<figure><img src="image_152.png" /></figure>

N-doping *in situ* by using ammonia solutions instead of water during the HTC process. Several examples of the electrochemical performances of carbonized, tannin-derived, HTC materials are described below.

N-doped materials were produced by HTC of mimosa tannin (Braghiroli et al., 2015a,b). Two functionalization routes were followed: (i) dissolution of tannin in an aqueous ammonia solution, evaporation at room temperature, then dissolution of the solid residue in water solution followed by HTC; and (ii) dissolution of tannin in an aqueous ammonia solution followed by HTC. The former resultant material was called EAT (for “evaporated aminated tannin”), and the latter was called AT (for “aminated tannin”). A reference material, prepared by direct HTC of tannin in water, called T, was also synthesized. HTC was carried out at temperatures ranging from 180 to 220°C for 24 h, and the recovered materials were carbonized at 900°C. The morphology radically changed from CNSs for the reference materials to CG structure for the EAT-materials, while the carbon obtained by the AT-route presented a mixed morphology, see **Figures 10A-C**. The carbon materials exhibited surface areas between 113 and 684 m² g⁻¹. AT- and T-materials had a high proportion of ultramicropores (pores of width < 0.7 nm), higher than 79%, while EAT-materials presented a majority of mesopores. The micropore volume remained around ~0.21 cm³

g⁻¹ in almost all cases. Regarding the presence of heteroatoms, the AT-route introduced the highest amount of nitrogen into the material with a maximum of 8.1 wt. % for HTC carried out at 220°C. The oxygen content decreased with the temperature, regardless of the functionalization route, remaining between 5.6 and 15.9 wt. %.

The electrochemical performances of carbon materials derived from HTC at 180 and 210°C were evaluated (Braghiroli et al., 2015b). Electrodes for symmetrical SCs were prepared by mixing the powdered carbon material with carbon black and PVDF (75:5:20 wt. %), and assembled in a T-type cell, using 4 M H₂SO₄ as electrolyte. CV tests were carried out using a potential window of 1 V. The obtained curves were asymmetric, indicating the existence of considerable resistance; examples are shown in **Figure 10D**. CV curves also showed humps characteristic of redox reactions occurring due to the high content of heteroatoms (N and O). Thanks to the pseudocapacitance contributions of these reactions, Cₑ reached values as high as 322 F g⁻¹ at 2 mV s⁻¹. However, as the scan rate increased, Cₑ dropped considerably. This behavior was attributed to the large size of the mesopores, 27–34 nm, which do not significantly account for the capacitance and do not successfully shorten the ion diffusion pathways as the narrower mesopores would. In order to study the effect of doping, the interfacial capacitance was calculated by
<table><caption>**TABLE 2** | Hazard statements for phenol, resorcinol, formaldehyde, and tannin with data from the Kim et al. (2018) and the National Institute for Occupational Safety Health (2018).</caption><thead><tr><th></th><th>Phenol</th><th>Resorcinol</th><th>Formaldehyde</th><th>Tannin</th></tr></thead><tbody><tr><th>PEL</th><td>TWA 5 ppm (skin)</td><td>Not reported</td><td>TWA 0.75 ppm<br>STEL 2 ppm</td><td>No adverse human health effects*<br>Does not have skin or eye irritation effects*</td></tr><tr><th>REL</th><td>TWA 5 ppm (skin)<br>STEL 15.6 ppm (skin)</td><td>TWA 10 ppm<br>STEL 20 ppm</td><td>TWA 0.016 ppm<br>STEL 0.1 ppm</td><td></td></tr><tr><th>GHS and NFPA hazards</th><td></td><td></td><td></td><td>Not reported for<br>profisetinidin,<br>prorobinetinidin,<br>prodelphinidin and<br>procyanidin</td></tr></tbody></table>

PEL, permissible exposure limit; REL, recommended exposure limit; TWA, time-weighted average over 8 h; STEL, short-term exposure limits over 15 min; GHS, globally harmonized system; NFPA, National fire protection association. *According to SilvaChimica (St Michelle Mondovi, Italy), provider of mimosa, quebracho, and tara tannins.

<table><caption>**TABLE 3** | Summary of methods, properties and electrochemical performance of tannin-derived carbon materials.</caption><thead><tr><th>Tannin precursor/ Synthesis method</th><th>Carbon material</th><th>Activation/ Doping</th><th>BET area (m² g⁻¹)</th><th>Cell type</th><th>Electrolyte</th><th>Potential window (V)</th><th>C<sub>e</sub> (F g⁻¹)</th><th>References</th></tr></thead><tbody><tr><td>Mimosa/Sol-gel</td><td>Cryo-CGs</td><td>—/—</td><td>399–1 420</td><td>T</td><td>4 M H<sub>2</sub>SO<sub>4</sub></td><td>1.0</td><td>30–109, at 2 mV s⁻¹</td><td>Amaral-Labat et al., 2012</td></tr><tr><td>Mimosa/HTC</td><td>CMSs and CG-like</td><td>—/N, O</td><td>442–684</td><td>T</td><td>4 M H<sub>2</sub>SO<sub>4</sub></td><td>1.0</td><td>181–322, at 2 mV s⁻¹</td><td>Braghiroli et al., 2015b</td></tr><tr><td>Mimosa/HTC</td><td>Aero-, cryo- and xero-CGs</td><td>—/N, O</td><td>496–864</td><td>T</td><td>4 M H<sub>2</sub>SO<sub>4</sub></td><td>1.0</td><td>231–388, at 2 mV s⁻¹</td><td>Braghiroli et al., 2015c</td></tr><tr><td>Pine/HTC</td><td>CMSs and CG-like</td><td>—/N, O</td><td>80–652</td><td>3-e<br>2-e</td><td>1 M H<sub>2</sub>SO<sub>4</sub></td><td>0.9</td><td>114–321, at 0.5 mV s⁻¹<br>112–258, at 0.1 A g⁻¹</td><td>Sanchez-Sanchez et al., 2017b</td></tr><tr><td>Quebracho/ Microwave-assisted</td><td>MCs</td><td>—/P, N</td><td>113–855</td><td>3-e</td><td>1 M H<sub>2</sub>SO<sub>4</sub><br>6 M KOH</td><td>0.8</td><td>172–272, at 5 mV s⁻¹<br>176–236, at 5 mV s⁻¹</td><td>Nasini et al., 2014</td></tr><tr><td>Mimosa/Hard- templating</td><td>OMCs</td><td>—/—</td><td>1006</td><td>3-e<br>2-e</td><td>1 M H<sub>2</sub>SO<sub>4</sub></td><td>0.8</td><td>~250, at 0.5 mV s⁻¹<br>~196, at 0.1 A g⁻¹</td><td>Sanchez-Sanchez et al., 2017a</td></tr><tr><td>Mimosa/ Phase separation</td><td>OMCs</td><td>CO<sub>2</sub>/—</td><td>563–1137</td><td>3-e<br>2-e</td><td>2 M H<sub>2</sub>SO<sub>4</sub></td><td>0.8</td><td>158–286, at 0.5 mV s⁻¹<br>Up to 244, at 0.1 A g⁻¹</td><td>Sanchez-Sanchez et al., 2018</td></tr><tr><td>Mimosa/ Mechanosynthesis</td><td>OMCs</td><td>CO<sub>2</sub>/—</td><td>567–2061</td><td>2-e</td><td>1 M H<sub>2</sub>SO<sub>4</sub><br>1 M Na<sub>2</sub>SO<sub>4</sub><br>1 M TEABF<sub>4</sub>/ACN</td><td>1.0<br>1.6<br>2.7</td><td>138–147, at 0.2 A g⁻¹<br>94–100, at 0.2 A g⁻¹<br>103–109, at 0.2 A g⁻¹</td><td>Castro-Gutiérrez et al., 2019</td></tr></tbody></table>

C<sub>e</sub>, specific electrode capacitance; HTC, hydrothermal carbonization; CGs, carbon gels; CMSs, carbon microspheres; MCs, mesoporous carbons; OMCs, ordered mesoporous carbons; T, T-type cell; 3-e, three-electrode cell; 2-e, two-electrode cell.

normalizing C<sub>e</sub> by the surface area and was plotted against the O and N contents (wt. %), see **Figures 10E,F**. It was found that the interfacial capacitance increased with the O content, but showed a maximum value for an N content between 3 and 6 wt. %. A large amount of heteroatoms is believed to block the pores, resulting in reduced electrochemical performance.

A follow-up study evaluated the electrochemical performances of N-doped CGs produced by HTC of EAT (Braghiroli et al., 2015c). In this case, aqueous solutions of different concentrations of EAT were submitted to HTC at 180°C for 24 h. The recovered products were then dried to produce organic aerogels, cryogels, or xerogels, and the latter materials were further carbonized at 900°C to obtain N-doped

CGs. The materials exhibited both micro- and mesoporosity in a proportion of ~40/60, and the highest surface areas were obtained for the highest concentration of EAT, reaching a maximum of 864 m² g⁻¹. For all the CGs, the N content remained around 2.5 wt. %, and an O content higher than 13.9 wt. % was observed. Electrodes for symmetrical SCs were prepared and assembled as described above for AT- and EAT-materials. Again, the CV curves were asymmetrical with the characteristic humps due to redox reactions, similar to those displayed in **Figure 10D**. At a scan rate of 2 mV s⁻¹, the ultramicropore volume was found to have more impact on the C<sub>e</sub> values than the surface area. The capacitance increased significantly up to an ultramicropore volume of 0.2 cm³ g⁻¹,
<figure><img src="image_153.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_154.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_155.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_156.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_157.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_158.png" /></figure>

**FIGURE 10** | SEM images of materials obtained after HTC at 210°C and subsequent pyrolysis at 900°C of tannin in: (A) water, or (B) ammonia solution, and (C) evaporated aminated tannin in water. (D) Examples of cyclic voltammetry curves obtained at 2 mV s⁻¹ from carbonized (900°C) HTC materials produced at 180°C (Adapted with permission of Elsevier, from Braghiroli et al., 2015b; permission conveyed through Copyright Clearance Center, Inc.). Normalized capacitance as a function of (E) nitrogen and (F) oxygen content of: carbonized HTC materials produced at 180 and 210°C, referred to as TM, and of EAT-derived CGs, referred to as CA, CC, and CX for carbon aerogels, cryogels and xerogels, respectively (Adapted with permission of Elsevier, from Braghiroli et al., 2015c; permission conveyed through Copyright Clearance Center, Inc.). The sample labels correspond to those used in their respective source references.

for which it reached a maximum value of ~400 F g⁻¹. A further increase in the volume did not affect the capacitance values. Contrary to what was expected, the values were more scattered than in the trends previously observed when comparing the effect of O and N doping on the interfacial capacitance with the results of Braghiroli et al. (2015b) (see again **Figures 10E, F**). Recalling that all CGs have a similar N content, this scattering might be due to their larger differences of pore texture. The enhancement in the capacitance due to N-doping could be positively or negatively influenced by, for example, a larger or lower surface area, explaining the scattering of the values in **Figure 10E**. An analogous reasoning would also explain the behavior observed for the effect of the O content in **Figure 10F**.

Another study proved that N-doped carbon materials can also be obtained by HTC treatment of aminated pine tannin (Sanchez-Sanchez et al., 2017b). The synthesis reported by Braghiroli et al. (2015b) was used by replacing mimosa tannin with pine tannin and carrying out the HTC process at 190°C. The tannin extraction from *Pinus pinaster* was carried out by two methods. In method (i), a mix of pine bark and water in weight ratio of 1:6 was heated up to 90°C for 90 min. In method (ii), the same mix used in (i) but in a 1:5 weight ratio was heated up to 75°C; then, an aqueous solution containing 2 and 0.5% of Na₂SO₃ and Na₂CO₃, respectively, was added to the

mixture while maintaining the temperature for an additional 60 min. The suspensions from methods (i) and (ii) were filtered, washed with water and spray-dried, leading to tannin powders referred as TW or TNa, respectively. The morphology of the carbon materials was similar to that obtained from the same treatment of mimosa tannin, i.e., showing a transition from spherical particles to a gel-like structure. Most of the materials presented a high microporosity that led to surface areas between ~300 and ~700 m² g⁻¹. However, samples obtained from the direct HTC of TNa and from the EAT-route did not develop any porous texture, which is the reason why they were excluded from the electrochemical tests.

