.0 mol/mol) and biomass loading (20 wt%).

**Figure 2.** Hydrolysis experiments on wet E cardoon sample at different reaction times. Experimental conditions: 190 ◦C, HCl as catalyst (1.4 wt%), sub/cat = 2.0 mol/mol, biomass loading = 20 wt% on dry basis, MW heating.

Only a weak time effect on the hydrolysis efficiency was observed, achieving the best LA yield of 49.9 mol% with the highest LA concentration of 62.1 g/L after 40 min of heating, adopting HCl in a low amount, 1.4 wt%.

#### *2.3. Hydrolysis of E Cardoon under Conventional Heating*

Based on the results discussed in the previous section, cellulose-rich steam-exploded cardoon (E sample) proved to be a particularly promising residual biomass for LA synthesis. Therefore, this hydrolysis reaction was also studied in a batch autoclave with conventional heating to verify the possible process intensification on an industrial scale. Taking into account that the heat transfer process in the autoclave is slower than the heating mechanism in the MW reactor, a longer reaction time (120 min) was applied in the autoclave, adopting two different biomass loadings, 10 and 15 wt% on dry basis. For comparison, MW hydrolysis reactions had been carried out for 20 and 40 min, adopting the same biomass loadings. The results and the compositions of the main reaction products are reported in Table 4 and Figure 3, respectively.

**Table 4.** Hydrolysis experiments on wet E cardoon sample adopting different heating systems, biomass loadings and substrate to catalyst molar ratios. Experimental conditions: 190 ◦C, HCl as catalyst.

a Determined on dry basis; b substrate to catalyst molar ratio: mol of anhydrous glucose unit in the starting biomass/mol of catalyst.

In the case of biomass loading of 10 wt%, similar LA molar yields were reached with the two heating systems, 49.7 and 53.1 mol% in MW and autoclave, respectively, while in the case of 15 wt% of biomass loading, heating in autoclave resulted in slightly lower yield, 47.1 mol%, compared to 51.8 mol% in MW, probably due to mass and heat transfer limitations. However, the comparable high LA concentrations reached both in the autoclave and in MW, 41.4 and 45.5 g/L respectively, demonstrate that both heating methodologies can be successfully applied for performing this process.

**Figure 3.** Compositions of the main reaction products of runs E3, E12, E13 and E14 reported in Table 3.

#### *2.4. MW-Assisted Alcoholysis of E Cardoon Sample*

In the second part of our investigation, the one-pot alcoholysis reaction of wet E cardoon in *n*-butanol to BL was studied in the MW reactor and the results are reported in Table 5. The presence of a significant amount of introduced water, due to the high humidity of the sample, is an unprecedented approach respect to the up to now reported alcoholysis studies, performed in the presence of alcohol alone as reactant/reaction medium. This procedure allowed us to convert efficiently the starting biomass, without the initial drying step, thus at the same time-saving time and significant resources.

**Table 5.** Butanolysis experiments on wet E cardoon sample, adopting different biomass loadings and catalyst amounts. Experimental conditions: 190 ◦C, H2SO4 as catalyst, MW heating.

a Determined on dry basis; b substrate to catalyst molar ratio: mol of anhydrous glucose unit in the starting biomass/mol of catalyst.

In the presence of low contents of the acid catalyst H2SO4 (1.3 and 2.4 wt%) with the same substrate to catalyst molar ratio, and applying only 15 min of heating, the butanolysis led to BL molar yields of 42.5 and 22.1 mol%, using respectively the biomass loading of 8 and 15 wt% on dry basis, runs AE1 and AE2 (Table 5). When the lower catalyst loading of 1.3 wt% was maintained with the biomass loading of 15 wt%, a significant decrease of BL yield (19.8 mol%) was ascertained, essentially due to the above-mentioned mass transfer issues. Taking into account that the best BL yield, as well as a good BL concentration (27.5 g/L), were obtained in the run AE1, the effect of duration was studied adopting the same conditions but prolonging the reaction time to 30 and 45 min (runs AE4 and AE5, respectively). The achieved results show that the prolonging of the reaction time does not improve the BL production, whereas slightly lower BL yields and BL concentrations were obtained, demonstrating the efficacy of MW irradiation within a short reaction time. Finally, to better evaluate the impact of introduced water on the ascertained performances, an explorative run adopting the dry cardoon E sample was performed employing the same reaction conditions of run AE1: in this case, the BL concentration of 17.9 g/L was achieved with BL ponderal and molar yields of 26.4 wt% and 38.3 mol% respectively, proving similar

to that ascertained on wet cardoon. In all the above runs only traces of levulinic acid were detected, thus confirming that under an excess of bioalcohol the hydrolysis reaction is irrelevant and also when a certain humidity is introduced with the wet biomass.

At the end of the reaction for every run, a significant amount of solid residue was also ascertained, whose characterization and potential valorization will be discussed below.

The results of this preliminary study demonstrate that direct BL production can be performed not only from conventional starting materials (LA, disaccharides, polysaccharides and furfuryl alcohol [24]) but also directly, using raw steam-exploded defatted cardoon as the starting feedstock, opening the way to the direct conversion of the cellulosic fraction of this cheap, residual biomass in a valuable intermediate/bio-fuel.

