3. The RO-ATMS and RO-AIRS inversion algorithms

#### 3.1 RO-ATM inversion algorithm

As mentioned above, MiRS is a MW inversion package used by NOAA to perform a physical-based microwave retrieval in all-weather scenarios over all landsurface types. The MiRS data products have been routinely assessed using independent measurements [9, 11, 12]. The MiRS package can be downloaded, for free, at http://mirs.nesdis.noaa.gov/download.php. In this study, we revised the current MiRS algorithm and use measurements from ATMS collocate with RO data to develop an enhanced RO-ATMS inversion algorithm (i.e., RO-MiRS). We included the RO refractivity forward operator (Eq. (1)) into the RO-MiRS algorithm.

#### 3.2 RO-AIRS inversion algorithm

Ref. [34] has detailed the information content for AIRS, RO, and the combined AIRS with RO for temperature and water vapor retrievals. Similar to the AIRS V6, the RO-AIRS inversion system is a 1D-var physical inversion system that retrieves the temperature and water vapor profiles sequentially. The updated fast and accurate AIRS transmittance model (Standard Alone AIRS-Radiance Transfer Algorithm package—SARTA [47]) is served as an AIRS forward operator. The configurations of background covariance matrix, error covariance matrix, and a priori used in the concurrent RO-AIRS retrieval are detailed in [48] and are not further descripted here.


**1. Introduction**

Vanadium and chromium are strategic transition elements that have been widely used in some fields such as steel-making, energy-storage, catalysts, the petrochemical industry, and green chemistry owing to their excellence hardness, high corrosion resistance, and other excellent physicochemical properties [1–5]. High-chromium vanadium slag (HCVS) is a by-product generated during the smelting process of high-chromiumvanadium-titanium-magnetite, and it is an important vanadium source in China [6–10]. During the smelting process, the vanadium and chromium are reduced into the molten enriched in the spinel, which is hard to destroy directly and restricted the large-scale utilization of HCVS [11]. Thus, some enhancement technologies were needed to recover vanadium and chromium efficiently.

To date, the basic recovery technology for vanadium has been sodium-roasting leaching technology, which was first proposed by Birck in 1912 and is widely used in the Chinese industries since the 1980s [12–14]. The vanadium-containing ores are mixed with the sodium salts (sodium carbonate (800–1000 ◦C), sodium sulfate (1200–1250 ◦C), sodium chloride (750–850 ◦C), and sodium hydroxide (400–800 ◦C)) at determined mole ratios and then roasted in a vertical kiln under a high temperature atmosphere with O2 [13,15,16]. The structure of vanadium-containing ores is destroyed in the high temperature and lowvalence vanadium is exposed and oxidized to a high valence. The high-valence vanadium

**Citation:** Peng, H.; Li, B.; Shi, W.; Liu, Z. Efficient Recovery of Vanadium from High-Chromium Vanadium Slag with Calcium-Roasting Acidic Leaching. *Minerals* **2022**, *12*, 160. https://doi.org/10.3390/ min12020160

Academic Editors: Jean-François Blais, Shuai Wang, Xingjie Wang and Jia Yang

Received: 10 January 2022 Accepted: 25 January 2022 Published: 28 January 2022

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

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

is formed as sodium vanadate and can be easily leached out with acid, alkaline, or waterleaching [17–20]. However, some environmental hazards (sulfur dioxide, chlorine, and hydrogen chloride) and large amount of wastewater have limited continuous large-scale industrial application with the high environmental standards of today. To overcome the above problems, a roasting technology called calcium-roasting technology was developed, in which the sodium salts are replaced by calcium salts [21–25]. The roasting process is similar to the sodium-roasting process; the vanadium-containing ores are mixed with lime, limestone, and calcium salts at fixed mole ratios and then roasted at high temperatures, which are higher than with sodium roasting. During the roasting process, the vanadium spinel is decomposed and reacted with calcium salts to form different kinds of calciumvanadate, which are determined by the mole ratio of the vanadium to calcium salts [26–30]. Usually, some leaching enhancing processes or multiple roasting processes accompany this process to achieve high recovery [16,31–34].

In this paper, direct acid leaching and calcium-roasting acid leaching technology were applied to leach out chromium and vanadium. The effects of experimental parameters including reaction time, liquid-to-solid ratio, reaction temperature, and concentration of H2SO4 on the leaching process were investigated. The leaching kinetics were also investigated.

#### **2. Materials and Methods**

#### *2.1. Materials*

The HCVS was collected from Pangang Group Co. Ltd., Panzhihua City, Sichuan Province, China. It was dried and ground to below 75 µm for further experiments. The elemental composition of HCVS was measured by XRF. The results displayed in Table 1 indicate that the vanadium and chromium were about 5.43 wt.% and 6.84 wt.%, respectively.

**Table 1.** Elemental accounts of the HCVS (wt.%).

#### *2.2. Experimental Procedure*

The batch experiments were conducted in a 300 mL glass beaker placed in a thermostatic mixing water bath. Firstly, the water bath was heated to a determined temperature. Then, a predetermined concentration of H2SO4 solution and a predetermined amount of HCVS or roasting HCVS were added to the beaker. Then, the beaker was placed in the water bath. Finally, the filtrate was collected by vacuum filtration after the required reaction time.

## *2.3. Analytical Methods*

The concentrations of chromium and vanadium in the filtrate were measured by inductively coupled plasma-optical mission spectrometry (ICP-OES, PerkinElmer Optima 6300DV, Kyoto, Japan.) and the leaching efficiency was calculated following Equations (1) and (2):

$$
\eta\_{\rm V} = \frac{\rm V \cdot C\_{\rm V}}{\rm m \cdot \omega\_{\rm V}} \times 100\% \tag{1}
$$

$$
\eta\_{\rm Cr} = \frac{\rm V \cdot C\_{\rm Cr}}{\rm m \cdot \omega\_{\rm Cr}} \times 100\% \tag{2}
$$

where CV and CCr, are the concentration of chromium and vanadium in the filtrate in g/L; V, is the volume of the filtrate in liters; ωV, and ωCr, are the percentages of chromium and vanadium in the HCVS; and m, is the mass of the HCVS used in the batch experiments in grams.

#### *2.4. Characterization*

The element percentages of HCVS were measured by XRF (Shimadzu Lab Center XRF-1800, Kyoto, Japan) and the main phases were measured by XRD (Shimadzu Lab Center XRD-6000, Kyoto, Japan). The valences of the vanadium and chromium in the HCVS were detected by UV-Vis DRS (Shimadzu Lab Center, Kyoto, Japan) and XPS (ESCALAB-250Xi, Thermo Fisher Scientific, New York, NY, USA). The thermo-gravimetric analysis was conducted by TG-DSC (Shimadzu Lab Center DSC-60H, Kyoto, Japan) with a heating rate of 10 ◦C/min from 0 ◦C to 900 ◦C.