A three-electrode configuration was used to evaluate the electrochemical performance of the pine tannin-derived materials in a potential window of 0.9 V in 1 M H₂SO₄. Electrodes were prepared by mixing the porous carbon, carbon black and PTFE (85:5:10 wt. %). Faradaic peaks were observed on the CV curves at low scan rates (0.5 mV s⁻¹). Remarkably, the TW-materials retained a quasi-rectangular CV curve for a high scan rate of 1 V s⁻¹, but the deviations from the ideal rectangular shape suggest that the diffusion of ions in the narrow micropores was hindered. At low scan rate, Cₑ of TW-materials was above 200 F g⁻¹ due pseudocapacitance contributions. However, for a scan rate of 20 mV s⁻¹, Cₑ has already decreased to ~40%
of its original values because redox reactions do not occur at faster scan rates. Capacitance then continued to decrease but at a considerably slower rate, almost reaching a plateau at 1 V s⁻¹. In addition, a linear increase in interfacial capacitance with the surface concentration of heteroatoms (N and O) was observed for the TW-materials. As the scan rate increased, the slope of the fitted line decreased, becoming almost a horizontal line at 1 V s⁻¹. This implies that, for fast scan rates, the heteroatom content does not contribute to the capacitance, in agreement with the previous statements. TNa-A sample, obtained from aminated TNa, exhibited the lowest performance, whatever the scan rate and despite a surface area and density of surface functionalities similar to those of other materials from the TW-series. Furthermore, TNa-A exhibited the lowest value of interfacial capacitance, indicating that a large part of the surface of the material was not accessible to the electrolyte ions. The high amount of surface functionalities was not able to compensate the lack of charge storage sites resulting in the poor performance observed. In addition, GCD and EIS measurements were carried out using a two-electrode cell. The calculation of C~e~ from the GCD curves gave values similar to those obtained from the CV curves, and a drastic drop in capacitance was observed as the applied current increased. Again, TNa-A had the lowest performance. In general, the doped materials from the TW-series had the highest values of capacitance, confirming the beneficial role of surface modification. However, there are still some drawbacks to the doping procedure followed, as the EIS and GCD tests also revealed that the doped materials had larger EDR values and lower stability after continuous cycling than their non-doped counterparts.

## Microwave-Assisted Synthesis

P- and N-doped carbons (PNDCs) have been successfully produced by a microwave technique using quebracho tannin as carbon precursor (Gopal Bairi et al., 2013). Melamine and hexamine were used as N source and polyphosphoric acid as P source. The synthesis was carried out by dissolving melamine and tannin in water, then a solution of hexamine was added and the whole mixture was heated to its boiling point. Finally, the resultant material was mixed with polyphosphoric acid and subjected to microwaves for 30 min. The temperature reached was estimated to be between 1,200 and 1,400°C during the process carried out in a 1.25 kW–2.4 GHz microwave oven. The physicochemical properties and performances of the PNDCs obtained from three different tannin:melamine:hexamine molar ratios as electrodes for SCs were further studied by the same research group (Nasini et al., 2014). The samples were referred to as PNDC-x, where x = 1, 2, or 3: the higher the value of x, the lower the amount of melamine and hexamine used for synthesis.

A large amount of heteroatoms was successfully introduced on the surface of the carbon. However, this was achieved by sacrificing considerably the available surface. The sample PNDC-1, having the highest content of surface functionalities (N 9.7 at. %, P 9.0 at. %, and O 14.9 at. %), thus had the lowest BET area, 113 m² g⁻¹ due to an accumulation of heteroatoms that could hinder access to the pores. Moreover, the higher contents of N and P are the consequence of the use of large quantities

of hexamine and polyphosphoric acid, both substances reducing the amount of gases released during pyrolysis, increasing the carbon yield, but also reducing the development of the porous texture. Decreasing the level of doping increased the surface area and increased the volumes of micro- and mesopores. The sample PNDC-3 thus had the lowest content of heteroatoms and exhibited a surface area of 855 m² g⁻¹ with a total pore volume of 0.65 cm³ g⁻¹. Intermediate values of heteroatom content and textural properties were found for PNDC-2. PNDCs are mesoporous materials, and most of their pore volume is due to narrow mesopores (of width ~3–4 nm) at the levels of 99, 78, and 89% for PNDC-1, PNDC-2, and PNDC-3, respectively.

The electrochemical performance of electrodes composed of PNDC, carbon black and PTFE (90:5:5 wt. %) was evaluated in a three-electrode cell using either 1 M H₂SO₄ or 6 M KOH as electrolytes. The CV curves of all materials in acidic medium revealed the typical peaks associated with redox reactions. At 5 mV s⁻¹, the calculated C~e~ for PNDC-2 and PNDC-3 was 272 and 246 F g⁻¹ and, despite its low surface area, PNDC-1 exhibited a capacitance of 172 F g⁻¹ mainly due to pseudocapacitance contributions. As expected, C~e~ decreased for higher scan rates. A faster drop in capacitance was observed for PNDC-2 than for PNDC-3. At 100 mV s⁻¹, PNDC-2 lost 30% of its capacitance, while PNDC-3 lost only 12%. The higher content of heteroatoms of PNDC-2, compared to PNDC-3, implies that at low scan rate, the contributions of pseudocapacitance are larger. However, at faster rates, redox reactions no longer occur, which reduces the performance of the material. On the other hand, the more stable performance of PNDC-3 is explained by the strong presence of mesopores and a low content of heteroatoms. Thus, for this material, the charge is mainly stored by an EDL mechanism that is not affected by an increase in the scan rate because the mesopores facilitate the diffusion of ions. In alkaline medium, PNDC-3 exhibited a behavior similar to that in acidic medium. However, lower values of C~e~ were reached; this decrease in capacitance might be caused by the larger size and lower mobility of ions in KOH, compared to those in H₂SO₄ (see Table 1). PNDC-2 exhibited the best performance of the series across the range of scan rates, attributed to a combination of sufficiently high surface area, pore volume and pseudocapacitance contribution of the surface functionalities that are reactive in alkaline media. Subsequent studies have extended this synthesis method to produce PNDCs from aminated quebracho tannin (Bairi et al., 2015) and Si- and P-doped carbons from quebracho tannin (Ramasahayam et al., 2015). In both studies, behaviors and values of capacitance similar to those detailed above were found.

## Hard-Templating

OMCs have been produced by hard-templating using mimosa tannin and other tannin-related molecules such as phloroglucinol, gallic acid, and catechin (Sanchez-Sanchez et al., 2017a). SBA-15 silica template was synthesized using a mixture of tetraethyl orthosilicate (TEOS), Pluronic® P123, HCl, and water (molar ratio 1:0.017:6.04:145.8) that was stirred at 40°C for 4 h and aged at 125°C for 72 h. The product, recovered by filtration, was heat-treated in air at 550°C for 6 h. An ethanol solution of
the corresponding precursor (0.01 mol/40 mL) was added to the SBA-15, previously degassed under vacuum (150°C, 2 h). Ethanol was evaporated under vacuum and, if necessary, the obtained solid was washed with deionized water to eliminate the excess of precursor deposited on the silica particles. These composites were carbonized under nitrogen flow according to a two-step temperature program: heating from room temperature to 100°C (1°C min⁻¹) and holding the temperature for 2 h in order to remove moisture and polymerize the precursors, then heating up to 900°C (2°C min⁻¹) and holding the temperature for 1 h. The template was removed from the carbonized composites with HF, washed with deionized water and finally dried at 80°C for 12 h. The OMCs were labeled according to their precursors as TanC, PhC, GaC, and CatC for tannin, phloroglucinol, gallic acid, and catechin, respectively.

**Figure 11A** shows the schematic synthesis process and TEM images of the produced OMCs. TEM images revealed some discontinuities in the structure for CatC and TanC, indicating that catechin and mimosa tannin permeated the template heterogeneously. The large size of these molecules could block some of the pores, preventing the complete filling of the template and resulting in the gaps observed. XRD measurements revealed that all materials exhibited a structure in the *p6mm* space group (2D hexagonal), and that the long-range order decreased as the size of the precursor molecule increased, in agreement with TEM results. Nevertheless, TanC had the advantage of achieving the highest carbon yield (80%) among the carbon precursors used for this study. The textural characterization showed that all the OMCs exhibited BET surface areas of ~1,000 m² g⁻¹ and total pore volumes higher than 0.87 cm³ g⁻¹, of which 75–87% came from mesopores. Chemical characterization by elemental analysis and XPS revealed oxygen as the main heteroatom in the material, which was expected given its abundance in the precursor molecules.

The electrochemical performance was evaluated in 1 M H₂SO₄. CV tests were carried out in a three-electrode cell while GCD and EIS measurements were done in a two-electrode cell. The electrodes, prepared by mixing the OMC, carbon black and PTFE (85:5:10 wt. %), were spread onto graphite foil and pressed at 5 MPa. Rectangular CV curves were observed up to a scan rate of 100 mV s⁻¹. At 0.5 mV s⁻¹, Cₑ reached values higher than 200 F g⁻¹, which are in the same range of other carbons with similar textural properties but that required additional oxidation post-treatments (Jurewicz et al., 2004; Tanaka et al., 2015). GCD tests led to quasi-triangular curves whose slight curvature suggests the occurrence of redox reactions due to the presence of oxygen on the surface. For both CV and GCD tests, as the charging rate increased (scan rate or applied current), Cₑ decreased. In particular, TanC presented a capacitance retention of 42 % at 100 mV s⁻¹ and 51 % at 12 A g⁻¹, higher than the ~30% retention reported for the pine tannin-derived HTC carbons described above and tested under similar conditions (Sanchez-Sanchez et al., 2017b). The enhanced performance of OMCs as electrodes for SCs confirms the beneficial role of a hierarchical porous structure as a means of improving ion diffusion in the carbon material.

# Soft-Templating

## Phase Separation

Mimosa tannin was successfully used as precursor to produce OMCs by an easy and simple phase-separation method (Braghiroli et al., 2016). The synthesis only required the mixing, at room temperature, of two solutions: one of mimosa tannin, the other of Pluronic® F127. Immediately after mixing, a phase-separation occurred and after 2 days, complete sedimentation of the resultant mesophase was observed. The recovered mesophase was dried at 80°C and carbonized at temperatures ranging from 400 to 900°C. The pH of the tannin solution was varied from 0 to 4.2 but no significant change in the carbon yield or the porosity were observed by modifying this parameter. The synthesis process is shown in **Figure 11B**, and TEM images of OMCs obtained for different pH values are presented. Thermogravimetric analysis showed that the Pluronic® F127 degrades at 400°C, so carbonization at higher temperatures is required to remove it completely from the material. Higher temperature treatments resulted in more developed microporosity and consequently higher surface areas: a maximum of ~700 m² g⁻¹ BET area was reached. These values of surface area are ~200 m² g⁻¹ higher than those obtained from an EISA method also using mimosa tannin as precursor and carbonizing the mesophase at a maximum temperature of 850°C (Schlienger et al., 2012).

Some modifications to the phase-separation method were made in a following study that also investigated the effect of the assembly temperature (Sanchez-Sanchez et al., 2018). The pH was varied from 1 to 4.5 and the assembly temperature ranged from 10 to 30°C. The mixed tannin/F127 solutions were kept at a constant assembly temperature for 72 h, and then they remained at room temperature for 5 days until complete phase-separation. The retrieved resins were dried at 60°C and carbonized in two consecutive steps: increasing the temperature up to 400°C, holding it for 4 h, and then heating until 900°C and holding for 1 h. In agreement with the previous work of Braghiroli et al. (2016), it was found that for a fixed temperature, the pH did not change considerably the porous texture. On the contrary, for a given pH, an effect of the assembly temperature was observed, where the greatest change occurred at pH 3 after increasing the temperature from 20 to 30°C. The solution of pH 3 assembled at 30°C gave the carbon material, called F3/30, having the highest values of surface area and micro- and mesopore volumes, 750 m² g⁻¹, 0.24 and 0.49 cm³ g⁻¹, respectively. Therefore, F3/30 was selected for CO₂ activation to improve its porous texture further.