#### *2.5. Characterization of Postreaction Solid Residues*

The solid residues recovered from the best MW-assisted hydrolysis and alcoholysis reactions to LA and BL, runs E7 and C7 for hydrolysis and run AE1 for alcoholysis, amounted respectively 30.6, 31.4 and 27.4 wt% of the starting biomass, calculated on a dry basis. All these samples were analyzed by elemental analysis and Table 6 reports the obtained results, compared with the C and E dry cardoon samples starting feedstocks.

**Table 6.** Results of the elemental analysis for the starting C and E dry cardoon samples, for the solid residues at the end of runs C7 and E7 (hydrolysis reactions) and for the solid residue at the end of run AE1 (alcoholysis reaction).

a Oxygen content was calculated by difference: O (%) = 100 (%)–C (%)–H (%)–N(%)–S(%).

The above data related to the starting feedstocks C and E confirm only a limited beginning of carbonization for the latter sample, as shown by the slight increase in its carbon content, occurred as a consequence of the mild steam-explosion treatment, aimed at the breakdown of the biomass matrix (cross-linking lignin), the bulk solubilization of the hemicellulose fraction and the removal of smaller hydrocarbon molecules (volatiles and gases) [38]. Instead, more advanced carbonization has occurred as a consequence of the acid-catalyzed hydrothermal treatment. H/C and O/C molar ratios of both solid residues at the end of runs C7 and E7 fall within the range reported in the literature for the hydrochars (H/C: ~0.8–1.4 and O/C: ~0.3–0.5) [39] and also in agreement with our previous work [40]. Therefore, the differences in the carbon content of the two different starting feedstocks (cardoon C and E, both as starting feedstocks in Table 6) have been attenuated by the acid-catalyzed hydrothermal treatment (solid residue of run C7 and E7, respectively, in Table 6), demonstrating the progressed carbonization occurred with this technology. Lastly, the postalcoholysis residue (solid residue of run AE1 in Table 6) does not show significant compositional differences respect to the posthydrolysis ones (solid residues of run C7 and E7 in Table 6), thus highlighting the similarity between the performed hydrolysis/alcoholysis treatments. These conclusions are also evident from the Van Krevelen diagram shown in Figure 4 which plots the molar ratios of the H/C and O/C for both the starting biomasses, cardoon samples C and E, and for the solid residues at the end of runs C7 and E7 for hydrolysis and run AE1 for butanolysis.

**Figure 4.** Van Krevelen diagram of starting biomasses, cardoon sample C7 and E7, and solid residues at the end of runs C7 and E7 for alcoholysis and run AE1 for butanolysis.

The positions of the starting feedstocks and the corresponding chars in the Van Krevelen diagram confirm that, in all cases, dehydration is the main allowed path, leading to the formation of carbonaceous material and the H/C and O/C ratios of all the obtained residues fall within the range reported in the literature for this kind of biomaterial (H/C: ~0.8–1.4 and O/C: ~0.3–0.5) [40,41]. In addition, the higher heating value (HHV) was calculated from elemental analysis (Table 6): the ascertained values significantly increase going from the starting feedstock to the corresponding chars, thus resulting comparable with that of the traditional lignite coal, in agreement with the conclusions gathered from the Van Krevelen plot [41].

Furthermore, FT-IR characterization of the postreaction solid residues was performed and Figure 5 shows the FT-IR spectra registered in ATR mode both for the starting crude cardoon, both C and E samples, and for the solid residues recovered after hydrolysis reactions, runs E7 and C7.

**Figure 5.** FT-IR spectra registered in ATR mode for the starting crude cardoon, both C and E samples, and for the solid residues recovered after hydrolysis reactions, runs E7 and C7.

The spectrum of crude sample C shown in Figure 5a shows characteristic signals of the three biopolymers, cellulose, hemicellulose and lignin: a broad band at about 3330 cm−1 assigned to vibration mode of O–H bonds and a band at 2910 cm−1 assigned to C–H stretching of methyl and methylene groups. The peak at 1734 cm−1 is due to C=O stretching in the acetyl group and carboxylic acid of hemicellulose, while the signals at 1600 and 1510 cm−1 are attributed to aromatic C=O stretching and C=C vibration of lignin [41,42]. Weak peaks are observed at 1422, 1375 and 1318 cm−1 that can be assigned to the C–H asymmetric mode of CH2 in cellulose, aromatic C–H and C–O in lignin, respectively [43,44]. The band at 1239 cm−1 is due to the C–O stretching of alcoholic, phenolic and ether groups [44]. The peak at 1155 cm−1 is assigned to C–O–C asymmetric stretching in cellulose and hemicellulose, the intense peak at 1030 cm−1 to C–O–H stretching and the one at 900 cm−1 to anomeric vibration at the β-glycosidic linkage, while the band at 590 cm−1 can be due to aromatic C–H bonds [40,45]. As expected, the peaks at 1734 and 1600 cm−1 are not noticeable in the spectrum of crude sample E showed in Figure 5b, since the steam-explosion pretreatment degrades hemicellulose and lignin. The FT-IR spectroscopy in ATR mode allows the characterization of functional groups on the material surface and the analysis is in agreement with the bulk composition determined by the NREL procedure for the samples of raw cardoon. In the case of the solid residues recovered after the posthydrolysis reactions 7E and 7C, reported again in Figure 5a,b respectively, peaks characteristic of cellulose, such as the intense peak at about 1030 cm−1 and the peak at about 900 cm−1, are strongly decreased in accordance with the fact that cellulose was efficiently converted to LA. However, signals that may be assigned to byproducts such as humins appear: a peak at about 1700 cm−1 due to the C=O stretching, another one at about 1600 cm−1 due to stretching vibration of C=C bonds of furanic rings and the last one at about 795 cm−1 due to aromatic bending off the plane of the C–H bond [38,44] are evident in the spectrum of both post-hydrolysis residues. Broad absorbance in the 1300–1100 cm−1 region could be ascribed to multiple C–OH stretching bonds. However, the intense signal at 1208 cm−1 evident in the posthydrolysis residues 7C and 7E could arise also from the presence of ether bonds [46]. Humins can result from condensation reactions between sugars, HMF and intermediates during the dehydration of carbohydrates [47–50] and their formation can be competitive with the rehydration of HMF to LA. Moreover, humins are complex and recalcitrant carbonaceous materials that can cover the substrate surface, making it less accessible to acid attack.