#### **3. Results**

#### *3.1. Characterization of HCVS*

The XRD pattern showed in Figure 1a shows that the main crystal structures in the HCVS were Fe2O3, FeCr2O4, MgFe2O4, Fe2VO4 and Fe2SiO4 [27,28,30]. The vanadium and chromium mainly existed as spinel structures (FeCr2O4 and Fe2VO4), which are hard to destroy. Therefore, the leaching efficiency of vanadium and chromium may not be high and some enhancing technologies are needed in the further experiments. *Minerals* **2022**, *12*, x 4 of 12

**Figure 1.** Characterization of HCVS. (**a**) XRD pattern of HCVS; (**b**) UV-Vis DRS of HCVS; (**c**) XPS of vanadium; (**d**) XPS of chromium. **Figure 1.** Characterization of HCVS. (**a**) XRD pattern of HCVS; (**b**) UV-Vis DRS of HCVS; (**c**) XPS of vanadium; (**d**) XPS of chromium.

*3.2. Direct-Acid-Leaching Process*  The direct acid leaching process was conducted to leach out vanadium and chromium from the HCVS. Figure 2 summarizes the effects of the liquid-to-solid ratio, reaction time, concentration of H2SO4, and reaction temperature on the leaching process. The leaching efficiencies of vanadium and chromium were relatively low (<35% for vanadium and 8% for chromium), which is consistent with the results of XRD. Figure 2a shows that the leaching efficiency of vanadium increased with the increase of the concentration of H2SO4. During the leaching process, the H+ attacks the spinel and The UV-Vis DRS of HCVS was conducted to help understanding the composition of HCVS; the result is displayed in Figure 1b. The original spectrum signal was analyzed and the peaks were fitted to four main peaks: 280 nm, 380 nm, 482 nm, and 542 nm. The peak at 280 nm was assigned to Fe (III) and confirmed the existence of Fe2O3 and MgFe2O4 [35,36]. The peak at 380 nm was assigned to Cr (III) [37], which corresponds to the FeCr2O4 phase. The peak at 482 nm was assigned to V=O stretching, which confirmed the existence of V (IV) and V (V) [38,39].

destroys the spinel structure to release vanadium and chromium. With an increasing of concentration of H2SO4, the corrosion process of the spinel by the highly concentrated H+

33.90%, as the concentration of H2SO4 increased from 10 vt.% to 50 vt.%. As the chromium spinel was more stable than the vanadium spinel, the chromium was harder to leach out, with a leaching efficiency below 8%. Otherwise, the leaching efficiency showed no obvious increase when the concentration of H2SO4 increased from 40 vt.% to 50 vt.%, and high concentrations bring high impurities [40]; thus, the concentration of 40 vt.% was selected

The results showed in Figure 2b indicate that only 10.40% vanadium and 2.18% chromium can be leached out at 30 ℃. Higher temperatures enhances the activity of vanadium and chromium ions and further favors the reaction intensity [41]. The leaching efficiency increased to 33.89% for vanadium and 7.56% for chromium at 90 °C. In other words, high

reaction temperature was beneficial to the leaching process.

as optimal for further experiments.

The XPS results showed that most vanadium existed as V(III) and V(IV) (515.7 eV and 516.4 eV), and the Cr (III) accounted for about 82.84% of the HCVS (576.1 eV, 577.3 eV and 586.3 eV), while there were no V (III) and V (V) phases in the XRD pattern. According to our previous study, V(III), V(IV), and V(V) co-exist in HCVS, which means that some V(III) and V(V) compounds in the HCVS exist amorphously and could not detected by XRD [28]. contribution to the leaching process; thus, the reaction time of 3 h was selected in the following experiments. During the leaching process, the HCVS particles was ground fine enough for good contact with the concentrated H2SO4 solution. The leaching process was most controlled by parameters such as the reaction temperature and concentration of H2SO4, but less by

Usually, in order to produce more products, long reaction times are utilized. Figure 2c shows that the leaching efficiency of vanadium and chromium increased with the reaction time, but the increase amplitude was slow. Longer reaction times may not make any

#### *3.2. Direct-Acid-Leaching Process* the liquid-to-solid ratio, as the liquid-to-solid ratio had no obvious effect on the leaching efficiency (seen in Figure 2d).

The direct acid leaching process was conducted to leach out vanadium and chromium from the HCVS. Figure 2 summarizes the effects of the liquid-to-solid ratio, reaction time, concentration of H2SO4, and reaction temperature on the leaching process. The leaching efficiencies of vanadium and chromium were relatively low (<35% for vanadium and 8% for chromium), which is consistent with the results of XRD. As the spinel structure was hard to destroy directly, the leaching efficiencies of vanadium and chromium were 33.89% and 7.56%, respectively, at the selected optimum conditions: reaction time of 3 h, liquid-to-solid ratio of 4 :1 mL/g, concentration of H2SO4 of 40 vt.%, reaction temperature of 90 °C, and stirring rate of 500 rpm.

*Minerals* **2022**, *12*, x 5 of 12

**Figure 2.** Effect of parameters on leaching efficiency of vanadium and chromium: (**a**) concentration of H2SO4; (**b**) reaction temperature; (**c**) reaction time; (**d**) liquid-to-solid ratio. **Figure 2.** Effect of parameters on leaching efficiency of vanadium and chromium: (**a**) concentration of H2SO4 ; (**b**) reaction temperature; (**c**) reaction time; (**d**) liquid-to-solid ratio.

*3.3. Characterization of Roasting HCVS*  In order to achieve efficient leaching performance of HCVS, calcium-roasting technology was applied to oxidize the low valence compounds. The obtained TG-DSC curves shown in Figure 3a indicate that there was a dehydration step, with weight loss of 1.23% from 0 °C to 400 °C, and an obvious exothermic peak of the DSC curve at 400 °C was observed, which corresponds to the decomposition of the spinel structure. After the temperature increased to 620 °C, a dramatic mass gain of 6.46% was obtained due to the oxidative decomposition of the vanadium spinel phase. This means that the oxidative roasting of vanadium spinel should be conducted above 620 °C. Thus, the calcium-roasting Figure 2a shows that the leaching efficiency of vanadium increased with the increase of the concentration of H2SO4. During the leaching process, the H+ attacks the spinel and destroys the spinel structure to release vanadium and chromium. With an increasing of concentration of H2SO4, the corrosion process of the spinel by the highly concentrated H+ was intensified and the leaching efficiency of vanadium was increased from 17.87% to 33.90%, as the concentration of H2SO4 increased from 10 vt.% to 50 vt.%. As the chromium spinel was more stable than the vanadium spinel, the chromium was harder to leach out, with a leaching efficiency below 8%. Otherwise, the leaching efficiency showed no obvious increase when the concentration of H2SO4 increased from 40 vt.% to 50 vt.%, and high concentrations bring high impurities [40]; thus, the concentration of 40 vt.% was selected as optimal for further experiments.

process was conducted at 650–850 °C and the HCVS was mixed with CaO at a mole ratio

of n(CaO)/n(V2O5) = 1.1

The results showed in Figure 2b indicate that only 10.40% vanadium and 2.18% chromium can be leached out at 30 ◦C. Higher temperatures enhances the activity of vanadium and chromium ions and further favors the reaction intensity [41]. The leaching efficiency increased to 33.89% for vanadium and 7.56% for chromium at 90 ◦C. In other words, high reaction temperature was beneficial to the leaching process.