As expected, for the resultant activated carbon, labeled C3/30, an increased surface area (~1,100 m² g⁻¹) and a widening of pores were observed. Three- and two-electrode cells were used for its electrochemical characterization, using 2 M H₂SO₄ as electrolyte and electrodes composed of a mixture of the studied carbon, carbon black and PTFE (85:5:10 wt. %). It was found that the main mechanism of storage of energy was the EDL, since the contribution of pseudocapacitance from heteroatoms was negligible. In addition, CV and GCD tests showed that the activation process increased the capacitance of the material by 1.5 times, probably due to the widening and
<figure><img src="image_159.png" /></figure>

opening of previously blocked pores, which resulted in improved connectivity of the pore network and surface accessibility to electrolyte ions. Even more, the electrochemical tests showed that the rate capabilities of C3/30 were comparable to those measured for the hard-templated TanC obtained from the same precursor, mimosa tannin, but through a less eco-friendly synthesis method (Sanchez-Sanchez et al., 2017a), thus showing the advantage of the soft-template method.

## Mechanosynthesis

Mechanosynthesis methods, for which mechanical energy is supplied to the materials by shearing, stretching, or grinding (James et al., 2012), are commonly used to produce ceramics, metal alloys, and organic materials (Kaupp, 2009, 2011; Wang, 2013). This type of solid-state synthesis has attracted interest for producing OMCs since they require little or no solvent, short synthesis times compared to other methods and fewer synthesis steps, which results in cheaper and more eco-friendly products (James et al., 2012; Wang, 2013; Zhu et al., 2016). Ball-milling has been found to be an appropriate method for synthesizing OMCs, as it can provide higher energy and repeatability than manual grinding, thereby increasing its potential for use on an industrial scale (Wang, 2013). OMCs were indeed obtained by ball-milling mimosa tannin, Pluronic® F127 and metal acetates used as crosslinkers. After washing, drying and carbonization, 2D hexagonal geometries were observed (Zhang et al., 2017).

More recently, another study reported an easy, one-pot mechanosynthesis method for preparing OMCs with a 2D hexagonal structure by ball-milling, where only mimosa tannin (T), Pluronic® F127 (P) and small amounts of water (W) were needed (Castro-Gutiérrez et al., 2018). This surfactant-water-assisted mechanochemical mesostructuration (SWAMM) method does not involve curing or drying steps before carbonization or the use of hazardous substances as crosslinkers. The influence of synthesis parameters such as milling time, pH, P:W weight ratio and carbonization temperature on the textural properties of the obtained carbon materials was analyzed. Optimal values of milling time (60 min) and pH (unchanged) to produce uniform PSDs were found and it was shown that by changing the P:W weight ratio, it was possible to produce either ordered or disordered mesoporous materials. Increasing the carbonization temperature from 900 to 1,100°C resulted in the development of micro- and mesoporosity, but higher temperatures, up to 1,500°C, did not seem to have a significant impact on the textural properties of the OMCs. Nonetheless, Raman spectra revealed that despite being in the non-graphitizable regime, higher carbonization temperatures yielded materials with improved long-range order at the nanoscale. Several applications were explored with promising results, such as the adsorption of hydrocarbons from water, the adsorption of CO₂ and, after activation, the storage of energy in SCs.
Energy storage in SCs was further investigated in a follow-
up study, where the electrochemical performances of activated
OMCs (AOMCs) produced by the SWAMM method were
evaluated (Castro-Gutiérrez et al., 2019). The OMC was placed
inside a tubular furnace and heated up to 900°C (5°C min⁻¹)
under N₂ flow, then N₂ was switched to CO₂ to physically
activate the samples for durations ranging from 15 to 120 min.
The samples were labeled At-CTPW with *t* standing for
the time in minutes. TEM images of as-synthesized and A90-CTPW
materials are shown in **Figure 11C** with a scheme of the synthesis
method. It was observed that the ordered mesoporous structure
was maintained up to a burn-off of 78% (A90-CTPW). The
surface area of the AOMCs increased with burn-off, reaching a
maximum of ~2,000 m² g⁻¹ for a burn-off of ~80%. Higher
activation times resulted in a sudden decrease of surface area
due to pore widening and merging, evidenced by the PSDs and
the derived calculations of ultramicro-, supermicro- (0.7 < pore
width < 2 nm) and mesopore volumes. Electrodes were prepared
with samples A30- to A-90-CTPW that were mixed with carbon
black and PTFE (85:5:10 wt. %) to form a paste from which
electrodes with carbon loads of ~10 mg cm⁻² were cut and
assembled in a two-electrode cell.

CV tests performed in 1 M H₂SO₄ revealed rectangular curves at 5 mV s⁻¹, with Cₑ values between 136 and 160 F g⁻¹, the maximum being reached by A75-CTPW. Only A60- and A75-CTPW retained more than 60% of their initial capacitance at 500 mV s⁻¹, which was attributed to a combination of high surface area originating from a high micropore volume (<0.50 cm³ g⁻¹), providing multiple charge storage sites, and a suitable PSD, favoring ion diffusion at high charging rates. A further characterization was then carried out on these samples, using also 1 M TEABF₄/ACN as electrolyte. Examples of CV curves in aqueous and organic electrolytes are shown in **Figures 12A,D**. During GCD tests, A60- and A75-CTPW exhibited triangular curves in the aqueous electrolyte. However, A75-CTPW performed better once the applied current increased, showing a capacitance retention, Cₘ, of 70%, whereas that observed for A60-CTPW was only 51%, both at a high current of 80 A g⁻¹, see **Figure 12B**. In the organic electrolyte, A60- and A75-CTPW behaved in a similar way and a more rapid drop of Cₘ was observed compared to that in the aqueous electrolyte (**Figure 12E**). This was ascribed to the larger size and lower mobility of the ions in the organic electrolyte that increased the resistance of the SC cell, thereby decreasing its performance. In all cases, the tannin-derived AOMCs presented superior or comparable rate capabilities than those previously reported for similar materials (also displayed in **Figures 12B,E**) with the advantage of being produced by an easy and eco-friendly method. Even more, A60- and A75-CTPW exhibited great long-term stability over time and after continuous cycling at 5 A g⁻¹. More precisely, in the aqueous electrolyte, Cₘ remained at ~98% after 10,000 cycles and ~94% after 50 h of potentiostatic hold at 1 V, while in the organic electrolyte Cₘ was above 90% after 8,000 cycles and reached 86% after 72 h of potentiostatic hold at 2.7 V.

As expected, the values of *E* and *P* in organic electrolyte
are considerably higher than in aqueous electrolyte, see
**Figures 12C,F**. Indeed, a maximum *E* of 28 Wh kg⁻¹ (at a *P*

output of 138 W kg⁻¹) was attained in the organic electrolyte,
more than 5 times the maximum *E* of 5 Wh kg⁻¹ (at a *P* output
of 44 W kg⁻¹) reached in the aqueous electrolyte. For the organic
electrolyte, it is important to note that, despite the drastic drop
in *E* observed, the selected AOMCs were still able to store ~13
Wh kg⁻¹ at a high *P* of 12 kW kg⁻¹. Finally, the electrochemical
performances of the selected AOMCs were also evaluated in 1 M
Na₂SO₄, a neutral aqueous electrolyte. The results are shown in
**Figures 12G-I**. In general, lower values of *C*ₐ and *C*ₚet were found
compared to those obtained in acidic medium (H₂SO₄). The
lower performance of the SCs in Na₂SO₄ can be related to a larger
size and lower mobility of the Na⁺ ion. The larger size results
in fewer charge storage sites, leading to a lower capacitance
while the lower mobility hinders diffusion at high charging
rates. Nevertheless, the advantage of the neutral electrolyte lies
in the possibility of extending the operating potential window.
Consequently, an increase of ~60% of the maximum *E* was
observed with respect to that reached in acidic medium.

SUMMARY AND PERSPECTIVES

SCs are devices with high power outputs, rate capability,
cyclability, and long lifetimes. However, the amount of stored
energy in SCs is lower than that found in batteries. In this
sense, research in recent decades has focused on the development
of new materials that can improve SC performance, not only
by increasing the storage capacity but also by increasing the
rate capability and the long-term stability. Porous carbons
have proven to be adequate materials for SC electrodes and,
indeed, ACs are already widely used in commercial devices.
Multiple synthesis methods have been developed to control
the textural and chemical properties of porous carbons; in
particular, templated carbons, such as OMCs, are promising
electrode materials because they can be produced with different
morphologies, high surface area and adjusted hierarchical
porosity. The possibility of tuning the pore size is of significant
importance as it allows full accessibility of the electrolyte into
the material, while the hierarchical structure provides channels
facilitating the diffusion of ions toward the smallest accessible
pores. The modification of the surface chemistry of porous
carbons presents another route to increase SC performance, and
doping with oxygen, nitrogen or other heteroatoms can induce
redox reactions that increase the energy stored in the device.
Even if all the surface functionalities do not always induce redox
reactions, doping can also improve other characteristics of the
material such as electrical conductivity, wettability, or stability.

Besides, the quest for environmentally friendly technologies
has pushed research to find substitutes for synthetic and
hazardous molecules commonly used as carbon precursors
or crosslinkers. Among the various biosourced precursors,
tannins are excellent alternatives to petrochemical carbon sources
because of their phenolic nature. In addition, condensed tannins
have a reactivity similar to that of phenol or resorcinol, so tannins
can replace them in already established synthesis methods. The
autocondensation property of condensed tannins also offers
the opportunity of avoiding the use of crosslinkers, which
Aqueous electrolyte ($H_2SO_4$)

<figure><img src="image_160.png" /></figure>

<figure><img src="image_161.png" /></figure>

<figure><img src="image_162.png" /></figure>

Organic electrolyte (TEABF₄ / ACN)

<figure><img src="image_163.png" /></figure>

<figure><img src="image_164.png" /></figure>

<figure><img src="image_165.png" /></figure>

Aqueous electrolyte ($Na_2SO_4$)

<figure><img src="image_166.png" /></figure>

<figure><img src="image_167.png" /></figure>

<figure><img src="image_168.png" /></figure>

**FIGURE 12** | Electrochemical performances of activated OMCs derived from mimosa tannin: (A,D,G) CV curves; (B,E,H) capacitance retention as a function of applied current; and (C,F,I) specific energy as a function of specific power (Ragone-like graphs) calculated from the GCD curves. The tests were carried out in (A–C) 1 M $H_2SO_4$ aqueous electrolyte (acidic medium), (D–F) 1 M TEABF₄/ACN organic electrolyte and (G–I) 1 M $Na_2SO_4$ aqueous electrolyte (neutral medium). (Adapted with permission from Castro-Gutiérrez et al., 2019. Copyright 2019 American Chemical Society).

makes them suitable precursors for self-assembly methods. As described in the last section of this review, several studies have demonstrated the versatility of tannins to produce porous carbon materials through “green” processes reducing or even eliminating the use of toxic or hazardous substances and thus decreasing their negative environmental impact. In addition, tannins are extracted industrially and marketed on a large scale, because they are essential for the leather, wine and food industry. Therefore, they are readily available at low cost. Thus, the production of

porous carbons from tannin will provide and add value to this precursor derived from biomass.

In general, the main disadvantages of producing advanced porous carbon materials are their complex multi-step procedures and their tedious synthesis. Therefore, future materials research efforts for SCs should continue to focus on the development and optimization of easy and environmentally friendly procedures such as one-pot synthesis methods that allow for *in situ* doping and/or activation of biomass or biosourced precursors.
Simplifying and reducing the number of steps would reduce costs and increase scalability, resulting in a use of biosourced carbons that is more widespread in commercial SC devices.

## AUTHOR CONTRIBUTIONS

JC-G performed writing original draft and conceptualization. AC performed writing—review and editing. VF performed conceptualization, supervision, funding acquisition, and writing—review and editing. All authors contributed to the article and approved the submitted version.

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## FUNDING

This study was partly funded by the French PIA project Lorraine Université d'Excellence, reference ANR-15-IDEX-04-LUE and the TALiSMAN project, funded by FEDER (2019-000214).

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JC-G gratefully acknowledges CONACYT-SENER (601021/438978) for the scholarship awarded to support her Ph.D. studies, resulting in the work presented herein.