Figure 6 reports the FT-IR spectra of the solid residue recovered after the butanolysis reaction AE1 together with the starting crude E sample.

**Figure 6.** FT-IR spectra registered in ATR mode for the starting cardoon E sample and that of the solid residue recovered after alcoholysis reaction AE1.

In Figure 6, the FT-IR spectrum of the postalcoholysis char is shown, resulting very similar to that obtained after hydrothermal processing (Figure 5). In particular, the absorption bands at 1700 and 1600 cm−1 (C=O and C=C stretching vibrations, respectively) are visible also in the postalcoholysis char, as well as that at 1030 cm−1 (C–O–H stretching in cellulose), even if in this case of lower intensity respect to the crude E feedstock, thus confirming the occurred cellulose decomposition, as previously stated. In addition, the absorption bands at 1208 cm−1 (C–O stretching), and 795 cm−1 (C–H bending off the plane) further confirm the similarity between the chars produced by hydrolysis/alcoholysis. At this stage of the investigation, the severity of the performed treatments dampens any differences deriving from the use of different reaction solvents, otherwise reported by some authors very recently [51].

Moreover, the recovered residues and the starting biomasses were also characterized by thermogravimetric analysis and weight loss and weight loss thermograms are depicted in the Supplementary Section (Figure S8).

Regarding the devolatilization behavior of the starting feedstocks (Figure S8, thermograms (a) and (b)), the first peak is found below 100 ◦C and is due to the loss of humidity. The degradation of the cardoon starts at higher temperatures. In particular, a shoulder is found at about 250 ◦C, present only in the C cardoon starting feedstock, ascribed to hemicellulose fraction, overlapped with that of cellulose, in the range 300–350 ◦C [52,53]. Lastly, lignin degradation is very slow, occurring for the whole temperature range of the thermogravimetric analysis [52]. The comparison between the thermograms of the starting feedstocks, C and E cardoon samples ((a) and (b)), confirms the effectiveness of the steam-explosion treatment, which has allowed the selective removal of the hemicellulose fraction. Moreover, the E cardoon starting feedstock shows a very weak shoulder at about 400 ◦C, which is due to the lignin component [52], which proved more accessible and reactive as a consequence of the occurred steam-explosion pretreatment. On the other hand, the three residues deriving from hydrolysis/alcoholysis treatments ((c), (d), (e)) show analogous thermal profiles, thus confirming their chemical similarity, also in agreement with the previous characterization data. Again, the humidity loss of these samples occurs below 100 ◦C, then a degradation step was found at about 200 ◦C, due to the release of some organic compounds (such as LA and FA), trapped into this porous bio-material [54]. The absence of the degradation steps of hemicellulose and cellulose fractions confirms the effectiveness of the performed hydrolysis/alcoholysis treatments, whereas the main peak at about 400 ◦C is attributed to the decomposition of volatile lignin/furanic structures [54,55]. In all cases of the synthesized residues, a final weight loss of ~50 wt% was ascertained, thus revealing increased thermal stability above the starting biomass and demonstrating similar carbonization occurred [40], also in agreement with the previous characterization data.

The characterization of the obtained solid residues opens the way to their uses. Regarding this aspect, certainly the most immediate use is the combustion for the energy recovery, but this choice is currently considered as the last option, preferring, when possible, its reuse within the scopes of the circular economy [56]. For agricultural uses, the application of biochar to the soil can mitigate climate change by promoting carbon sequestration and decrease greenhouse gas emissions [57]. Moreover, char has been advantageously proposed as a growing medium, to be used in combination with other components (vermicultite, clays, etc.) to improve physicochemical soil properties, such as increase in cation exchange capacity, water holding capacity, available water, improvement of soil structure, reduction in soil acidity, microbiological activity, quality, and yield of the crops [57]. Use of the char as a soil amendment for the recovery of contaminated soils, including stabilization of organic and inorganic contaminants, has been proposed and seems attractive [57]. Lately, more added-value char-based products are under development in many research fields, such as adsorption, catalysis and electrochemical energy storage (lithium-ion batteries, lithium-sulfur batteries, sodium-ion batteries and supercapacitors), after having properly tuned its porosity and functional groups, by choosing the appropriate starting feedstocks and optimizing the reaction conditions [58].