Usually, in order to produce more products, long reaction times are utilized. Figure 2c shows that the leaching efficiency of vanadium and chromium increased with the reaction time, but the increase amplitude was slow. Longer reaction times may not make any contribution to the leaching process; thus, the reaction time of 3 h was selected in the following experiments.

During the leaching process, the HCVS particles was ground fine enough for good contact with the concentrated H2SO4 solution. The leaching process was most controlled by parameters such as the reaction temperature and concentration of H2SO4, but less by the liquid-to-solid ratio, as the liquid-to-solid ratio had no obvious effect on the leaching efficiency (seen in Figure 2d).

As the spinel structure was hard to destroy directly, the leaching efficiencies of vanadium and chromium were 33.89% and 7.56%, respectively, at the selected optimum conditions: reaction time of 3 h, liquid-to-solid ratio of 4: 1 mL/g, concentration of H2SO4 of 40 vt.%, reaction temperature of 90 ◦C, and stirring rate of 500 rpm.

## *3.3. Characterization of Roasting HCVS*

In order to achieve efficient leaching performance of HCVS, calcium-roasting technology was applied to oxidize the low valence compounds. The obtained TG-DSC curves shown in Figure 3a indicate that there was a dehydration step, with weight loss of 1.23% from 0 ◦C to 400 ◦C, and an obvious exothermic peak of the DSC curve at 400 ◦C was observed, which corresponds to the decomposition of the spinel structure. After the temperature increased to 620 ◦C, a dramatic mass gain of 6.46% was obtained due to the oxidative decomposition of the vanadium spinel phase. This means that the oxidative roasting of vanadium spinel should be conducted above 620 ◦C. Thus, the calcium-roasting process was conducted at 650–850 ◦C and the HCVS was mixed with CaO at a mole ratio of n(CaO)/n(V2O5) = 1.1

The XRD pattern was used to analyze the phase changes during the calcium-roasting process. The results showed in Figure 3b indicate that some new peaks appeared, corresponding to the new phases of Ca2V2O5, CaFe (Si2O6), and Ca2V2O7. These three new phases appeared at 650 ◦C, and the crystal structures became more stable as the roasting temperature increased from 650 ◦C to 850 ◦C. During the calcium-roasting process, the Fe2VO4 decomposed (seen in Equation (3)) to form V2O4 at nearly 400 ◦C according to DG-TSC results, and then reacted with CaO to form Ca2V2O5. With the increasing roasting temperature, partial Ca2V2O5 was further oxidized to Ca2V2O7, which means that in the calcium roasting of HCVS, the V(IV) and V(V) co-existed.

After roasting, some V(III) and V(IV) were oxidized to V(V). The XPS results indicate that only 9.55% V(III) was retained in the roasted HCVS, while Cr(III) still accounted for about 80.32%. As the Cr spinel was more stable than the V spinel [13], the Cr was not oxidized and still existed in FeCr2O4, according to the XRD results. It was concluded that the chromium was still hard to leach out.

$$\text{2Fe}\_2\text{VO}\_4 \to \text{4FeO} + \text{V}\_2\text{O}\_4\tag{3}$$

$$\text{V}\_2\text{O}\_4 + \text{CaO} \rightarrow \text{CaV}\_2\text{O}\_5\tag{4}$$

$$2\text{CaV}\_2\text{O}\_5 + \text{O}\_2 + 2\text{CaO} \rightarrow 2\text{Ca}\_2\text{V}\_2\text{O}\_7\tag{5}$$

cium roasting of HCVS, the V(IV) and V(V) co-existed.

the chromium was still hard to leach out.

**Figure 3.** Characterization of roasting HCVS. (**a**) TG and DSC; (**b**) XRD pattern of roasted HCVS; (**c**) XPS of vanadium; (**d**) XPS of chromium. **Figure 3.** Characterization of roasting HCVS. (**a**) TG and DSC; (**b**) XRD pattern of roasted HCVS; (**c**) XPS of vanadium; (**d**) XPS of chromium.

The XRD pattern was used to analyze the phase changes during the calcium-roasting process. The results showed in Figure 3b indicate that some new peaks appeared, corresponding to the new phases of Ca2V2O5, CaFe (Si2O6), and Ca2V2O7. These three new phases appeared at 650 °C, and the crystal structures became more stable as the roasting temperature increased from 650 °C to 850 ℃. During the calcium-roasting process, the Fe2VO4 decomposed (seen in Equation (3)) to form V2O4 at nearly 400 °C according to DG-TSC results, and then reacted with CaO to form Ca2V2O5. With the increasing roasting temperature, partial Ca2V2O5 was further oxidized to Ca2V2O7, which means that in the cal-

After roasting, some V(III) and V(IV) were oxidized to V(V). The XPS results indicate that only 9.55% V(III) was retained in the roasted HCVS, while Cr(III) still accounted for about 80.32%. As the Cr spinel was more stable than the V spinel [13], the Cr was not oxidized and still existed in FeCr2O4, according to the XRD results. It was concluded that

**2Fe VO 4FeO + V O 2 4 24** → (1)

**V O + CaO CaV O 24 25** → (2)

**2CaV O + O +2CaO 2Ca V O 2 5 2 22 7** → (3)

## *3.4. Acid Leaching for Roasting HCVS*

The acid-leaching experiments with calcium roasting of HCVS were conducted to investigate the effect of calcium roasting on the leaching process under the same reaction conditions as the direct acid leaching process described above. As the Cr spinel was still stable under the roasting temperature, the leaching efficiency of chromium showed no obvious increase compared with the direct acid leaching process; therefore, the leaching behavior of chromium is not discussed in this part.