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Castro-Gutiérrez, Celzard and Fierro. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
<figure><img src="image_169.png" /></figure>

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# Non-precious Melamine/Chitosan Composites for the Oxygen Reduction Reaction: Effect of the Transition Metal

B. Aghabarari¹, M. V. Martínez-Huerta²*, M. C. Capel-Sánchez² and M. J. Lázaro³*

¹ Department of Nanotechnology and Advanced Material, Materials and Energy Research Center (MERC), Tehran, Iran, ² Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain, ³ Instituto de Carboquímica, CSIC, Zaragoza, Spain

The development of active and low-cost electrocatalysts for the oxygen reduction reaction (ORR) is crucial for the sustainable commercialization of fuel cell technologies. In this study, we have synthesized Me/Mo₂C (Me = Fe, Co, Cu)-based composites embedded in N- and P-dual doped carbon by means of inexpensive industrial materials, such as melamine and chitosan, as C and N sources, and the heteropolyacid H₃PMo₁₂O₄₀ as P and Mo precursor. The effect of the transition metal (Fe, Co, and Cu) on the ORR in alkaline medium has been investigated. The physicochemical properties of the electrocatalysts were performed by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and transmission electron microscopy (TEM). Activity towards ORR was carried out in a three-electrode cell using a ring-disk electrode in 0.1M NaOH. The results obtained clearly show the important role played by each transition metal (Fe, Co, and Cu) in the electrochemical activity. Among them, Fe gives rise to the best performing composite in carrying out the oxygen reduction reaction. The formation Fe₃C/ Mo₂C species embedded in N- and P-dual doped carbon seems to be the determining role in the increase of the ORR performance.

**Keywords:** oxygen reduction, composites, chitosan, melamine, electrocatalyst, heteropolyacid

**OPEN ACCESS**

**Edited by:**
Emilia Morallon,
University of Alicante, Spain

**Reviewed by:**
Jialiang Wang,
University of Wisconsin-Madison,
United States
Dongsheng Geng,
University of Science and Technology
Beijing, China

*Correspondence:
M. V. Martínez-Huerta
mmartinez@icp.csic.es
M. J. Lázaro
mlazaro@icb.csic.es

**Specialty section:**
This article was submitted to *Energy Materials*, a section of the journal *Frontiers in Materials*

**Received:** 30 June 2020
**Accepted:** 05 October 2020
**Published:** 26 November 2020

**Citation:**
Aghabarari B, Martínez-Huerta MV, Capel-Sánchez MC and Lázaro MJ (2020) Non-precious Melamine/ Chitosan Composites for the Oxygen Reduction Reaction: Effect of the Transition Metal. *Front. Mater.* 7:578518. doi: 10.3389/fmats.2020.578518

## INTRODUCTION

The oxygen reduction reaction (ORR) is the main electrochemical reaction occurring at the cathode of polymer electrolyte fuel cells. Due to the inherently slow reaction kinetics of the ORR, platinum precious metal is required as an electrocatalyst because of its high activity and durability (Stephens et al., 2012; Shao et al., 2016; Martínez-Huerta and Lázaro, 2017). However, Pt must be substituted or dramatically reduced in the ORR cathode in order to reduce the cost and to ensure sustainable development for commercial applications. The development of active and precious-metal-free catalysts thereby becomes an imperative issue in the field of electrocatalysis (Jaouen et al., 2011; Wang et al., 2019).

One of the most significant ways to reduce the cost and improve the kinetics of the ORR is the utilization of an alkaline environment. In this condition, oxygen is reduced to water at the cathode via two possible reaction pathways: the direct four-electron (4e⁻) pathway and the energetically undesirable two-stage (2e⁻) pathway with a peroxide intermediate (Martínez-Huerta and Lázaro, 2017). The elementary reaction routes in alkaline media are (Eqs 1, 2, and 3):
$$ \text{Direct four-electron pathway: } O_2 + 2H_2O + 4e^- \rightarrow 4OH^- \quad (1) $$

$$ \text{Two-stage pathway: } O_2 + 2H_2O + 2e^- \rightarrow HO_2^- + OH^- \quad (2) $$

$$ HO_2^- + OH^- + 2e^- \rightarrow 3OH^- \quad (3) $$

Among non-precious metal catalysts, N-doped carbon with transition metals (for example, Fe or Co) have proven to have remarkably enhanced electrocatalytic activity for ORR in an alkaline environment (Wu et al., 2011; Palaniselvam et al., 2016; Shao et al., 2016; Wang et al., 2019; Yasuda et al., 2016; Wang et al., 2019). Conventionally, these catalysts are synthesized by pyrolysis of mixtures containing a carbon material, a metal salt, and a nitrogen-rich organic compound in the presence of an inert gas. Additionally, N species can be incorporated into the carbon nanostructures by pyrolysis of abundant eco-friendly raw material sources, which has benefits such as low cost or easy synthetic conditions (Chung et al., 2013; Li et al., 2014a; Das and Yurtcan, 2016; Pan et al., 2016; Qiao et al., 2016).

In this study, N-doped carbon matrix is obtained from two low-cost industrial materials, chitosan and melamine. Chitosan is a plentiful, harmless and low cost biopolymer, which is a waste product derivative of the crabbing and shrimp canning industry (Marguerite, 2006). Chitosan has been chosen as both carbon and N sources due to its abundant N-bearing groups (Wu et al., 2015; Xie et al., 2015; Aghabarari et al., 2017; Aghabarari et al. 2013). Qiao et al. prepared graphene/nitrogen-doped porous carbon sandwiches for the metal-free oxygen reduction reaction using chitosan as a nitrogen source (Qiao et al., 2016). Nitrogen doping increased the electrical conductivity and the number and the nature of the active sites for the ORR. Melamine is also a nitrogen-rich organic molecule and low-cost raw material in the chemical industry. Melamine possesses a three-dimensional monoclinic structure which is similar to $C_3N_4$. Its decomposition favors the incorporation of nitrogen in carbonaceous materials obtained by pyrolysis (Sheng et al., 2011; Liu et al., 2013; Li et al., 2014b; Peng et al., 2014). Rybarczyk et al. also studied the formation of nitrogen-doped porous carbonaceous materials by thermal decomposition of a mixture of chitosan and melamine in an inert atmosphere (Rybarczyk et al., 2015; Khan et al., 2020). They found that, in addition to the entire nitrogen content and the kind of nitrogen group (pyridinic N or quaternary N), the content of carbon “kinks” and/or surface roughness also deeply influenced the ORR activity.

Transition metal carbides are highly attractive since they exhibit electronic and catalytic properties similar to Pt (Guil-López et al., 2010; Roca-Ayats et al., 2014; Zhong et al., 2015). Recently, a number of research groups have demonstrated that molybdenum carbides can be active electrocatalysts for the oxygen reduction reaction (Liao et al., 2014; Fan et al., 2017; Zhang et al., 2017; Li et al., 2019). The physicochemical properties of molybdenum carbides, such as mechanical hardness, thermal stability, singular metal-carbon chemical bonds, and noble-metal-like d-state density around the Fermi level, make them very promising materials for obtaining advanced ORR composites.

Herein, we have synthesized $Me/Mo_2C$ (Me = Fe, Co, Cu)-based composites embedded in N- and P-dual doped carbon using a facile synthesis strategy developed by our research group (Aghabarari et al., 2019). For this preparation, the corresponding metallic precursor and molybdophosphoric acid (HMoP) are pyrolyzed together with the mixture chitosan/melamine as carbon and nitrogen sources. HMoP was used as a source of P and Mo. The effect of the transition metal (Fe, Co, and Cu) on the structure and ORR activity has been investigated. The physicochemical properties of the electrocatalysts were performed by Raman spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). Activity towards ORR was carried out in a three-electrode electrochemical cell using a ring-disk electrode in 0.1M NaOH.

## EXPERIMENTAL

### Composites Synthesis

The preparation of the composites follows the synthetic strategy developed by our research group (Aghabarari et al., 2019) and is schematically illustrated in Figure 1. For the synthesis of composites, an appropriate amount of the chloride salt precursor (Sigma-Aldrich Company) of Co, Fe, or Cu (20 wt % with respect to the composite melamine/chitosan) was dissolved in water and then was added to a solution of chitosan in acetic acid solution. After that, a solution of melamine was added drop by drop to the mixture and stirred vigorously (weight ratio melamine/chitosan = 1/1). In the next step, the appropriate amount of the $H_3PMO_{12}O_{40}$ solution (10 wt % with respect to the composite melamine/chitosan) was added to the mixture and, after 2 h stirring, the solution was dried at 70°C in a vacuum oven. The sample was pyrolized in a tubular quartz furnace at 800°C over 3h. The product was washed with 10 wt% HCl solution. The acid-treated solid sample was filtered and washed with water until free of chlorine ions. Finally, the sample was dried at 70°C in a vacuum oven to obtain the final catalysts. The three catalysts were labeled as Fe/Mo$_2$C/NPC, Co/Mo$_2$C/NPC, and Cu/Mo$_2$C/NPC. For comparison, melamine/chitosan (NPC) composite was also prepared using a similar route but without adding any metal and heteropolyacid into the composite.

### Characterization of the Electro catalytic Materials

X-ray diffraction analyses of the composites were carried out on a PANalytical X'Pert Pro X-ray diffractometer with a Cu $K_α$ source. X-ray Photoelectron Spectroscopy (XPS) data was analyzed with a PHOIBOS 150 9MCD spectrometer with Multi-Channeltron detector fitted with a Mg $K_α$ radiation ($hν = 1,253.6$ eV) and an XRC-1000 X-ray gun. TEM images were obtained using a high resolution of transmission electron microscopy (HRTEM) JEOL 2100F operating at an accelerating voltage of 200 kV. Raman spectroscopy was carried out with a Renishaw in Via Raman
<figure><img src="image_170.png" /><figcaption>**Figure 1** | Schematic illustration of the formation of composites.</figcaption></figure>

<figure><img src="image_171.png" /><figcaption>**Figure 2** | X-ray diffraction (XRD) patterns of composites.</figcaption></figure>
<table><caption>TABLE 1 | Surface elemental contents examined by XPS (wt.%)</caption><thead><tr><th>Catalyst</th><th>C 1s</th><th>N 1s</th><th>Fe 2p</th><th>Co 2p</th><th>Cu 2p</th><th>P 2p</th><th>Mo 3d</th></tr></thead><tbody><tr><td>Fe/Mo<sub>2</sub>C/NPC</td><td>65</td><td>2.7</td><td>8.8</td><td>—</td><td>—</td><td>0.6</td><td>3.6</td></tr><tr><td>Co/Mo<sub>2</sub>C/NPC</td><td>70</td><td>6.7</td><td>—</td><td>5.0</td><td>—</td><td>0.3</td><td>6.7</td></tr><tr><td>Cu/Mo<sub>2</sub>C/NPC</td><td>47</td><td>5.0</td><td>—</td><td>—</td><td>25</td><td>0.9</td><td>7.9</td></tr></tbody></table>

Microscope spectrometer equipped with a laser beam emitting at 532 nm and 5 mW output power.

The electrochemical characterization of the composites was performed in a three-electrode cell system at room temperature controlled by an Autolab PGSTAT302N potentiostat-galvanostat. A glassy carbon rod and a reversible hydrogen electrode (RHE) were used as the counter electrode and the reference electrode, respectively. For the working electrode, a rotating ring-disk electrode (RRDE, PINE) with a glassy carbon disk with 0.196 cm² of area and a Pt ring was employed. The supporting electrolyte was 0.1M NaOH aqueous solution. The working electrode was prepared by adding 30 µL of catalytic ink. For the catalytic ink, 4 mg of composite was dispersed in 385 µL of Isopropyl alcohol (IPA) and water, IPA:H2O (1:1) solution and 15 µL of Nafion® (5 wt.%, Sigma-Aldrich) as a binder. For all measurements, N₂ (99.99%, Air Liquid) was employed to make an inert environment, and O₂ (99.99%, Air Liquid) was used for ORR measurements. In order to analyze the H₂O₂ yield, the ORR activity was performed while keeping the Pt ring at 1.2 V.