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

#### *3.1. Materials*

Two types of cardoon waste residues after seeds removal were investigated: nonpretreated cardoon (C) and steam-explosion pretreated cardoon (E). Catalysts and chemicals were purchased from Sigma-Aldrich and employed as received: hydrochloric acid (HCl, 37 wt%), sulfuric acid (H2SO4, 95 wt%), 5-hydroxymethyl-2-furaldehyde (HMF, 98%), levulinic acid (LA, 98%), formic acid (FA, 98%), glucose (Glu, 99.5%), acetic acid (AA., 99%), furfural (99%), diethyl ether (98%), water for HPLC; n-butanol (94,5%), n-dodecane (99%), n-butyl levulinate (BL, 98%). Both C and E cardoon samples were used as received and/or after a drying step carried out at 105 ◦C in an oven until a constant weight was reached.

The biomass loading and the catalyst amount were calculated according to the following equations, respectively:

Biomass loading (wt%) = employed dry biomass (g)/[employed biomass (for dry or wet samples) (g) + solvent (g)] × 100;

Catalyst (wt%) = catalyst (g)/[catalyst (g) + employed biomass (for dry or wet samples) (g) + solvent (g)] × 100.

#### *3.2. Steam-Explosion Process Conditions*

The steam-explosion pretreatment was carried out on the untreated Cynara cardunculus L. sample C using the CRB/CIRIAF equipment [30]. In particular, 447.50 g of dry cardoon sample was treated at 165 ◦C and 200 bar for 10 min, employing the severity factor Log R0 equal to 2.91.

#### *3.3. Compositional Analysis of Raw Biomass*

For both C and E cardoon residues, the contents of cellulose, hemicellulose and lignin were determined according to the NREL protocol, as well as extractives and ash [59–61]. The content of humidity was estimated according to the NREL procedure [62].

#### *3.4. Acid-Catalyzed Hydrolysis of Cardoon*

Hydrolysis reactions were carried out in deionized water, using HCl or H2SO4 as catalyst, in the single-mode MW reactor CEM Discover S-class System (maximum pulsedpower 300 W, 35 mL pyrex vial). The reaction slurries were mixed with a magnetic stirrer and irradiated up to the set-point temperature for the selected rection time. During the reaction, pressure and temperature values were continuously acquired with the software and controlled with a feedback algorithm to maintain the constant temperature. Batch experiments were carried out also in an electrically heated 600 mL Parr zirconium madefixed head autoclave equipped with a P.I.D. controller (4848). The reactor was pressurized with nitrogen up to 30 bar and the reaction mixtures were stirred using a mechanical overhead stirrer. The reactions were carried out at the selected temperature for the chosen time. At the end of each reaction, the reactors were rapidly cooled at room temperature by blown air, the hydrolyze was separated from solid residuals by vacuum filtration, filtered with a PTFE filter (0.2 μm) and analyzed through high-pressure liquid chromatography (HPLC) with a refractive index detector.

#### *3.5. Acid-Catalyzed Alcoholysis of Cardoon*

Alcoholysis reactions were carried out in n-butanol, using H2SO4 as catalyst, in the MW reactor CEM Discover S-class System at 190 ◦C for the selected reaction time. At the end of each reaction, the slurry was filtered under vacuum on a crucible and the liquid samples were diluted with acetone and analyzed by a gas chromatograph coupled with a flame ionization detector (GC-FID).

#### *3.6. Analytical Techniques*

Liquid samples deriving from hydrolysis were analyzed by an HPLC system (Perkin Elmer Flexer Isocratic Platform) equipped with a Benson 2000-0 BP-OA column (300 mm × 7.8 mm) and coupled with a Waters 2140 refractive index detector. A 0.005 M H2SO4 aqueous solution was adopted as mobile phase, maintaining the column at 60 ◦C with the flow rate of 0.6 mL/min. The concentrations of hydrolysis products were determined from calibration curves obtained with external standard solutions. Each analysis was carried out in duplicate and the reproducibility of this analysis was within 3%.

Liquid samples from alcoholysis were analyzed by a GC-FID instrument (DANI GC1000 DPC) equipped with a fused silica capillary column–HP-PONA cross-linked methyl silicone gum (20 m × 0.2 mm × 0.5 μm). The injection and flame ionization detector ports were set at 250 ◦C. The oven temperature program was set at 90 ◦C for 3 min and then increased at the rate of 10 ◦C/min till it reached 260 ◦C, where it was maintained for 5 min and up to 280 ◦C with the rate of 10 ◦C/min and maintained for 3 min. Nitrogen was used as the carrier gas, at the flow rate of 0.2 mL/min. The quantitative analysis of alcoholysis products was performed by calibration using n-dodecane as internal standard. Each analysis was carried out in duplicate and the reproducibility of this analysis was within 5%.

The molar and the ponderal yields of the compounds of interest were calculated according to the following equations, respectively:

Molar Yield (mol%) = [product (mol)/anhydrous glucose unit in starting biomass (mol)] × 100;

Ponderal Yield (wt%) [product (g)/dry starting biomass (g)] × 100.