Figure 4a shows that the calcium roasting made a great contribution to the leaching process. The leaching efficiency of vanadium was increased by nearly 40 percentage (up to 57.54%) after roasting, compared with the direct acidic leaching process (at the concentration of 10 vt.% H2SO4). During the roasting process, most V(III) and V(IV) were oxidized to V(V), making a contribution to the great leaching performance of roasting HCVS. The leaching efficiency increased quickly at the beginning and then smoothly with the increase of H2SO4 concentration. The highest leaching efficiency was up to 90.12% at a concentration of 50 vt.%, which showed nearly a 58% improvement compared to the direct acid leaching process. Compared to our previous study, the leaching efficiency might be increased more with some enhancing technologies, such as oxidative leaching and electro-oxidative leaching [28]. Otherwise, the formation of the by-product CaSO4, which is a villous particle, might adsorb on the surface of leaching residue and have negative effects on the leaching process [40]; thus, a too high concentration of H2SO4 is not suitable for leaching while calcium roasting HCVS. Meanwhile, the leaching efficiency showed little increase as the concentration increased from 40 vt.% to 50 vt.%; thus, a concentration of H2SO4 of 40 vt.% was selected for further experiments.

**Figure 4.** Effect of parameters on leaching efficiency of vanadium and chromium: (**a**) concentration of H2SO4; (**b**) reaction temperature; (**c**) reaction time; (**d**) liquid-to-solid ratio. **Figure 4.** Effect of parameters on leaching efficiency of vanadium and chromium: (**a**) concentration of H2SO4 ; (**b**) reaction temperature; (**c**) reaction time; (**d**) liquid-to-solid ratio.

*3.5. Leaching Kinetics*  In order to understand the reaction mechanism, the leaching kinetics of vanadium were analyzed (leaching out chromium was very difficult; thus, it is not analyzed here). Usually, the leaching kinetics followed the shrinking core model described in Equation The same phenomenon can also be observed in Figure 4b. The leaching process was greatly enhanced by calcium roasting; the leaching efficiency was increased from 10.4% to 89.12% as the reaction temperature increased from 30 ◦C to 90 ◦C. Usually, metal ions have high solubility at high temperatures, accompanied by high activity; thus, 90 ◦C was chose in further experiments.

(6), which was used to describe the liquid-solid reaction [40,42–45]: 1/3 [(1 ) 1] 1 / 3 Ln (1 ) k t − −η − + ⋅ −η = ⋅ (6) where *η* is the leaching efficiency of vanadium, in percentage. The experimental data was fitted to Equation (6) and the results are shown in Figure The results displayed in Figure 4c,d indicate that the liquid-to-solid ratio and reaction time showed similar effects on the leaching process, and a suitable liquid-to-solid ratio and a long reaction time could achieve high leaching efficiency. As can be seen, the calciumroasting process can oxidize low valence vanadium to high valence vanadium and enhance the leaching process to achieve high leaching efficiency of vanadium, but has no influence on the change trend of leaching efficiency affected by the experimental parameters.

5. Based on the fitting results, the Ea for vanadium leached out was calculated following the Arrhenius equations (Equation (7)). Figure 6 shows that the Ea for vanadium leached out was 58.95 kJ/mol for the direct acid leaching process and 62.98 kJ/mol for the calciumroasting acid leaching process, which indicates that the controlling step for vanadium leaching is the surface chemical reaction [40,43–45]. Compared with the references [40,43,45,46], the Ea was much larger, indicating that the vanadium in the HCVS was hard To summarize, low valence vanadium in V spinel was decomposed and oxidized to V(V) during the calcium-roasting process, but Cr spinel was too stable to decompose. For vanadium, 89.12% was leached out under the optimal reaction conditions: reaction time of 3 h, reaction temperature of 90 ◦C, liquid-to-solid ratio at 4:1 mL/g, concentration of H2SO4 at 40 vt.%, and stirring rate at 500 rpm. Most chromium existing as FeCr2O4 was hard to leach out and was retained in the leaching residue.

#### to leach out by both direct acid leaching technology and calcium-roasting acid leaching *3.5. Leaching Kinetics*

mole gas constant.

technology. In order to improve the leaching efficiency and enhance the leaching process, some more efficient pre-treatment technologies are needed. Lnk LnA Ea / RT = − ( ) (7) where Ea is the apparent activation energy, A is the pre-exponential factor, and R is the In order to understand the reaction mechanism, the leaching kinetics of vanadium were analyzed (leaching out chromium was very difficult; thus, it is not analyzed here). Usually, the leaching kinetics followed the shrinking core model described in Equation (6), which was used to describe the liquid-solid reaction [40,42–45]:

$$\frac{1}{2}\left[\left(1-\eta\right)^{-1/3}-1\right]+1/3\cdot\text{Ln}(1-\eta)=\mathbf{k}\cdot\mathbf{t}\tag{6}$$

where η is the leaching efficiency of vanadium, in percentage.

The experimental data was fitted to Equation (6) and the results are shown in Figure 5. Based on the fitting results, the Ea for vanadium leached out was calculated following the Arrhenius equations (Equation (7)). Figure 6 shows that the Ea for vanadium leached out was 58.95 kJ/mol for the direct acid leaching process and 62.98 kJ/mol for the calciumroasting acid leaching process, which indicates that the controlling step for vanadium leaching is the surface chemical reaction [40,43–45]. Compared with the references [40,43,45,46], the Ea was much larger, indicating that the vanadium in the HCVS was hard to leach out by both direct acid leaching technology and calcium-roasting acid leaching technology. In order to improve the leaching efficiency and enhance the leaching process, some more efficient pre-treatment technologies are needed.

$$\text{Lnk} = \text{LnA} - \text{Ea/(RT)}\tag{7}$$

where Ea is the apparent activation energy, A is the pre-exponential factor, and R is the mole gas constant. *Minerals* **2022**, *12*, x 9 of 12 *Minerals* **2022**, *12*, x 9 of 12

**Figure 5.** Plot of leaching kinetics of vanadium at various reaction temperatures. (**a**) direct acid leaching process; (**b**) calcium-roasting acid leaching process. **Figure 5.** Plot of leaching kinetics of vanadium at various reaction temperatures. (**a**) direct acid leaching process; (**b**) calcium-roasting acid leaching process. **Figure 5.** Plot of leaching kinetics of vanadium at various reaction temperatures. (**a**) direct acid leaching process; (**b**) calcium-roasting acid leaching process.