## RESULTS AND DISCUSSION

X-ray diffractograms of electrocatalysts are shown in **Figure 2**. All composites show diffraction peaks corresponding to Mo₂C (JPCDS 00-035-0787), while MoN (JPCDS 01-077-1999) is observed in Co and Fe composites. The diffraction peak at 26.6° confirms the correct graphitization of carbon in the Fe and Co composites. However, the highest crystallinity of Cu₂O (JPCDS 01-077-0199) and Cu (JPCDS 00-001-1242) contrasts with the low crystallinity of the carbon in the Cu/Mo₂C/NPC composite. It seems that copper promotes a lower graphitization degree of the carbon during the pyrolysis process. Co/Mo₂C/NPC shows diffraction peaks assigned to Co (JPCDS 00-001-1255) and CoO (JPCDS 01-072-1474). A diffraction pattern of Fe₃C (JPCDS 00-034-0001) was also observed in the Fe/Mo₂C/NPC composite.

During the pyrolysis process, chitosan and melamine gradually decompose to several carbon-nitrogen intermediates. Functionalization chitosan biopolymer with melamine becomes effective due to the strong interaction between the pyranose units and the amine groups, which results in the crosslinked networks (Rybarczyk et al., 2015). It is also reported that the pyrolysis of melamine generates a series of intermediate condensed phases, such as so-called melem (C₆N₇(NH₂)₃) or graphitic carbon nitride materials (g-C₃N₄), and then further releases some chemically reactive hydrogen-, carbon-, and nitrogen-containing atomic species, such as C₃N₃⁺, C₂N₂⁺ and CN₂H⁺, at higher temperatures (Huang et al., 2015). These reactive intermediates can behave as powerful reducing agents to

reduce different metal oxides to metal carbide, nitride, or metallic particles during the pyrolysis treatment. Furthermore, the simultaneous formation of C=C species from intermediates is considered as the carbon source of graphite (Li et al., 2014b; Yang et al., 2015).

The surface elemental compositions and electronic configurations of the atoms in the composites were investigated by employing XPS. **Table 1** summarizes the chemical surface composition in weight % of the catalysts. It is important to note that the copper content on the surface far exceeds the Fe and Co surface content of the other two composites and is also more than twice the nominal value, which would indicate a poor dispersion or higher segregation of copper on the carbon matrix. The transition metal used (Fe, Co, or Cu) affects the amount of N, P, and Mo incorporated into the composite. Cobalt composite shows the higher surface N content (6,7 wt.%), and copper composite the higher amount of P (0.9 wt.%) and Mo (7.9 wt.%). All samples display low P content, being lower than 1 wt.%.

The high-resolution N1s and P2p XPS spectra of the composites were shown in **Figures 3A** and **B**, respectively. The N1s peak deconvolution of the three catalysts proposes three nitrogen components with the binding energies centered at about 398, 400, and 401 eV, corresponding to pyridinic N, pyrrolic N, and quaternary N, respectively. Additionally, in **Figure 3A**, the peak at the lower binding energy at 397 eV is assigned to Mo3p in all catalysts (Li et al., 2016). However, this peak could also be assigned to nitride species, such as MoN in cobalt and iron composites, according to XRD analysis. The P2p XPS spectra display a broad and low intensity peak centered at 133-133.5 eV, assigned to P-C (Razmjooei et al., 2014; Razmjooei et al., 2015). These XPS results indicate that N and P are incorporated into the carbon matrix in the three catalysts.

The spectra for Fe 2p, Co2p, and Cu2p core level are shown in **Figure 4**. The Fe2p spectra in **Figure 4A** shows peaks at 711.1 and 724.9 eV, which are assigned to Fe2p3/2 and Fe2p1/2 respectively Fe₃O₄ (Wagner et al., 1979). The surface oxidation of the iron samples does not allow the analysis of the inner layer, and therefore the detection of the iron carbide observed by XRD. Co2p shows binding energies of Co2p3/2 and Co2p1/2 at 782.3 and 798.1 eV, respectively, and the respective “satellite” peaks, which can be attributed to Co²⁺-like species (Wagner et al., 1979; Luque-Centeno et al., 2020). The Cu2p core level spectra is displayed in **Figure 4C**. The main Cu2p3/2 signal is composed of two contributions at 932.7 and a shoulder at 934.4 eV, where the former is assigned to surface Cu⁰ and the latter to Cu²⁺ with the respective satellite peaks (Wagner et al., 1979; Ania et al., 2015). In a similar way, the Mo3d core level spectra shows the thin oxidized layer on the molybdenum carbide surface and MoO₃ is only detected by XPS (not shown).

The TEM images of the NPC samples are displayed in **Figure 5**. All composites show metal particles embedded in an amorphous graphite structure. In Fe/Mo₂C/NPC, the nanoparticles are uniformly dispersed in the carbon matrix. This dispersion decreases in Co/Mo₂C/NPC and gets much worse in Cu/Mo₂C/NPC. Iron and cobalt composites show the presence of particles with average particle sizes between 10 and
<figure><img src="image_172.png" /><figcaption>**Figure 3** | High-resolution N1s XPS spectra (A) and P2p (B) of composites.</figcaption></figure>

<figure><img src="image_173.png" /><figcaption>**Figure 4** | High-resolution Fe 2p (A), Co 2p (B), and Cu 2p (C) XPS spectra.</figcaption></figure>

30 nm cover of a thickness of the external carbon sheet. However, Cu/Mo~2~C/NPC shows the presence of copper aggregates in the carbon matrix with an irregular morphology, and some metal nanoparticles outside of the carbon matrix (Figure 5C).

The Raman spectra of the composites show the characteristic peak corresponding to the D and G bands at 1350 cm⁻¹ and 1580 cm⁻¹, respectively (Figure 6). The D band is assigned to the Alg breathing mode of disordered graphite structure and is due to the defect sites in
<figure><img src="image_174.png" /><figcaption>**Figure 5** | TEM images of Fe/Mo2C/NPC (A), Co/Mo2C/NPC (B), and Cu/Mo2C/NPC (C).</figcaption></figure>

<figure><img src="image_175.png" /><figcaption>**Figure 6** | Raman spectra of the composites. The calculated $I_D/I_G$ values are also presented.</figcaption></figure>

the hexagonal framework of graphite materials. The G band corresponds to the E2g stretching mode of graphite structure and denotes the sp² hybridized carbon atom structure (Yang et al., 2018). Both Raman bands have their corresponding overtones between 2500 and 3000 cm⁻¹, respectively. The height of the D peak usually increases upon chemical alteration of carbon structure. The ratio of D and G bands ($I_D/I_G$) is a good indicator of the disorder degree, graphitization, and crystallinity of the composites (values are indicated in Figure 6). It is observed that the $I_D/I_G$ ratio is lower for the Cu (0.91) composite with respect to Co and Fe composites (ca. 0.99), which indicate a higher disorder degree and defects of the graphite formed in the latter catalysts. Certainly, several studies have established the relevance of defects, which seem to be one of the most

challenging targets, in ORR electrocatalysis (Quílez-Bermejo et al., 2020). It is also observed that the intensity of the two-dimensional (2D) peak (~2690 cm⁻¹) increase in the Co composite and, particularly, in the Fe composite, revealing better graphitization of the carbon framework (Cheng et al., 2017). This suggests that the doping and graphitic structure of carbon matrix is not too dependent on the Co and Fe metal transition, but it changes with the copper metal.

The electrocatalytic activity of the composites was studied in alkaline media using a rotating ring-disk electrode (RRDE) technique with a rotation rate of 1,600 rpm. The key values for evaluating the ORR catalytic activity are the onset potential (obtained at -0.1 mA cm⁻²), half-wave potential ($E_{1/2}$) and the diffusion-limiting current. The ORR activity and H₂O₂ yields of the electrocatalysts are displayed in **Figures 7A** and **B**, respectively. NPC was added for comparison. As evidenced in **Figure 7A** and **Table 2**, Fe/Mo₂C/NPC exhibits the best performance with an onset potential of 0.922 V (vs RHE) at -0.1 mA cm⁻², and limiting current density of -4.39 mA cm⁻² at 0.2 V. In all other composites, the onset potential and limiting current gradually decreases from Co/Mo₂C/NPC (0.850 V, -3.57 mA cm⁻²), NPC (0.714 V, -1.86 mA cm⁻²), to Cu/Mo₂C/NPC (0.683 V, -1.64 mA cm⁻²). It is observed that the plateau is inclined in all composites, which could indicate some diffusion limitations or inhomogeneous distribution of active sites on the electrode. The $E_{1/2}$ values can embody the capacity that a fuel cell cathode needs, holding a high working potential to provide high energy conversion efficiency. From **Table 2**, it can be seen that the Fe composite shows the highest $E_{1/2}$ value (0.811 V), further indicating the highly active nature of the Fe/Mo₂C/NPC towards ORR. These results demonstrate that the transition metal considerably affects the ORR activity in the Mo₂C/NPC
<figure><img src="image_176.png" /><figcaption>**Figure 7** | ORR (A) and H₂O₂ yield (B) polarization curves for all catalysts at 1,600 rpm in 0.1 M NaOH, 10 mV s⁻¹. The applied potential at the ring electrode was 1.2 V for the ORR.</figcaption></figure>

<table><caption>**TABLE 2** | Electrochemical parameters for ORR of the composites</caption><thead><tr><th>Catalyst</th><th>E<sub>onset</sub><br>(V) at -0.1 mA cm<sup>-2</sup></th><th>E<sub>1/2</sub>(V)</th><th>J<sub>L</sub>(mA cm<sup>-2</sup>) at 0.2 V</th></tr></thead><tbody><tr><td>Fe/Mo<sub>2</sub>C/NPC</td><td>0.922</td><td>0.811</td><td>4.39</td></tr><tr><td>Co/Mo<sub>2</sub>C/NPC</td><td>0.850</td><td>0.710</td><td>3.57</td></tr><tr><td>Cu/Mo<sub>2</sub>C/NPC</td><td>0.683</td><td>0.576</td><td>1.64</td></tr><tr><td>NPC</td><td>0.714</td><td>0.502</td><td>1.86</td></tr></tbody></table>

The polarization curves indicate that the Fe/Mo₂C/NPC catalyst has very high ORR activities and is comparable with conventional Pt/C catalysts, in which the onset potential is usually identified ca. 0.990 V (Jiang et al., 2015). Therefore, the gap between the onset potential of Fe/Mo₂C/NPC and the standard Pt/C is only 32 mV.

Based on the ring current generated from the oxidation of H₂O₂, we can observe that the Cu/Mo₂C/NPC catalyst produces a higher level of H₂O₂ (ca. 50%) than iron and cobalt composites do. The peroxide formations of the latter two are lower than 13% in all potential ranges studied. These results reveal that the Cu composite follows a combined 2-3 e⁻ electron-transfer number. In addition, the RRDE measurements reveal that the production of the corrosive H₂O₂ is highly suppressed on Fe/Mo₂C/NPC and Co/Mo₂C/NPC, promoting a 4e⁻ pathway. It is also observed that NPC produced a higher level of H₂O₂ compared to Fe- and Co-based composites. This may be due to how in free-metals-doped carbons, the carbon atoms next to heteroatoms exhibit excellent bonding ability to adsorb OOH, thus assisting the easy formation

of the peroxide intermediate (Brouzgou et al., 2016; Sun et al., 2018).

Therefore, Fe/Mo₂C/NPC exhibits a higher onset potential and limiting current density, and has a selectivity of about four electrons in alkaline media, while Cu/Mo₂C/NPC has the lowest electron transfer number and worst ORR performance. These results indicate that Fe/Mo₂C/NPC composite is a promising electrocatalyst for H₂O₂ production.

It is known that nitrogen and phosphorous dual-doped carbons improve the catalytic activity for ORR due to the synergetic effects (Choi et al., 2012; Li et al., 2015; Qiao et al., 2015). DFT simulations have demonstrated that quaternary nitrogen, along with phosphorus groups, are responsible for the improvement in the electrocatalytic activity in alkaline solution (Li et al., 2017). Also, the higher disorder degree and defects promoted by doped-carbons have revealed its significance in ORR activity (Quílez-Bermejo et al., 2020). According to our results, N and P are incorporated into the carbon matrix in the three composites. However, it has been shown that the transition metal is essential to obtain optimal carbon framework for ORR, with the copper being the one that promotes the formation of a less graphitized and defective carbon matrix during the synthetic process.