Analysis of impurities was performed by GC-MS and HPLC-MS. When GC-MS analysis was carried out, the starting aqueous hydrolysate was extracted with diethyl ether and the diluted extract (about 0.1–0.2 μL) was qualitatively analyzed by the instrument Hewlett-Packard HP 7890 (Palo Alto, CA, USA), equipped with an MSDHP 5977 detector and with a G.C. column Phenomenex Zebron with a 100% methyl polysiloxane stationary phase (column length 30 m, inner diameter 0.25 mm and thickness of the stationary phase 0.25 μm), in splitless mode. The transport gas was helium 5.5 and the flow was 1 mL/min. The temperature of the injection port was set at 250 ◦C, carrier pressure at 100 kPa. The oven was heated at 50 ◦C for 1 min, then the temperature was raised at 3 ◦C/min up to 250 ◦C and held for 5 min. When HPLC-MS analysis was carried out, a Sciex X500 qTOF mass spectrometer (Sciex, Darmstadt, Germany) was coupled to an HPLC Agilent 1260 Infinity II (Agilent, Waldbronn, Germany) and operated in ESI negative mode (spray voltage of −4500 V). HPLC system was equipped with a diode-array detector (DAD), which operated at 210, 250 and 280 nm. Chromatographic separation was achieved on a Zorbax SB-C18 column 150 mm × 4.6 mm, particle size 3.5 μm (Agilent, California, United States) as the stationary phase, using water with 0.1% (*v/v*) of formic acid (A) and methanol (B) as the eluents. The mobile phase flow rate was 0.7 mL/min, and the column oven temperature was set at 25 ◦C. The gradient was programmed as follows: 0–22.0 min at 95% (A), 22.0–30.0 min at 5% (A), 30.0 min at 95% (A), held for 5 min. The sample was filtered and 10 μL were injected into the HPLC system, after proper dilution (1:1) and filtration.

Fourier transform infrared (FT-IR) spectra for raw biomasses and solid residues after reactions were recorded in attenuated total reflection (ATR) mode with a Spectrum-Two Perkin-Elmer spectrophotometer. The acquisition of each spectrum provided 12 scans, with a resolution of 8 cm−1, in the wavenumber range between 4000 and 450 cm−1.

Thermogravimetric analysis (TGA) of starting feedstocks and solid residues after reactions was performed with a Mettler Toledo TGA/SDTA 851 apparatus in high purity N2. The sample was heated from 30 ◦C up to 900 ◦C, at a rate of 10 ◦C/min, under nitrogen atmosphere (60 mL/min). Both weight loss and weight loss rate were acquired during each experiment.

Elemental analysis (C, H, N, S) of starting feedstocks and solid residues after reactions was performed by a commercially available automatic analyzer Elementar Vario MICRO Cube (Elementar, Germany). These elements were quantified adopting a thermal conductivity detector (TCD). Lastly, oxygen content was calculated by difference: O (%) = 100

(%)–C (%)–H (%)–N (%)–S (%). HHV was calculated according to the following correlation proposed by Channiwala and Parikh [63]:

HHV (MJ/Kg) = 0.3491 C(%) + 1.1783 H(%) + 0.1005 S(%)−0.1034 O(%)−0.0151N (%)−0.0211 Ash(%).

#### *3.7. Purification of the Crude Mother Liquor*

Crude mother liquor was extracted by continuous liquid/liquid extraction of the aqueous mixture with 2-methyltetrahydrofuran as the extraction solvent. In a typical procedure, 20 mL of the crude hydrolyzate was treated with 60 mL of 2-methyltetrahydrofuran in a continuous liquid/liquid extractor apparatus for 4 h and then, once the organic fraction was separated, the extract was subjected to a fractional distillation, in order to remove the solvent and recover the LA as pure product, according to the general procedure previously patented [64]. Briefly, the extract was subjected to a first distillation step, in order to remove the solvent, working under atmospheric pressure, until the bottom temperature was 100 ◦C. The bottom product was further distilled, in order to remove any lights, using a 50 cm Vigreux column, working at 100 mbar, progressively increasing the temperature of the oil bath from 75 to 130 ◦C (corresponding temperatures of the bottom zone of the distiller in the range 40–115 ◦C), and the distillation was stopped when no vapors reached the top of the distillation apparatus. Lastly, the bottom product was further distilled, working at 5 mbar, increasing the oil bath temperature from 185 to 195 ◦C (corresponding temperatures of the bottom zone of the distiller in the range 135–145 ◦C). The isolated top fraction was dried by a mechanical pump and the dried fraction was characterized by HPLC and GC chromatography. The LA purity grade of 93% was ascertained and on the basis of weighted amount and taking into account the LA purity grade, the isolated yield was determined.

#### **4. Conclusions**

In this paper, the acid-catalyzed hydrolysis and alcoholysis of waste cardoon residues from agroindustry to give strategic platforms LA and BL, respectively, were investigated. After removal of oil seeds, the cardoon was employed directly and after a steam-explosion pretreatment, this last affording a cellulose-rich feedstock. This positive effect of the SE pretreatment can be applied to a larger scale only if its economic sustainability is verified: in this sense a specific cost-benefit analysis should be developed case by case, considering that the more expensive subsection of the SE facility is the vapor generator.