**Figure 6.** Natural logarithm of reaction rate constant versus reciprocal temperature of vanadium. **Figure 6.** Natural logarithm of reaction rate constant versus reciprocal temperature of vanadium. reaction, with an *Ea* of 58.95 kJ/mol for the calcium-roasting acid leaching process. **Figure 6.** Natural logarithm of reaction rate constant versus reciprocal temperature of vanadium.

were conducted. The following conclusions were obtained:

(1) The chromium and vanadium existed as spinel structure in the HCVS, which are too stable to destroy directly; only 33.89% of vanadium and 7.56% of chromium could be leached out at the selected conditions during the direct acid leaching process: reaction time of 3 h, liquid-to-solid ratio at 4:1 mL/g, concentration of H2SO4 at 40 vt.%, reaction temperature of 90 °C, and stirring rate at 500 rpm. The *Ea* of the vanadium leached out was 62.98 kJ/mol, which indicates that the vanadium was hard to leach out directly; (2) Most low valence vanadium could be oxidized to high valence during the calciumroasting process, and the leaching efficiency could achieve 89.12% under the optimal conditions: reaction time of 3 h, liquid-to-solid ratio at 4:1 mL/g, reaction temperature of 90 °C, concentration of H2SO4 at 40 vt.%, and stirring rate at 500 rpm. The leaching behavior followed the shrinking core model well, and the controlling step was the surface chemical

(1) The chromium and vanadium existed as spinel structure in the HCVS, which are too stable to destroy directly; only 33.89% of vanadium and 7.56% of chromium could be leached out at the selected conditions during the direct acid leaching process: reaction time of 3 h, liquid-to-solid ratio at 4:1 mL/g, concentration of H2SO4 at 40 vt.%, reaction temperature of 90 °C, and stirring rate at 500 rpm. The *Ea* of the vanadium leached out was 62.98 kJ/mol, which indicates that the vanadium was hard to leach out directly; (2) Most low valence vanadium could be oxidized to high valence during the calciumroasting process, and the leaching efficiency could achieve 89.12% under the optimal conditions: reaction time of 3 h, liquid-to-solid ratio at 4:1 mL/g, reaction temperature of 90 °C, concentration of H2SO4 at 40 vt.%, and stirring rate at 500 rpm. The leaching behavior followed the shrinking core model well, and the controlling step was the surface chemical reaction, with an *Ea* of 58.95 kJ/mol for the calcium-roasting acid leaching process.

**4. Conclusions**

**4. Conclusions**


*3.1. Microscopic Examination*

A significant number of honeydew elements were detected in all honeys. The great majority of the samples also contained fungal spores which are present solely in pine honeys [48]. The HDE/P ratio showed a great variation and was on average 8.23 ± 16.98, while the total number of pollen grains in 10 g of honey was 57 871 ± 55 867 (Tables 1 and S1). In about half of the samples, the HDE/P ratio was below 3, which is the minimum value for honeydew honeys proposed in the past [49]. However, later studies have shown that the HDE/P ratio of honeydew honeys may vary and Greek pine honeys have relatively few honeydew elements and large numbers of pollen grains [39,48,50–53]. Thus, the samples were considered typical Greek pine honeys.

**Table 1.** Mean values of physicochemical parameters.

1 RSA: Radical Scavenging Activity. SD: Standard Deviation.


Thus, we aimed to evaluate the changes in plasma levels of these bioactive lipids in healthy runners performing a half-marathon, at the end of the race and after 24 h recovery, and their associations with new recently proposed and common biomarkers of immune activation, which are:

### **2. Materials and Methods**

### *2.1. Characteristics of the Participants*

The studied population included 13 healthy Caucasian trained runners belonging to the "Gruppo Podistico Rossini" enrolled during the 2018 edition of the Pisa half-marathon. Inclusion criteria of athletes were regular training and absence of cardiovascular disease or any other systemic disorder. Preliminarily to the race, each participant was submitted to a questionnaire to obtain demographic and clinical data and training history. Body mass index (BMI) was calculated by height and weight, measured in each subject.  The study was conducted according to the guidelines of the Declaration of Helsinki, and was approved by the Pisa Ethics Committee, Italy (protocol number for study acceptance 2805). Informed consent was obtained from all subjects enrolled in the study.

### *2.2. Sample Collection, Preparation and Evaluation of Lipids and Inflammatory Markers*

Three blood samples were collected from the peripheral vein of the athletes (in fasting conditions): (1) the day before the race (baseline), (2) within 20 min after the end of the half-marathon (post) and (3) 24 h after the run (24 h, recovery period). Blood samples were immediately centrifuged at 2500 *g* for 10 min and stored at −80 ◦C until assayed. Ceramides, SM and DAG were evaluated from 20 μL of plasma (upon being deproteinized using 200 μL of cold methanol) in 13 healthy Caucasian trained runners.

An aliquot of 20 μL of a mix of internal standards SM (d18:1/17:0), ceramide (d18:1/17:0), (Avanti Polar Lipids, Alabaster, AL, USA); DAG (17:0/17:0) (Larodan Solna SE) was added to the sample before deproteinization. The extract was injected in high performance liquid chromatography (Agilent UHPLC 1290, Santa Clara, CA, USA), coupled with a quadrupole time-of-flight mass spectrometry QTOF (QTOF-MS, Agilent 6540, Santa Clara, CA, USA), equipped with electrospray ionization source (ESI). For liquid chromatography analysis, we used ZORBAX Eclipse Plus C18 2.1 × 100 mm 1.8-micron column at 50 ◦C (Agilent, Santa Clara, CA, USA). The mobile phase A was made by water with 0.1% formic acid and the mobile phase B was made by isopropanol/acetonitrile (1:1, *v*:*v*) with 0.1% formic acid. Injection volume was 1 μL. Lipids were measured in positive electrospray ionization and identified using an internal spectral library. The data were normalized by internal standard representative lipids present in the sample and the analysis of the peaks was performed with Agilent MassHunter software program [11]. DAG species founded in plasma were 32:0, 32:1, 34:1, 34:2, 36:3 and 36:4, while ceramide species evaluated in plasma sample were 18:1/16:0, 18:1/18:0, 18:1/25:0, 18:1/26:0, 18:1/22:0, 18:1/24:1, 18:1/24:0, 18:0/24:0. The metabocard for ceramide species was reported as supplementary material (supplemental data). Fractalkine CX3CL1, VEGF, IL6 and TNFα were analyzed in 25 μL of plasma by a specific assay (MILLIPLEX MAP Millipore corporation, Billerica, MA, USA) using an integrated multi-analyte detection platform (high-throughput technology MagPix system, Luminex xMAP technology) with combined analyst software (MILLIPLEX®) for the biomarkers quantification developing new curve fitting algorithms and optimizing mathematical methods to minimize fitting errors.

### *2.3. Statistical Analysis*

Data were expressed as mean ± SD. **Repeated-measures ANOVA** was used for compared data from the same subjects measured more than once (baseline, post, 24 h). Correlation analysis was performed by Spearman parametric test to assess the relationship between continuous variables. Post Δ and 24 h Δ values from baseline (differences) and % change at different successive time periods with respect to values observed at baseline were calculated for fractalkine (pg/mL), IL-6 (pg/mL), TNFα (pg/mL) and VEGF-α (pg/mL). Owing to skewness, log transformation of fractalkine and TNFα was used for statistical analyses. Log-transformed values were then back-transformed for data presentation.

Data statistical analyses were performed with the Statview statistical package, version 5.0.1 (SAS Institute, Abacus Concept, Berkeley, CA, USA). A *p* value of <0.05 was considered statistically significant. Heatmapping was performed using MetaboAnalyst R 1.0.3 (XiaLab at McGill University, Montreal, QC, Canada). The data in heatmaps were analyzed by t test, the algorithm used was the average method and the measure of distance was Euclidean.