Furthermore, previous studies by our group show that the presence of the heteropolyacid HPMo can improve the ORR activity of graphene/chitosan composites compared to HPMo-free catalysts (Aghabarari et al., 2017). However, it should be pointed out that the order of surface N, P, and Mo amounts are not consistent with the catalyst's ORR performance. The Cu-composite has the highest Mo and P amounts and the second highest N amount, but it
has the lowest ORR performance. Clearly, the results obtained provide evidence of the important role played by each transition metal in the formation of active sites for ORR.

The Fe₃C encapsulated in dual-doped carbon materials seems to be an important active site (Yang et al., 2015; Ren et al., 2016). The outer protective graphitic layers stabilize the carbide nanoparticles, while the Fe₃C/Mo₂C nanoparticles should play a synergetic role in activating the outer surface of the graphitic layers towards the ORR. Such an enhanced catalytic activity has been explained from the large amounts of oxygen vacancies created by Mo₂C and Fe₃C as electron donors. On the other site, the outstanding ORR performance of Fe₃C-based ORR catalysts can be also attributable to the factors of the N dopants and Fe–Nₓ coordination species (Jiang et al., 2015). Furthermore, in this complex system we cannot rule out the presence of electrochemically active Fe-P interactions either. Recently, Razmjooei et al. found that the introduction of Fe on P-functionalized graphene caused remarkable synergistic effects, which were beneficial for ORR (Razmjooei et al., 2015). Therefore, these results highlighted the advantage of the facile synthetic approach to obtain a complex formed by Fe₃C along Mo₂C embedded in N- and P-dual doped carbon, which leads to significant ORR activity. However, the active sites of the Fe-related species need to be further elaborated, which is under progress.

## CONCLUSIONS

In summary, a facile and cost-effective strategy for the production of Me/Mo₂C (Me = Fe, Co, Cu)-based composites embedded in N- and P-dual doped carbon matrices has been developed. For this end, inexpensive industrial materials, such as melamine and chitosan, as C and N sources and the heteropolyacid H₃PMO₁₂O₄₀ as P and Mo precursor were successfully used. Among transition

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## DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

## AUTHOR CONTRIBUTIONS

B.A. conceptualization, methodology, investigation, materials. M.-C.S. analysis, interpretation. M.L. review, funding acquisition, suggestions. M.M.-H. supervision, logical interpretation, writing—review and editing.

## ACKNOWLEDGMENTS

The authors acknowledge the Ministerio de Ciencia, Innovación y Universidades (MCIU), and FEDER for the funding received for the project, with references ENE2017-83976-C2-1-R, Spanish National Research Council COOPB20202, and MERC project 721399002.

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Lazaro, Martínez-Huerta, Aghabarari and Capel-Sánchez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Environmental Material

<figure><img src="image_177.png" /><figcaption>Elisa Franzoni</figcaption></figure>

Prof. Elisa Franzoni has a Ph.D. in Materials Engineering and is an Associate Professor in Materials Science and Technology at the University of Bologna, where she leads the research group Materials and Technologies for Cultural Heritage. In 2020, she coordinated the project HAP4MARBLE and participated in both ROCK and SHELTER. She was in charge of research projects funded by the industry and has collaborated on research with national and international universities, private companies, and public authorities in the field of materials for cultural heritage. She is a member of RILEM and an Associate Editor of Materials and Structures (IF 2.90). She is the author of more than 150 scientific publications (90 in Scopus, H-index 27) and the creator of one patent.
<figure><img src="image_178.png" /></figure>

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# Experimental Study on an Innovative Biopolymeric Treatment Against Salt Deterioration of Materials in Cultural Heritage

Mattia Bassi¹, Enrico Sassoni¹,² and Elisa Franzoni¹,²*

¹Interdepartmental Centre for Industrial Research in Building and Construction (CIRI Building and Construction), University of Bologna, Bologna, Italy, ²Department of Civil, Chemical, Environmental and Materials Engineering (DICAM), University of Bologna, Bologna, Italy

Salt crystallization is one of the harshest deterioration mechanisms affecting heritage materials, causing impressive decay patterns and the loss of a high thickness of original materials. Although salt damage has been widely investigated in the literature from the theoretical and experimental points of view, the solutions to mitigate this problem are still extremely limited. In the present paper, a new biopolymeric treatment based on chitosan was tested on two kinds of porous limestones widely used in historic architecture, aiming at inhibiting the crystallization of sodium sulphate inside the stone and promoting the formation of salt efflorescence over the surface, rather than harmful subflorescence inside the pore network. The treatment was applied to the bare stone and also after an inorganic pre-treatment based on the formation of hydroxyapatite in the stone. Hydroxyapatite was recently proposed for the consolidation and protection of carbonate stones and here it is expected to provide an effective anchoring layer for the chitosan coating on the pores surface, and also to prevent the calcite washout from the stone and hence the removal of chitosan. The effect of hydroxyapatite alone was also tested, for comparison's sake. Treated and untreated stone specimens were subjected to two different accelerated salt crystallization tests, one based on crystallization cycles (wetting-drying cycles) and the other one based on continuous capillary absorption of a saline solution ("wick effect"), evaluating the results in terms of weight loss, efflorescence formation, and changes in porosity and mechanical properties. The results showed that all the treatments are compatible with the stones, and the combined treatment (hydroxyapatite + chitosan) is extremely promising for the prevention of salt damage.

**Keywords:** chitosan, hydroxyapatite, salt inhibitor, sodium sulphate (mirabilite and thenardite), biopolymer, efflorescence, limestone, salt crystallization tests

## INTRODUCTION

One of the major causes of deterioration and damage of cultural heritage is the presence of salts within historic building materials such as stone, brick, mortars and plasters, and also in more recent materials such as concrete (Charola, 2000; Sandrolini and Franzoni, 2007; Doehne and Price, 2010; Espinosa-Marzal and Scherer, 2010; Franzoni, 2014; Charola and Bläuer, 2015; Charola and Wendler, 2015; Lubelli et al., 2018; Andreotti et al., 2019). Damage is caused by crystallization

## OPEN ACCESS

**Edited by:**

Guang-Ling Song,
Xiamen University, China

**Reviewed by:**

Dusan Losic,
University of Adelaide, Australia
Peng Wang,
Harbin Medical University, China
Pagona-Noni Maravelaki,
Technical University of Crete, Greece

**\*Correspondence:**

Elisa Franzoni
elisa.franzoni@unibo.it

**Specialty section:**

This article was submitted to
*Environmental Materials,*
a section of the journal
*Frontiers in Materials*

**Received:** 15 July 2020

**Accepted:** 08 January 2021

**Published:** 18 February 2021

**Citation:**

Bassi M, Sassoni E and Franzoni E (2021) Experimental Study on an Innovative Biopolymeric Treatment Against Salt Deterioration of Materials in Cultural Heritage. *Front. Mater.* 8:583112
doi: 10.3389/fmats.2021.583112
of salts, due to evaporation and/or cooling of saline solutions, and in particular by the solubilization/crystallization cycles, due to the strong sensitivity of salts to the thermo-hygrometric conditions. This leads to formation of internal mechanical stress in the material, protracted over time. In most cases, this stress can easily overcome the tensile strength of building materials, usually quite low, causing formation and propagation of cracks, which affect structural and decorative elements of buildings (Flatt, 2002; Scherer, 2004).

Salts can enter building materials in different ways, the main one being certainly the capillary absorption of moisture from the soil (Charola, 2000; Doehne and Price, 2010; Franzoni, 2014; Charola and Bläuer, 2015; Charola and Wendler, 2015). In this case, water coming from rain, underground water sources or broken pipes, filters through the ground dissolving the salts contained in it. The saline solution is absorbed by the wall by capillarity, up to a height that depends on the evaporation rate, related to thermo-hygrometric conditions (temperature, T, and relative humidity, RH) and materials porosity (Sandrolini and Franzoni, 2007; Charola and Bläuer, 2015; Charola and Wendler, 2015). As long as the water supply is greater than the evaporation rate, the wall will appear wet on the surface and salts will crystallize over the surface as efflorescence, involving mainly aesthetical damage. When the evaporation rate increases, the evaporation front will move inwards, where the salt solution will reach the supersaturation necessary for the crystallization of salts within the porous network of the materials, causing formation of potentially harmful subflorescence (Franzoni, 2014; Andreotti et al., 2019). In addition to capillary rise, salts can be autochthonous in the materials (for example, in Portland cement or in bricks) or can come from external sources such as deicing salts used in roads, from chemical products used in agriculture, from marine spray in the case of buildings near the coast or from the chemical reactions between building materials and air pollutants (Charola and Bläuer, 2015; Charola and Wendler, 2015).

Over the last decades, the phenomenon of damage caused by salt crystallization has been widely studied and many theories have been formulated. It is now clear that, following evaporation or cooling of a salt solution, supersaturation conditions are reached, i.e., conditions where the concentration of salt dissolved in solution is greater than the saturation one, at the same temperature: in this case, salt crystals precipitate in the pores of the material (Scherer, 2004; Doehne and Price, 2010). As a crystal grows and approaches the inner pore wall, repulsive forces arise to prevent the direct contact between the two surfaces: this "disjoining pressure" is attributed to repulsive Van der Waals forces in the case of ice or to electrostatic forces in the case of salt crystals (Scherer, 1999; Houck and Scherer, 2006; Andreotti et al., 2019). Therefore, the two surfaces organize the orientation of the water molecules and ions in different ways, to minimize their surface energy, and this prevents the direct contact between the crystal and the pore wall, allowing the persistence of a thin liquid film (a few nanometers thick) (Scherer, 1999). Internal tensile forces are generated in this liquid film and, if this layer did not exist, the crystal would not damage the material. However, the energy necessary to overcome repulsive forces (energy that would

allow direct contact between the surfaces) is usually greater than the tensile strength of the material and this causes the formation of cracks and the subsequent failure of the material itself (Houck and Scherer, 2006; Espinosa-Marzal and Scherer, 2010). The amount of damage will depend on various factors, first of all the degree of supersaturation reached, which in turn is influenced by the type of salt, material porosity, pore size distribution, etc. (Scherer, 2004; Angeli et al., 2007).

Increasing the tensile strength of decayed building materials by application of consolidating materials can indirectly reduce their deterioration due to physical-mechanical processes, such as ice and salt damage. However, the goal of consolidation is restoring the cohesion of decayed materials and not preserving them from future deterioration processes, hence alternative strategies have been explored to mitigate salt damage. Of course, controlling the environmental conditions (T and RH) and eliminating water sources (first of all, capillary rise) can help reduce damaging of walls. However, it is impossible to control temperature and relative humidity in outdoors environment and also the elimination of rising damp is definitely challenging (Flatt, 2002; Franzoni, 2014; Franzoni, 2018; Andreotti et al., 2019). In fact, although there are different methods to prevent rising damp, many of these methods are not fully effective and compatible, or simply are difficult to apply in historic structures (Franzoni, 2014). Some interventions, such as the use of hydrophobic treatments or the application of scarcely porous layers on the masonry surfaces, are even counterproductive, leading to a higher rise of damp and, often, to more severe damage caused by moisture and salts (Franzoni, 2014).