MW-assisted hydrolysis reactions on this last biomass led to high values of LA molar yield and concentration, up to 49.9 mol% and 62.1 g/L, respectively. The hydrolysis was also performed in a traditional batch autoclave, reaching LA yield and concentration of 47.1 mol% and 41.4 g/L, respectively, demonstrating that such a process can be successfully intensified, by switching to traditional industrial reactors. The achieved results are really interesting, highlighting that eco-friendly reaction conditions, such as high biomass to acid catalyst ratio, water as the reactant/reaction medium and energy-saving heating, can be adopted for the conversion of low-cost residual cardoon, implying that sustainable exploitation of such biomass can be developed. Moreover, a preliminary study on the acidcatalyzed butanolysis of steam-exploded cardoon led to good BL yields, up to 42.5 mol%, proving that the direct one-pot conversion of the cellulosic fraction of this waste biomass can supply a wider range of value-added products, including bio-fuels. Moreover, the characterization of the solid residues recovered from both processes has allowed us to envisage their possible exploitation, in a perspective of the complete valorization of cardoon biomass.

The proposed approaches represent alternative solutions to reduce consumption of fossil resources and carbon dioxide emissions and recycle massive amounts of agricultural residues, in agreement with the concept of third-generation biorefinery.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/catal11091082/s1, Figure S1: Total ion chromatogram (TIC) of the diethyl ether extract and corresponding mass spectra of the identified compounds, Figure S2: RI-and UV-HPLC chromatogram of the crude hydrolysate at 284 nm, Figure S3: UV-HPLC chromatograms of the crude hydrolysate at 205 and 284 nm, Figure S4: TIC related to all ions of all detected masses and corresponding UV chromatograms (280, 250 nm, superimposed) and 205 nm, Figure S5: XIC chromatogram obtained from selected compounds of interest, according to Gli ´nska et al. For numbering and corresponding assignments, see Table S2, Figure S6: XIC chromatogram obtained from selected compounds of interest, according to Ibáñez et al. For numbering and corresponding assignments, see Table S3, Figure S7: Monomeric/oligomeric (as dimeric/trimeric) compounds of 5-HMF and corresponding XIC chromatogram, Figure S8 and Table S1: Chromatographic data of the main detected compounds reported in Figure S4, Table S2: Chromatographic data of selected compounds of interest for hydrolyzates of biomass source, according to Gli ´nska et al. (see also XIC chromatogram of Figure S5), Table S3: Chromatographic data of selected compounds of interest for hydrolyzates of biomass source, according to Ibáñez et al. (see also XIC chromatogram of Figure S6).

**Author Contributions:** A.M.R.G., C.A., S.C., and D.L. conceived the experiments; A.M.R.G., S.C., D.L., N.D.F. and C.A. designed the experiments; N.D.F., S.C., V.C. and D.L. performed the experiments and analysis; all the authors analyzed the data; A.M.R.G., C.A., S.C., V.C. and D.L. wrote the paper; N.D.F. and F.C. revised and supervised the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the project VISION PRIN 2017 FWC3WC\_002 funded by MIUR and by the project PRA\_2018\_26 funded by University of Pisa.

**Acknowledgments:** The project VISION PRIN 2017 FWC3WC\_002 and the project PRA\_2018\_26 of University of Pisa are gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

## *Article* **Hydrogenative Cyclization of Levulinic Acid to** γ**-Valerolactone with Methanol and Ni-Fe Bimetallic Catalysts**

**Ligang Luo 1,\*,**†**, Xiao Han 1,**† **and Qin Zeng 1,2**

Received: 26 August 2020; Accepted: 14 September 2020; Published: 21 September 2020

**Abstract:** A series of Ni-Fe/SBA-15 catalysts was prepared and tested for the catalytic hydrogenation of levulinic acid to γ-valerolactone, adopting methanol as the only hydrogen donor, and investigating the synergism between Fe and Ni, both supported on SBA-15, towards this reaction. The characterization of the synthesized catalysts was carried out by XRD (X-ray powder diffraction), TEM (transmission electron microscopy), H2-TPD (hydrogen temperature-programmed desorption), XPS (X-ray photoelectron spectroscopy), and in situ FT-IR (Fourier transform–infrared spectroscopy) techniques. H2-TPD and XPS results have shown that electron transfer occurs from Fe to Ni, which is helpful both for the activation of the C=O bond and for the dissociative activation of H2 molecules, also in agreement with the results of the in situ FT-IR spectroscopy. The effect of temperature and reaction time on γ-valerolactone production was also investigated, identifying the best reaction conditions at 200 ◦C and 180 min, allowing for the complete conversion of levulinic acid and the complete selectivity to γ-valerolactone. Moreover, methanol was identified as an efficient hydrogen donor, if used in combination with the Ni-Fe/SBA-15 catalyst. The obtained results are promising, especially if compared with those obtained with the traditional and more expensive molecular hydrogen and noble-based catalysts.

**Keywords:** biomass ester derivatives; solvothermal processing; levulinic acid; γ-valerolactone; Ni-Fe bimetallic catalysts