### **3. Results**

### *3.1. Demographic and Training Characteristics*

The characteristics of the runners are summarized in Table 1. Each athlete was regularly engaged in marathon training for more than 3 years, 3–7 times/week in 1–2 h/session. Athletes performed a running race over the distance of 21.0975 km. Race time ranged between 1.33 and 1.46 h. No correlation of inflammatory parameters or sphingolipids with demographic and training characteristics was observed. No gender-related differences were observed for all the biomarkers evaluated.

**Table 1.** Anthropometric measurement of runners and physical activity.

 Lipids Levels and Race-Related Trends*

### 3.2.1. Total Ceramides, DAG and SM

Total ceramides, total DAG and SM at baseline, post and 24 h after the race are reported in Figure 1 (panel A, B and C, respectively). When plasma levels of total ceramides were considered, a significant decrease was observed after the race and after 24 h compared to baseline (12.25 ± 3.0, 8.68 ± 0.62, *p* = 0.011 vs. baseline; 9.70 ± 0.51, μmol/L, *p* = 0.059 vs. baseline; baseline, post-race, 24 h, respectively), Figure 1A. DAG total analysis evidenced a significant decrease post-race compared to baseline (53.26 ± 21.83 vs. 36.07 ± 18.75 μmol/L, post-race vs. baseline, *p* = 0.04; and 51.56 ± 34.25 24 h μmol/L, not significant), Figure 1B. Instead, SM tended to decrease after the race and at 24 h, although not significantly (145.66 ± 44.83; 100.2 ± 8.9; 105 ± 7.31 μmol/L), Figure 1C.

### 3.2.2. Ceramides, DAG and SM Species

The specific DAG and SM ceramide species at baseline, post and 24 h after the race are shown by heatmaps that provide intuitive visualization of the data table. Each colored cell on the map corresponds to a concentration value in the data file, with samples in rows and compound in columns (supplemental data).

Trends of DAG classes at baseline and after the race are reported in Figure 2. The greatest changes were attributed to the DAG 36:3 that decreased significantly post-race (25.56 ± 10.6 basal vs. 14.89 ± 8.14 post-race *p* = 0.01; 22.44 ± 8.14 24 h μmol/L). The other DAG species followed the same pattern but did not significantly change: DAG 32:0 (1.06 ± 0.26; 2.32 ± 4.6; 1.21 ± 0.71 μmol/L, baseline, post-race, 24 h, respectively); 32:1 (2.07 ± 0.71; 1.64 ± 0.94; 2.24 ± 1.62 μmol/L); 34:1 (8.22 ± 2.68; 5.85 ± 3.32; 8.66 ± 4.54 μmol/L); 34:2 (12.1 ± 4.85; 8.45 ± 4.76; 12.76 ± 9.39 μmol/L) and 36:4 (4.24 ± 4.13; 2.92 ± 2.04; 4.24 ± 3.95 μmol/L) (Figure 2).

**Figure 1.** Bar chart reporting mean and SD of total ceramides, total DAG and SM (panel **A**, **B** and **C**, respectively) at baseline, post and 24 h after the race.

**Figure 2.** Bar chart reporting the trend of DAG classes as mean and SD at baseline, post and 24 h after the race.

 Trends of ceramide species at baseline and after the race are reported in Figure 3. Ceramide species containing long chain fatty acids (18:1/16:0; 18:1/18:0) transiently increased after the race, to significantly decrease at 24 h (0.16 ± 0.06; 0.20 ± 0.05; 0.18 ± 0.04 μmol/L, baseline, post-race, 24 h, respectively); (0.18 ± 0.07; 0.18 ± 0.03; 0.16 ± 0.03 μmol/L) (Figure 3). Instead, ceramides containing very long chain fatty acids, i.e., 18:1/25:0; 18:1/26:0; 18:1/22:0; 18:1/24:1; 18:1/24:0; 18:0/24:0 significantly decreased after the race and at 24 h when compared to baseline (0.82 ± 0.21; 0.55 ± 0.13; 0.62 ± 0.15 μmol/L, baseline, post-race and 24 h, respectively); (0.16 ± 0.05; 0.11 ± 0.03; 0.13 ± 0.03 μmol/L); (1.52 ± 0.35; 1.91 ± 0.50; 1.46 ± 0.43 μmol/L); (3.29 ± 0.91; 2.36 ± 0.60; 2.61 ± 0.38 μmol/L); (5.56 ± 1.4; 1.46 ± 0.43; 4.35 ± 0.95 μmol/L) and (0.17 ± 0.05; 0.12 ± 0.04; 0.13 ± 0.38 μmol/L), Figure 3. SM did not significantly change.

**Figure 3.** Boxplots with indication of the median, interquartiles and error bars reporting the temporal profile of ceramides are given.

### *3.3. Inflammatory Levels and Race-Related Trends*

Basal plasma levels, post and after 24 h of TNFα, IL-6, fractalkine CX3CL1 and VEGF-A in all runners are reported in Table 2.

**Table 2.** Plasma values of chemokine and cytokines in runners.

Cytokines and chemokines at baseline and after the race are reported in Figure 4. IL-6 and fractalkine CX3CL1 significantly changed soon after the race, decreasing towards baseline values after 24 h (Figure 4A,B). TNFα had the same trend but the increment after the race was not significant (Figure 4C). VEGF-A tended to decrease after the race (Figure 4D), but these changes were not significant when compared to baseline. Table 3 shows the exercise-induced change from baseline, as Δ values (% change) for each variable.

**Figure 4.** Bar chart reporting mean and SD of cytokines and chemokines at baseline, (**A**) IL-6 (pg/mL), (**B**) Fractalkine (pg/mL), (**C**) TNFα (pg/mL), (**D**) VEGF-A (pg/mL), post and 24 h after the race.

**Table 3.** Exercise-induced change from baseline, Δ values (% change) in fractalkine, IL-6, TNFα and VEGF-A.

Post Δ and 24 h Δ are differences from baseline. % change at different successive time periods with respect to values observed at baseline is reported in brackets.

Interestingly, a strong correlation between basal fractalkine CX3CL1 and TNFα was found at baseline and during recovery (Rho = 0.91 *p* = 0.0017), Figure 5.

**Figure 5.** Regression of fractalkine and TNFα in runners (Rho = 0.91 *p* = 0.0017 24 h). Green dots—baseline, blue dots—post-race, red dots—24 h post-race.

### **4. Discussion**

This is the first study which assessed levels of DAGs and ceramides, and evaluated their relationship with inflammatory parameters in athletes before and after a halfmarathon race. Data evidenced the decrease of specific lipid classes, and the lack of relationship between lipids with inflammatory biomarkers, suggesting their possible contribution to exercise-related beneficial effects.