Several studies have focused on the use of modifiers of salt crystallization, which can act during both steps of the crystallization process (nucleation and growth of crystals) and can be distinguished in two different groups: inhibitors and promoters (Rodriguez-Navarro et al., 2002; Granneman et al., 2019; Bracciale et al., 2020). Crystal nucleation inhibitors are modifiers that increase the degree of supersaturation necessary to allow for the nucleation of crystals, thus increasing the time between the formation of supersaturation condition and the crystallization of salt. This results in the transport of the supersaturated salt solution over longer distances, thus favoring the formation of efflorescence rather than more harmful subflorescence. On the other hand, at higher level of supersaturation, if conditions for crystallization of the supersaturated solution occur, more severe damage will be experienced (Granneman et al., 2019; Bracciale et al., 2020). Crystal growth inhibitors are modifiers that retard crystal growth, covering the whole crystal surface and preventing the addition or the detachment of salt units (Granneman et al., 2019). Nucleation promoters work in the opposite way, increasing the salt nucleation rate that occurs at lower supersaturation (near or at the saturation condition), thus causing less damage to the substrate (Granneman et al., 2019). Different substances were tested in the last years as crystallization modifiers, like ferrocyanides (Rodriguez-Navarro et al., 2002; Selwitz and Doehne, 2002; Lubelli and van Hees, 2007; Rivas et al., 2010; Gupta et al., 2012) as inhibitors for sodium chloride, phosphonates (Lubelli and van Hees, 2007; Ruiz-Agudo et al.,
2012) as inhibitors for sodium sulphate and borax (Smith et al., 2010; Granneman et al., 2017) as promoter for sodium sulphate, just to name the most promising ones. A different strategy, adopted for example in (Houck and Scherer, 2006), is based on eliminating the crystallization pressure between the growing salt crystal and the inner pore wall by deposition of a thin polymeric layer. If the material surface could be modified so that the salt crystal does not repel it, the crystal itself would be able to grow in direct contact with the pore wall: in this case, the liquid film will not be maintained and, therefore, no pressure or damage will be generated in the material (Houck and Scherer, 2006). All of these products have different effectiveness and give different results depending on the type of salt, the porous substrate on which they are applied, the concentration used, etc., so that an improvement in the resistance to salt crystallization is usually found, but sometimes an increase in damage is experienced. The development of these crystallization modifiers is still mostly in the experimental phase, with many laboratory tests and few onsite applications, so more studies seem necessary (Granneman et al., 2019; Bracciale et al., 2020). In recent years, biomass-derived inhibitors were developed for the prevention of salt crystallization damage caused by different types of salt, focusing mainly on sodium sulphate and sodium chloride due to their destructive potential. It is the case, for example, of phosphocitrate, that was already tested with good results on different materials in laboratory and in the field (Cassar et al., 2008; Franceschini et al., 2015; Bracciale et al., 2020). These products, besides being derived from renewable sources, are non-toxic and usually soluble in water and/or alcohol, thus avoiding the use of harmful solvents (Bracciale et al., 2020), giving benefits to environment and human health. It is in this perspective that, in the present paper, a new bio-based protective material against salt crystallization (chitosan) was employed to improve the resistance of two porous carbonate stones to salt damage, alone and combined with a recently developed consolidant (hydroxyapatite). The present paper follows a previous preliminary study, in which this combined treatment was proposed for the first time, with encouraging results (Andreotti et al., 2019). An overview of the main properties of hydroxyapatite and chitosan is provided in the following.

Since more than a century, various organic and inorganic consolidating materials have been proposed for stone and other porous materials, with different results. Among these, the use of hydroxyapatite (HAP) and other calcium phosphate phases (CaP) has been studied since 2011 for consolidation and protection of carbonate stones (Sassoni et al., 2011). The CaP-based treatment has high potential because it provides significant mechanical consolidation in a short time, without altering porosity or transport properties of the substrate (Naidu et al., 2011; Sassoni et al., 2011; Sassoni et al., 2016a; Sassoni et al., 2016b; Sassoni, 2018). Hydroxyapatite, which is the main inorganic constituent of human teeth and bones, is a good consolidant for carbonate substrates as it is much less soluble than calcite and, at the same time, has a similar crystalline structure (Sassoni et al., 2011). As reported in the literature, the formation of HAP *in situ* is expected to occur by reaction between a carbonate-rich substrate, which provides calcium ions

through dissolution, and an aqueous solution of a phosphate salt, usually diammonium hydrogen phosphate ((NH₄)₂HPO₄, indicated as DAP), which provides PO₄³⁻ ions (Naidu et al., 2011; Sassoni et al., 2011; Sassoni, 2018), according to the following reaction:

$$10\text{CaCO}_3 + 5(\text{NH}_4)_2\text{HPO}_4 \rightarrow \text{Ca}_{10}(\text{PO}_4)_6\text{CO}_3\cdot6(\text{OH},\text{CO}_3)_2 + 5(\text{NH}_4)_2\text{CO}_3 + 3\text{CO}_2 + 2\text{H}_2\text{O}$$

One of the advantages of the use of the DAP-treatment is that all the by-products of the reaction, i.e., ammonium carbonate, carbon dioxide and water, are innocuous and volatile and, therefore, unwanted residues are not expected to remain in the stone.

In very recent years, the use of natural and biodegradable substances, mainly biopolymers, has been proposed in the field of conservation of building materials, as well as in other sectors. The idea backgrounding this new class of conservation materials is that they can potentially provide some interesting properties for a certain number of years, then naturally disappearing without leaving unwanted residues (Ocak et al., 2009; Ocak et al., 2015; Pedna et al., 2016; Andreotti et al., 2018). This requires that the treatment is applied again after some years, during maintenance works, but it also involves some advantages in comparison with traditional polymers used in conservation for many decades. In fact, traditional polymers are subject to unavoidable aging processes and performance loss in a limited number of years, but they are also hard to remove once they transformed during aging (by cross-linking, chain scission, crystallization, oxidation, etc.), thus influencing, and sometimes jeopardizing, the possibility to re-treat the materials with new consolidating and/or protective substances. In fact, the application of different polymeric treatments to heritage materials caused severe problems during last decades. In this context, some biopolymer-based water-repellents have been proposed (Ocak et al., 2009; Ocak et al., 2015; Pedna et al., 2016; Andreotti et al., 2018) for the protection of heritage materials against rain absorption, and chitosan was proposed as a treatment to fight salt damage (Andreotti et al., 2019).

Chitosan is already widely used in agriculture, in food sector, in environmental protection and in various biomedical applications, where its antibacterial and antioxidant properties are exploited (Muxika et al., 2017). Chitosan is produced from chitin, a marine origin polysaccharide that is widely available, being the second most abundant natural polymer after cellulose (Muxika et al., 2017; Philibert et al., 2017). Chitin is the main structural component in the exoskeleton of marine invertebrates and insects, as well as molluscs and crustaceans. When combined with calcium carbonate, like in shells of crustaceans and insects, chitin produces a strong composite, exhibiting improved hardness and stiffness compared to pure chitin and also a greater tenacity and ductility than pure CaCO₃ (Muxika et al., 2017; Andreotti et al., 2019). Chitin is a linear polysaccharide, consisting in N-acetyl-D-glucosamine units, connected by β—1.4 bonds (Muxika et al., 2017; Philibert et al., 2017; Andreotti et al., 2019), which is mostly obtained from industrial waste in the processing of marine shells, shrimps and similar, hence it is abundantly available. Despite its outstanding characteristics such
as biocompatibility, biodegradability and high mechanical
strength, its exploitability is limited by its very low solubility
(Muxika et al., 2017; Philibert et al., 2017; Andreotti et al., 2019).
This turns the attention to chitosan, the main derivative of chitin.
Chitosan is a copolymer obtained from the alkaline deacetylation
of chitin, a process involving the treatment of chitin with
hydroxides at high temperature. It consists in D-glucosamine
and N-acetyl-D-glucosamine units, connected by β—1.4
glycosidic bonds (Muxika et al., 2017; Philibert et al., 2017;
Andreotti et al., 2019). The ratio between the two units is
called degree of deacetylation: for values ≥50%, chitosan
becomes soluble in acidic media and, in this case, the aminic
groups and the polymer become cationic, allowing them to
interact with different molecules. This positive charge is
believed to be responsible for its antimicrobial activity,
allowing it to interact with negatively charged cellular
membranes of microorganisms (Muxika et al., 2017).

The research in (Andreotti et al., 2019) was the first example of
study on the behavior and the effects of chitosan applied to
porous building materials. The study addressed prevention of salt
crystallization damage by covering the pore surface with a
chitosan coating, which can inhibit salt crystallization inside
the pore network of the material. Chitosan was selected for
some interesting properties: presence of functional groups in
the molecular structure, acting as anchoring groups towards the
mineral surfaces; film-forming capacity, to generate a continuous
coating on the pore walls; highly flexible chains that allow for the
interaction with any type of salt. Moreover, this polymer is not
hydrophobic and does not alter the water transport properties of
the treated substrate, which is beneficial for salt damage resistance
and for preventing other deterioration processes (Houck and
Scherer, 2006). In addition, it is bio-based and biodegradable,
thus providing a minimum toxicity and complying with the new
concept of “reversibility of treatment” explained above. Three
different experimental setups were used in (Andreotti et al., 2019)
and the results are briefly reported in the following. A first
crystallization test was carried out by cooling a saturated
aqueous solution of sodium sulphate and chitosan, showing
that the polymer has a strong inhibiting effect on the salt
crystallization, basically blocking it up to 2 °C, while in
absence of chitosan salt crystallization occurred at 13.1 °C. The
fact that chitosan inhibits sodium sulphate crystallization in the
solution is thought to be due to its polyelectrolytic nature,
providing a high number of positively charged sites that
sequester sulphate ions. However, it was also reported that
some polymers that inhibit salt crystallization when in
solution might favor it when they are adsorbed on a solid
surface, due to some polymer chain orientation (Ruiz-Agudo
et al., 2006), so this aspect was specifically taken into account. A
second test was carried out on different powdered substrates
treated with chitosan. The powders, thanks to their large specific
surface, allowed to demonstrate the remarkable film-forming
ability of chitosan, which was adsorbed on the solid surface,
creating a coating. Notably, the DAP-based consolidating
treatment, applied before the polymer, caused an increase in
the amount of chitosan adsorbed, probably thanks to the
roughness of CaP phases that act as anchoring surfaces. When

subjected to water flow, the treated powders (both with and
without the DAP pre-treatment) exhibited initial solubilization of
the thick chitosan layer, after which the adsorbed polymer was
not further leached in water. In the final test, limestone samples
were treated to investigate whether the inhibiting effect of
chitosan observed in solution actually delay salt crystallization
inside the stone, thus promoting formation of efflorescence over
the surface, rather than development of mechanical stress inside
the stone. While the polymeric treatment alone caused greater
damage in the samples compared to the untreated references, as
the retarding action of chitosan caused a higher supersaturation
degree and a greater crystallization pressure in the zones of the
pore walls not covered by the chitosan film, in the samples treated
with DAP and then chitosan there was a remarkable benefit, with
promotion of efflorescence and increase in the resistance to
degradation.

While a preliminary evaluation of the adhesion and thickness
of the investigated coatings on controlled, flat and non-porous
surfaces of calcite and silica, and on powders of the two minerals,
was carried out in the previous study with encouraging results
(Andreotti et al., 2019), this paper focuses on the performance of
the treatments in real stones, selecting two different salt
crystallization tests aimed at producing a less drastic and
hence more easy-to-interpret deterioration in the stone, with
respect to the previous study. The two tests were carried out in
order to consider two different mechanisms presently adopted to
reproduce the damage by salts in laboratory, namely salt
crystallization cycles and “wick effect”. In this frame, it was
decided to use the same shape of the samples adopted in the
relevant standards and literature papers, i.e., cylinders for salt
crystallization cycles (EN 12370, 1999) and prisms for the “wick
test” (Goudie, 1986).

Starting from the preliminary findings briefly summarized
above, the present study aims at further investigating the behavior
of chitosan, by applying it to different substrates. In addition to
the polymeric treatment alone, pre-treatment by DAP (followed
by chitosan application) and treatment by DAP alone were also
tested. The CaP phases that form in the material after the DAP
treatment have been shown to provide good consolidating
effectiveness, thus providing some benefits also in terms of
resistance to salt crystallization pressure. Moreover, in the
double treatment (DAP solution followed by chitosan), the
CaP layer is expected to provide a good anchorage for the
formation of a more homogeneous and defect-free polymeric
layer, and also to protect calcite from dissolution in both neutral
and slightly acidic water (Sassoni, 2018), thus preventing possible
washout of the coating, which could cause a loss of effectiveness
(Andreotti et al., 2019).

MATERIALS

Stones

The lithotypes chosen for this research are two types of bioclastic
limestone, widely used in historic architecture of specific
geographical areas, but also representative of porous carbonate
stones used in many countries.
The first one is Globigerina limestone (labeled as “G”), which had been used also in the cited preliminary study (Andreotti et al., 2019). This stone is the major lithotype of the island of Malta, widely used in historic architecture and subject to severe deterioration by atmospheric agents and salts, leading to alveolization, flaking and pulverization (Sassoni et al., 2016a). It is an organogenic limestone composed mainly of calcite (about 95%), with traces of quartz and other impurities, and having a yellow-cream color.