#### **1. Introduction**

The world is highly dependent on the utilization of fossil resources to fulfill energy needs for the production of heat and power. Nevertheless, the surplus consumption of fossil fuels is escalating the concentration of atmospheric CO2, which causes serious global warming threats [1,2]. As a solution to the above primary challenges, considerable attention has been paid to the exploitation of renewable resources [1]. In this context, the conversion of the cellulose fraction of biomass into value-added platform chemicals, such as 5-hydroxymethylfurfural (5-HMF) and levulinic acid (LA), has been thoroughly investigated [2,3]. Moreover, the transformation of LA into more added-value bio-fuels and bio-chemicals is strategic, thanks to its reactive keto and carboxylic functional groups, which can be exploited for the synthesis of many valuable bio-chemicals, such as fuel additives, fragrances, solvents, pharmaceuticals, and plasticizers [4]. Among these high-value chemicals, γ-valerolactone (GVL) is receiving considerable attention to synthesize added-value bio-chemicals, such as food additives, solvents, and drug intermediates, as well as new bio-fuels [5,6]. Three pathways have been proposed for GVL production, as reported in Scheme 1. The first requires the high-temperature LA conversion in

the gas phase, occurring via its endothermic dehydration to angelicalactone, which is subsequently hydrogenated to GVL. The second occurs in the liquid phase, at lower temperatures, where the LA keto group is reduced to 4-hydroxyvaleric acid, which is in turn dehydrated to GVL. Lastly, the third possibility provides the esterification of LA to the less reactive alkyl levulinate, which undergoes hydrogenation and dehydration to GVL [6].

**Scheme 1.** Different pathways for the hydrogenation of levulinic acid (LA) to γ-valerolactone (GVL).

According to the above mechanisms, dehydration is generally favored by the presence of acid catalysts, whereas the hydrogenation is catalyzed by transition metals. Many homogeneous and heterogeneous catalysts have been employed for the hydrogenation of LA to GVL in the liquid phase [7]. However, given the well-known drawbacks of the homogeneous catalysts (the possible corrosion of the equipment, difficult recycle/reuse, and environmental concerns), heterogeneous ones have been certainly preferred [8]. Many noble metal catalysts have been tested for this reaction, in particular, Pt, Au, Pd, Rh, and Ru, in many cases achieving promising catalytic performances with respect to GVL [9–12]. For example, Son et al. have studied the catalytic performances of Ru/C, Ru/SBA, Au/ZrC, and Au/ZrO2 catalysts for the LA hydrogenation, highlighting the good catalytic performances and stability of the Au/ZrO2 catalyst, which can be reused five times, achieving a high GVL yield (90 mol %) [10]. However, although the noble metal catalysts show excellent performances for the hydrogenation of such carbonyl compounds, the high costs limit their further application in the industry. On this basis, non-noble metal catalysts remain the preferred choice for improving the sustainable catalytic transformation of LA to GVL [13,14]. In particular, Ni-based catalysts (Raney-Ni, Ni/MoOx/C, Cu/ZrO2, NiCu/Al2O3, and Mo2C) are active in this reaction [13–15]. For example, Mohan et al. have examined the influence of catalysts with Ni supported on SiO2, Al2O3, ZnO, ZrO2, TiO2, and MgO, highlighting the excellent catalytic performances of Ni/SiO2 towards the LA hydrogenation to GVL (0.8506 kg GVL kg catalyst−1 h−1 at 250 ◦C) [15]. The characterization of these catalysts by FT-IR spectroscopy has indicated that both Lewis and Brönsted acid sites play an important role in the dehydration of the intermediate 4-hydroxypentanoic acid [16], thus highlighting that the use of tunable bifunctional catalysts is preferred for improving the catalysis of this reaction. In this context, the addition of another metal could modify the electronic structure of Ni, decreasing the interaction between active metal and supports, thus enhancing the reaction activity [17]. For example, the Cu species had an important effect on the chemical environment of Ni species and acted as an electronic promoter on Ni surface active sites in Ni-Cu/Al2O3 bimetallic catalysts, achieving the highest GVL yield of 96 mol % (250 ◦C, 6.5 MPa, and 2 h) [18]. Regarding the possible hydrogen sources, LA hydrogenation has been carried out mainly with molecular H2 (0.8–6.5 MPa), both in batch autoclaves and in continuous flow reactors, generally obtaining high yields and selectivities to GVL [15–17]. However, this reaction requires large quantities of molecular H2, whose production is still mostly fossil based, which also raises cost and safety issues. In this context, formic acid and

alcohols have been proposed as alternative hydrogen donors, which should help one to avoid the usage of external H2 and, subsequently, the requirement for a high-pressure reactor [19]. Regarding formic acid, H2 is generated through the decarboxylation route (HCOOH → CO2 + H2), which is generally slow, unless noble metals, such as Ru, are used as catalysts [19,20]. However, Ruppert et al. have demonstrated that formic acid can be irreversibly adsorbed on the Ru/C catalyst, hampering the LA conversion to GVL [21]. Furthermore, agglomeration and metal leaching generally occur, which could greatly affect catalyst stability, thus significantly hampering its effective use [22]. Alternatively, it is possible to use alcohol as an H2 source, and perform a hydrogen transfer, which is known as the Meerwein–Ponndorf–Verley (MPV) reaction, which is catalyzed by Lewis acid sites, such as those of metal oxides or zeolites [23]. Two mechanisms have been proposed for explaining the hydrogen transfer in the presence of alcohols, both reported in Scheme 2 [23–25]. In the first one (path A, Scheme 2), the MPV reaction is catalyzed by the metal catalyst, which receives the hydrogen from the alcohol donor, through β-hydride elimination, and the activated hydrogen is then transferred from the metal to the acceptor molecule, in this case LA [24]. The second one involves an intermolecular hydrogen transfer, which occurs by adsorption of the hydroxyl group of the alcohol and the carbonyl group of LA (or its ester) on the catalyst surface [25]. This direct reaction is allowed thanks to the presence of Lewis and Brönsted acids, both leading to the formation of a six-membered ring transition state. Luo et al. have studied the MPV reaction of methyl levulinate to GVL in the presence of zeolites, confirming that the main rate-limiting step of this reaction is the hydride shift [25].