### *4.1. Demographic and Training Characteristics*

The population was similar in demographic and training characteristics; likely, for this reason, we did not observe any correlation concerning inflammatory parameters and sphingolipids. Regarding gender-related differences, it is not clear in literature if ceramides are different in males and females, and neither wheter there is a differential effect of exercise on ceramides in the two sexes. In our population, we did not observe gender-related differences for any of the biomarkers evaluated. Moreover, there is still a lack of a shared consensus on the optimal cutoffs and reference ranges of ceramides to be used. In this context, our data might suggest a lack of need to establish gender-specific reference ranges. However, considering the low number of subjects enrolled in the study, this issue merits further investigation.

Notably, values of inflammatory biomarkers of athletes at baseline did not differ from those observed in a group of 15 sedentary subjects (corresponding to 0.6 ± 3.1 pg/mL for IL-6, 164 ± 145 pg/mL for fractalkine CX3CL1, 23 ± 38 pg/mL for TNFα), except for VEGF-A, which was significantly lower in sedentary subjects (28 ± 61 pg/mL, *p* < 0.001) (unpublished data).

### *4.2. Lipids Levels and Race-Related Trends*

To the best of our knowledge, this is the first report describing half-marathon consequences on circulating sphingolipid metabolism. It is known that exercise may induce modification in plasma levels of numerous circulating biomarkers, inflammatory parameters and lipid metabolites in humans [12] Thus, these changes may affect sphingolipid metabolites as well. In recent years, plasma sphingolipids have attracted attention for their role in pathophysiology of cardiometabolic diseases. In fact, high plasma ceramides may induce endothelial dysfunction, which is closely correlated with aerobic capacity [13], and promote cell growth arrest, cytoskeleton rearrangements, senescence and death (e.g., activation of caspases), impairment of nitric oxide synthase (eNOS) activity and insulin signaling, increasing vascular permeability, oxidative stress and inflammation [14], thus

contributing to onset and development of atherosclerosis [15,16]. Accordingly, sphingolipids may represent an independent risk determinant for ischemic disease [17]. In particular, Cer(d18:1/16:0) appears to be independently correlated with the presence of more vulnerable coronary plaques in CAD patients [18]. Conversely, the Mediterranean diet shows the potential to reduce the adverse effects of high ceramide levels in the PRED-IMED trial (Prevención con Dieta Mediterranea, a prospective case-cohort study), which is a study including nearly 1000 elderly subjects at high cardiovascular risk [19].

Ceramide metabolism also appears responsive to the exercise stimulus. Better cardiopulmonary fitness correlated with low ceramide concentration in elderly coronary artery disease patients has been observed [20], whereas muscle ceramide levels decreased after chronic aerobic exercise [21]. Moreover, experimental data suggests that total content of ceramides decreased in the muscle in trained rats, contributing to the elevation of the glucose uptake observed in skeletal muscles after training [22]. Previous data suggested that 12 weeks of aerobic exercise training in obese or diabetic subjects [23] and 16 weeks of exercise in overweight/obese subjects may reduce sphingolipids (e.g., C18:0, C20:0 and C24:1) [24].

A decrease in ceramide concentrations hints at increased insulin sensitivity, which contributes, at least in part, to the beneficial exercise effects [25]. According to these previous results, we also observed a decrease in total ceramides after the race in trained subjects. Thus, all together these data suggest that sphingolipid species can represent valuable mediators of cardiovascular risk. Studies related to cardiac lipotoxicity showed that the lipotoxic species were primarily driven by ceramides and DAG and not triacylglycerol [26]. Regarding DAG species, Luukkonen et al. showed that DAG (32:1, 34:1, 36:2, 36:3) were significantly increased in subjects with high peripheral insulin resistance versus low peripheral insulin resistance subjects [27] In our study, DAG 36:3 decreased significantly after the race, indicating an improvement of lipotoxicity, attributable to that species after the acute exercise. Experimental data suggest that total content of ceramides decreases in the muscle of trained rats, contributing to the elevation of the glucose uptake observed in skeletal muscles after training [22]. In overweight, obese subjects or type 2 diabetes (T2D), there is a decrease in total ceramides, indicating that endurance training reduces total content of ceramides, likely improving glucose tolerance [22,25,27–29]. However, ceramide metabolism in patients and obese subjects could be dissimilar to that in lean, trained individuals. In fact, the increased lipid metabolism following the physical exercise reduces the substrate availability required for ceramide synthesis (e.g., palmitate, myristate). Moreover, the analysis of the lipid classes in its total assessment may be unsatisfactory, as the specific and complex relationship of different chains of fatty acids in ceramides with chronic and acute exercise demonstrates the complexity of these biological pathways, which require the assessment of increased, decreased or unchanged specific species. Accordingly, our data, for the first time, associate specific molecular species of lipids to exercise. In particular, Cer18:1/16:0 and Cer18:1/18:0 were very low at baseline in athletes and, after the race, the same ceramides increased, indicating a lower consumption of fatty acids contained in these species (palmitic and stearic acid), making these ceramides a non-preferential species for energy purposes. Mardare, Kruger et al. suggest that endurance training in mice is able to reduce long-chain fatty acids ceramides [28]. In particular, this experimental study suggests that the exercise-induced decrease of very long chain fatty acids ceramides (C24:0 and C24:1) may account, almost in part, for the reduced expression of blood inflammatory markers, as well as the increased glucose tolerance. An elevation in long chain fatty acids (C24:0, C24:1, C26:0, C25:0, C22:0) was associated with mitochondrial damage, apoptosis and cell necrosis [29]. It is not completely clear which are the mechanisms able to prevent the elevation in plasma ceramide level correlated with aerobic exercise. In addition to the possibility that lipid utilization during exercise reduces ceramide production by decreasing the availability of substrates required for ceramide synthesis, there are a number of hypothesis-generating ideas [25]; for example, aerobic exercise may accelerate ceramide degradation and clearance by increasing the expression of genes responsible for ceramide

clearance (e.g., acidic and alkaline ceramidase 1 and 3, glucosylceramide synthase and sphingosine kinase 1) [30].

### *4.3. Inflammatory Levels and Race-Related Trends*

IL-6, a pro-inflammatory cytokine, is known to increase after endurance exercise [31]. However, in this setting, IL-6 may have a role as a myokine, and as such, with antiinflammatory properties, rather than as an inflammatory facilitator [32]. In fact, the observed post-exercise elevations may be in line with exercise-related metabolic IL-6 effects (regulation of glucose homeostasis and fat oxidation) and adaptation to training [33]. Moreover, the release of IL-6 during exercise induces an increase in circulating anti-inflammatory cytokines (e.g., IL-10 and IL-1 receptor antagonist) and decrease of TNFα, or the release of cortisol from the adrenal glands, suggesting that the beneficial effects of exercise (IL-6-mediated) can be expressed, almost in part, through protection against TNFα-induced insulin resistance [34]. These responses, which probably mediate autocrine and paracrine benefits of training, are likely related to training levels, intensity and type of exercise and individual characteristics (e.g., sex and age), and thus may be different in different categories of subjects. Instead, relatively low information is known on the balance between IL-6 and other cytokines and inflammatory biomarkers under such conditions. TNFα did not increase significantly in our population, and this may be the effect of the anti-inflammatory cytokine cascade, which may oppose TNFα increase, giving protection against TNFα-induced damage, as previously observed [35].

CX3CL1 exists in two forms; one form anchors to the membrane, acting as an adhesion molecule, whereas the other form acts as a soluble chemoattractant. CX3CL1 A acts in acute skeletal muscle damage and regeneration through recruitment of macrophages and other immune cells involved in repair and growth of skeletal muscle, influencing the adaptive response to exercise [36,37]. As in our population, previous data suggested an increase of CX3CL1 related to exercise (e.g., cycling) [38]. The variation of circulating CX3CL1 is closely related to changes in muscle gene expression, and as such, might have significance in the adaptive response to exercise [38,39]. Moreover, local CX3CL1 synthesis and expression depend on many factors, including inflammatory cytokines (e.g., TNFα), giving evidence of the relationship that we observed between these two biomarkers [40]. Experimental data suggested that CX3CL1 stimulation of monocytes is associated with a marked increase of TNFα, which is known to stimulate satellite cell proliferation [41,42]. Accordingly, we observed a strong correlation between fractalkine and TNFα, suggesting that this effect could represent an indirect mechanism by which CX3CL1 acts as a mitogen for muscle cells. Moreover, CX3CL1 seems to have beneficial effects in muscle regeneration through a direct effect on myogenic cells [43]. Thus, CX3CL1 likely induces the monocytic and myogenic expression of different factors known to increase in human skeletal muscle after exercise, with a role in the adaptive response following an exercise burst.

Interestingly, in our population, inflammatory biomarkers did not influence the relationships between sphingolipids and exercise, not supporting the existence of a link between inflammation and sphingolipids in half-marathon response, although further studies are needed to verify this possibility. In this context, we also did not observe any relationship between total and species of SM, ceramides and DAG and reactive oxygen species (ROM, a biomarker of oxidative stress) (unshown data) [44].

### *4.4. Strengths and Limitations*

This study has limitations. First, the number of athletes enrolled is not high. However, a strength is that studied athletes were very similar according to training characteristics (day/week of training, Km/week, years of training). Moreover, each subject served as a control for him/herself, increasing the statistical power, and reducing the effects of confounding factors.

Second, insulin was not directly assessed in these subjects. This important issue merits further investigation in future studies to understand the link between species of lipids, inflammatory markers and insulin resistance (IR) during exercise. However, all our subjects had no history of IR and type 2 diabetes (T2D) and they showed normal body mass index; therefore, this relationship could be negligible. Nonetheless, it will be interesting to assess exercise effects on lipid-related biomarkers in IR, T2D or obese subjects in future studies.

### **5. Conclusions**

IL-6 and CX3CL1 transiently increased soon after the half-marathon. As they are promptly reversible, these changes might represent a physiological response to acute exercise rather than a damage-related response. The decrease in plasma ceramide concentration observed after the race and the lack of their relationship with inflammatory mediators suggested the involvement of lipids in exercise adaptation, as well as their possible role in mechanisms underlying beneficial effects of regular physical activity on disease prevention, especially regarding cardiometabolic disease. This opens up a new area of investigation for future research to establish whether the measurement of specific plasma ceramides could provide new biomarkers useful to assess exercise adaptation and to evaluate specific exercise interventions in different categories of healthy subjects and patients.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/app11104622/s1.

**Author Contributions:** All authors discussed the results and contributed to the final manuscript. M.G., C.V. and A.P.: study concept; L.S.: certified English revision; F.S., F.C., M.M., E.B., F.M. and L.S.: acquisition, measurement and analysis of data; M.G., C.V., A.P. and A.G.: interpretation of data; M.G. and C.V.: drafting of the manuscript. All authors contributed to the manuscript's intellectual content and gave approval for the final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Pisa Ethics Committee, Italy, (protocol number for study acceptance 2805).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data are available from authors upon reasonable request.

**Acknowledgments:** The authors acknowledge the athletes of "Gruppo Podistico Rossini (www.podisticarossini.it accessed on 15 May 2021)" for the fundamental contribution.

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

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*2.2. Di*ff*erential RNA Editing in Root Nodules*

RNA-seq reads were mapped to the mitochondrial genome. A total of 631 RNA editing sites, with at least 15% edited reads in all three biological replicates of any one of the three samples, were identified (Supplementary Table S2). The average editing degrees of the three groups (N, SR, and UR) were compared, and 74 sites with ≥ 15% differences in the editing degrees between any two of the three tissues were identified (Supplementary Table S3). Out of these 74 editing sites, 12 sites were intronic, 23 sites were synonymous, and 39 sites were non-synonymous. There were a few observations: (i) UR showed higher editing degrees in *atp1-1*, *atp1-2*, *atp1-3*, and *atp1-4* than the other two samples, whereas all these sites are synonymous; (ii) There were 29 differentially edited sites in *nad* transcripts (*nad1*, *nad2*, *nad4*, *nad4L*, *nad5* and *nad7*). Most of these sites were intronic (11 sites) or synonymous (nine sites). Only six and three sites in *nad1* and *nad4*, respectively, could lead to amino acid substitutions; (iii) There were 18 differentially edited sites in ribosomal protein transcripts (*rpl5*, *rps1*, *rps4*, *rps10* and *rps12*). Only one site was intronic (*rps10*), but all the other sites could lead to amino acid substitutions. Comparing N to UR, 16 sites in *rps 1*, *4*, *10*, and *12* had higher editing degrees, while two sites in *rpl5* had lower editing degrees.

#### *2.3. matR Transcripts Underwent Extensive RNA Editing in Root Nodules*

We identified 17 editing sites on *matR*, of which eight sites had been identified in *A. thaliana* [15] (Table 3) and 11 sites had been reported previously in uninoculated soybean by comparing genomic DNA and cDNA sequences [28]. Out of these 17 editing sites, the degrees of RNA editing of eight sites were higher in the N than UR and SR, of which seven could lead to amino acid substitutions. To confirm that these sites were actually edited, RT-PCR and Sanger sequencing were performed on three biological replicates of each sample (Supplementary Figure S1). In general, the Sanger sequencing data confirmed the next-generation sequencing (NGS) data, and both showed that N had a higher degree of editing than UR and SR in multiple editing sites (Figure 1 and Table 2).

**Table**

of editing between the samples were labelled by different letters.