**Scheme 2.** Catalytic transfer hydrogenation mechanisms of LA.

Many kinds of alcohol have been employed for the MPV reaction, highlighting the higher reaction rates of the secondary alcohols. Chia et al. employed 2-BuOH to study the MPV transfer hydrogenation, achieving a good GVL yield (84.7 mol %), over the ZrO2 catalyst (150 ◦C, 16 h) [24]. A strong correlation has been established between the reducing capacity of alcohols and MPV reduction, according to this order: MeOH < EtOH < 1-BuOH < 2-BuOH = 2-PrOH [26]. However, by using MeOH or EtOH as hydrogen donors, more efficient and integrated biomass utilization chains could be created, thanks to the high availability of these alcohols from biomass feedstock. Tang et al. have used EtOH as a hydrogen donor, achieving the maximum GVL yield of 64 mol % (250 ◦C, 3 h) [26]. Yi et al. have reported that no hydrogen transfer observed within 48 h in the presence of EtOH, whilst a GVL yield of 38 mol % was achieved with 1 MPa H2, after only2h[27]. Despite the achieved progress, it is definitely still necessary to develop new, efficient and cheap catalytic systems for the LA hydrogenation to GVL, by MPV catalytic transfer, in the presence of short-chain primary alcohols.

In this work, new bimetallic Ni-Fe/SBA-15 catalysts have been synthesized and tested for the LA hydrogenation to GVL by the MPV route, in the presence of MeOH as the only hydrogen donor, and the oxophilic promotion of Fe was exploited to decrease the reduction temperature of the NiO phase and improve the reducibility of mixed oxides. Ni, Fe, and bimetallic Ni-Fe catalysts with different Fe/Ni molar ratios have been prepared by impregnation on the SBA-15 support, which helps the LA conversion to GVL. Moreover, this support provides high specific surface area (ranging between 690 and 1040 m2/g), narrow pore size distribution (average pore diameter between 5 and 30 nm), and high thermal stability (wall thicknesses between 3 and 5 nm) [28,29]. To investigate the relationship between catalyst structure and activity, characterization of the bimetallic Ni-Fe catalysts was performed by X-ray powder diffraction (XRD), transmission electron microscopy (TEM), hydrogen temperature-programmed desorption (H2-TPD), hydrogen temperature-programmed reduction (H2-TPR), X-ray photoelectron spectroscopy (XPS), and in situ Fourier transform–infrared spectroscopy (FT-IR). Lastly, the effects of the main reaction parameters on the GVL production have been evaluated and discussed, starting from the results of preliminary catalytic tests.

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

#### *2.1. Characterization of Catalysts*

First, the catalysts have been characterized by the XRD technique to investigate the atomic structure of the particles (Figure 1). The XRD patterns of Ni/SBA-15 catalyst exhibit three diffraction peaks at 2θ values of 44.2◦, 51.8◦, and 76.3◦, which are assigned to (111), (200), and (220) reflections of metallic Ni, respectively. Fu et al. reported analogous diffraction peaks for the Ni/Al2O3 catalyst at 2θ angles of 44.3◦, 51.7◦, and 76.3◦, assigned to metallic Ni [15]. Nevertheless, the absence of the diffraction peak at 37.3◦, due to the NiO species, confirms the complete reduction of the Ni particles [15]. In the case of the monometallic Fe/SBA-15 catalyst, another diffraction peak was found at about 44.7◦, which can be assigned to the (111) Fe cubic planes [30]. However, in the case of the Ni-Fe/SBA-15 catalysts, the Fe peak is shifted to lower 2θ, indicating a deep interaction between Ni and Fe particles to form face-centered cubic structures (fcc) of the Ni-Fe alloy, favored by the larger atomic radius of Fe, which increases the lattice constants [16].

**Figure 1.** XRD (X-ray powder diffraction) patterns of the Ni/SBA-15, 5Ni-1Fe/SBA-15, 5Ni-3Fe/SBA-15, 5Ni-5Fe/SBA-15, and Fe/SBA-15 catalysts.

TEM characterization of the synthesized catalysts was carried out to better investigate their textural properties. As shown in Figure 2, Ni particles (black spots) are quite well dispersed on the porous SBA-15 support. For Ni/SBA-15, the average Ni particle size is about 20 nm, which is in agreement with data in the literature [31]. The addition of Fe decreases the average size of Ni particles, which are more uniformly distributed, especially in the case of 5Ni-3Fe/SBA-15 (Ni particle size of about 9 nm), indicating that the Fe doping promotes a more homogeneous Ni dispersion, minimizing the formation of aggregates. However, when both metals are used in the same amount (5:5), the average particle size becomes higher (16 nm), highlighting that the Ni:Fe molar ratio should be carefully tuned. Based on these preliminary characterization data, the optimum achieved Ni:Fe molar ratio is 5:3.

**Figure 2.** TEM (transmission electron microscopy) images of (**A**) Ni/SBA-15, (**B**) 5Ni-1Fe/SBA-15, (**C**) 5Ni-3Fe/SBA-15, and (**D**) 5Ni-5Fe/SBA-15 catalysts.