The second lithotype was Lecce stone (labeled as “L”), a limestone used in southern Italy and, in particular, in Salento, which was chosen because it is similar to Globigerina limestone, but different enough to verify the repeatability of the results on a different substrate. Lecce stone is an organogenic limestone too, yellow-greyish in color, with a quite homogeneous composition based on calcite (about 95%), including a strong presence of marine fossils of different sizes, and a minor presence of phosphatic minerals (fluorapatite). The nature of this stone, as well as that of Globigerina limestone, makes it very vulnerable to deteriorating agents and salts in particular (Calia et al., 2013).

The slabs of Lecce stone from Cursi-Zollino-Melpignano quarry, Lecce (supplied by Décor S. r.l.) and the slabs of Globigerina limestone from the Qrendi area, Malta (Franka type, supplied by Xelini Skip Hire and High-up Service), were cut into eight prisms (3 × 3 × 25 cm³, the longest side cut parallel to the stone bedding planes) and 21 cylinders (5 cm in diameter, 5 cm in height, core-drilled perpendicular to the bedding planes). The samples were gently washed with deionized water and placed in an oven at 60 °C until constant weight, then cooled to room temperature. Cylinders and prisms are indicated with the letters “C” and “P”, respectively, following the letter indicating the type of stone (L_C, L_P, G_C, G_P).

## Treatments

In the light of previous literature studies, where the application of DAP-based solutions was tested on different substrates, adopting different DAP concentrations and different additional compounds (Naidu et al., 2011; Sassoni et al., 2011; Franzoni et al., 2015; Sassoni et al., 2016a; Sassoni et al., 2016b; Sassoni, 2018), a solution containing 0.1 M DAP + 0.1 mM CaCl₂ in 10 vol % ethanol was used in the present study. DAP, CaCl₂·2H₂O and ethanol were purchased from Sigma-Aldrich (reagent grade). A low concentration of DAP is preferable to avoid formation of a too thick film over the pore surface, which would more prone to cracking during drying, as well as to prevent formation of metastable CaP phases and the possible persistence of unreacted DAP in the treated stone. Because the lower the DAP concentration, the lower the amount of PO₄³⁻ ions necessary to form HAP, ethanol and CaCl₂ were added to the DAP solution. Ethanol exerts a weakening action on the hydration sphere of phosphate ions in solution and hence promotes formation of CaP (Graziani et al., 2016; Sassoni et al., 2018), while CaCl₂ guarantees a better surface coverage by providing additional calcium ions, also preventing excessive dissolution of the substrate (Naidu and Scherer, 2014).

For the polymeric treatment, based on the results obtained in (Andreotti et al., 2019), a 0.05 wt% chitosan solution was

prepared. As mentioned in the Introduction, chitosan can be solubilized only in acidic media. For this reason, the pH of the solution was brought to ~3 with the addition of acetic acid, which was chosen because it does not leave any unwanted components in the solution. Once the chitosan was completely dissolved, the solution was brought back to neutrality (pH between six and 7), not to damage the stone substrate. pH adjustment was obtained by adding 15 ml of a 0.03% NaOH solution to 1 L of chitosan solution.

The labels and number of specimens per type of treatment are summarized in the following:

- two cylinders for each lithotype were not treated, remaining as reference material (labeled as “UT”);

- two prisms and three cylinders for each lithotype were not treated and were subjected to the crystallization tests (labeled as “UT_SALT”);

- two prisms and five cylinders for each lithotype were treated only with the chitosan solution (labeled as “CHIT”). Except for two cylinders, which were left non-degraded, all these samples were subjected to the crystallization tests (“CHIT_SALT”);

- two prisms and five cylinders for each lithotype were treated only with the consolidating solution (labeled as “HAP”). Except for two cylinders, which were left non-degraded, all these samples were subjected to the crystallization tests (“HAP_SALT”);

- two prisms and six cylinders for each lithotype were treated first with the consolidating solution and then with the chitosan one (labeled as “HAP + CHIT”). Except for two cylinders, which were left non-degraded, all these samples were subjected to the crystallization tests (“HAP + CHIT_SALT”).

# METHODS

## Characterization

The main characteristics of the stones used in the research were first investigated. The values of bulk density, open porosity and dynamic elastic modulus were measured not only in the untreated stone specimens, but also in subsequent phases, to check the effectiveness of the treatments and the extent of degradation induced by the salt crystallization tests.

The bulk density, ρ_g [g/cm³], of the stone samples was determined as the ratio of dry mass over volume, as average for all the specimens. The size of the specimens was measured with a caliper and the dry mass was measured after oven drying at 50 °C until constant mass and cooling to room temperature.

Water absorption test was carried out according to (EN 15801, 2009) on three cylinders (UT) of each lithotype and on all the prisms. The samples were placed vertically on a filter paper pack partially immersed in deionized water, checking that the water level did not exceed the height of the filter paper, so that the samples absorbed water by capillarity only from the base surface. The samples were weighed at increasing time intervals for a total of 24 h. The capillary water absorption coefficient, AC [kg/m²√s], is represented by the slope of the first linear part of
the curve, and the total water absorption after 24 h, WA<sub>24hrs</sub> [wt %], is calculated as follows: WA<sub>24hrs</sub> = (W<sub>24hrs</sub>−W<sub>dry</sub>)/W<sub>dry</sub>. This test was carried out on two cylindrical replicates for each treatment too (CHIT, HAP, HAP + CHIT), to evaluate if and how much the treatments altered the transport properties of the materials. In fact, porosity and transport properties are fundamental aspects to consider when investigating the crystallization of any saline solution. Moreover, any treatment in the conservation of heritage materials is required to cause very limited porosity alterations, to avoid damages and defects both in short and long time.

Tensile splitting test was carried out on cylinders after the salt crystallization cycles (2 replicates for each condition) and on UT samples, to determine the tensile strength σt [MPa] of the stone before and after degradation. In fact, salt degradation leads to a loss of cohesion between the stone particles, the detachment of an outer layer and the formation of internal micro-fractures: this leads to decrease in the tensile strength of the material. Consolidating substances, such as hydroxyapatite, are applied to restore the cohesion between the particles and bring back the tensile strength of the stone to its original value, thus increasing its resistance to degradation. For this reason, the splitting test on degraded samples is an indication of the effectiveness of the treatments applied. The test was carried out using an Amsler-Wolpert loading machine (maximum load 20 kN) at a constant displacement rate of 4 mm/min and cardboard couplers (1 mm thick) to allow for uniform compression along the sample. Before carrying out this test, the lateral surface of the samples was gently scratched with fine sandpaper to remove any possible protuberances.

Open porosity and pore size distribution of the two stones were determined by mercury intrusion porosimetry (MIP), using a Thermo Scientific Pascal 140 (macropores unit) and a Pascal 240 (micropores unit). In particular, two fragments of each lithotype were analysed, about 1 g in weight. This analysis was performed also on the treated and the salt deteriorated samples, collecting one fragment for each condition from cylindrical samples, to analyse the changes in porosity. The fragments of the salt deteriorated samples (described in § 4.2.1) were taken close to the top surface of the cylinders (at the center of such surface), excluding the external salt crust. Before the analyses, these fragments were desalinated with a poultice of cellulose pulp and deionized water (weight ratio 1 : 4), which was wrapped in a PE film and left to act for 24 h. Afterwards, the film was removed and the poultice was allowed to completely dry. Desalination was carried out to remove the salt crystals deposited in the pores, thus allowing to investigate the variation of the stone porosity due to deterioration of only.

The external surface of untreated and treated samples was observed using a Philips XL20 SEM scanning electron microscope (SEM), equipped with energy dispersive spectrometry (EDS, EDAX probe). The sample surface was made conductive by sputtering with aluminum before SEM observation.

The dynamic elastic modulus E<sub>d</sub> [GPa] was measured on the cylinders before and after treatments, and on the prisms at each stage of the research. Ultrasonic tests for the determination of dynamic elastic modulus were carried out with a Matest

instrument, with 55 kHz transducers and rubber couplers to improve the contact between surfaces. For cylinders, the measurement was performed across the height of each sample, while for prisms 10 elastic modulus values were measured for each sample, i.e., at 4 cm intervals along the height of the prism and in two orthogonal directions. In fact, as the prisms are 25 cm high, they could exhibit internal inclusions (for example, fossils in Lecce stone), causing a certain degree of heterogeneity and variability of the values. The dynamic elastic modulus for the prisms was obtained averaging all the 10 values obtained for each sample and the two samples of each condition. For the prisms, E<sub>d</sub> was measured in the original stone specimens, in the samples treated with consolidant and/or polymer, in the samples deteriorated by the salt crystallization test and in these latter samples after desalination. In fact, after the salt crystallization cycles, the prisms where subjected to three desalination procedures, by using the same poulticing method described above. Before any E<sub>d</sub> measurement, the samples were dried in oven at 50 °C for 24 h.

## Treatment Procedure

Both consolidating and biopolymeric solutions were applied to stone samples by capillarity. In the case of the consolidant, four prisms and 11 cylinders of each lithotype were placed in vessels under a 5 mm solution head, the prisms lying on a lateral face to allow for a better absorption. For the first 2 h, necessary refills were carried out and then, when the samples appeared completely wet, the vessels were covered, and the samples were left to absorb the solution up to a total of 24 h. Afterwards, a quick washing under deionized water was carried out, to eliminate unreacted DAP and any possible surface patina, and the samples were dried in room conditions until constant weight. The biopolymeric treatment was applied according to the same procedure. In particular, the chitosan solution was applied to two prisms and five cylinders of untreated stone and two prisms and six cylinders of stone that had been previously treated with the DAP-based consolidating solution and left to dry.

After each treatment application and subsequent drying, the samples were weighed to determine their weight increase, an indication of the amount of hydroxyapatite and/or polymer deposited in the stone. The dynamic elastic modulus was measured again on treated samples, after drying. The weights and the dynamic elastic modulus values were averaged for the five replicates of UT, CHIT and HAP cylinders, the six replicates of the HAP + CHIT cylinders and the two prismatic replicates of each type.

## Crystallization Tests

Sodium sulphate is normally used for accelerated weathering tests in laboratory because it is one of the most harmful salts in nature (Rodriguez-Navarro et al., 2000; Tsui et al., 2003; Steiger and Asmussen, 2008; Espinosa-Marzal and Scherer, 2009; Flatt et al., 2014). The peculiarity of this salt is its capacity to crystallize in eight different forms and, above all, to have two phases that can crystallize at normal environmental temperature and humidity: thenardite (anhydrous phase) and mirabilite (decahydrate phase) (Steiger and Asmussen, 2008; Flatt et al., 2014). The greatest
damage occurs when thenardite is dissolved and mirabilite precipitates: in this case, during the dissolution of thenardite, a solution highly supersaturated with respect to mirabilite is formed, which causes a strong crystallization pressure inside the material when mirabilite precipitates, which occurs with a large volumetric expansion (about 314%) (Flatt, 2002; Espinosa-Marzal and Scherer, 2009).

An accelerated salt deterioration test procedure is proposed in the European standard (EN 12370, 1999). This procedure involves cycles of total immersion in a saturated (14 wt%) sodium sulphate decahydrate solution for 2 h, followed by oven drying at 105 °C for at least 16 h and finally cooling to room temperature. For each new cycle, new saline solution with the same concentration is used, which leads to the accumulation of a very high amount of salt inside the samples. This procedure results extremely aggressive towards materials and it is not considered very representative of real salt damage mechanisms occurring in historic buildings (Lubelli et al., 2018), both due to the impressive amount of salt accumulated and high drying temperature. For this reason, a 14 wt% sodium sulphate decahydrate solution was used for the present crystallization tests, as in the EN standard, but it was decided to slightly modify the testing procedure.

In a first type of crystallization test, the cylindrical samples of both lithotype (3 replicates for UT, CHIT and HAP and four replicates for HAP + CHIT) were subjected to cycles of impregnation, followed by drying. During the first cycle, the samples were impregnated by partial immersion in the saline solution. The samples, divided according to the lithotype and type of treatment, were placed in separate vessels with 400 ml of saline solution and the solution level (about 5 mm) was kept constant by refilling with deionized water. The impregnation phase lasted 4 h, followed by drying for 44 h in laboratory conditions (T = 20–25 °C, RH = 45–80%). In all the subsequent cycles, the wetting phase (4 h) was carried out using deionized water only, in order to keep the total salt amount in the samples constant and to induce salt accumulation at the upper surface. After each drying phase (44 h), the samples were gently brushed to remove both efflorescence and debris or powder detached from the substrate. For each cycle, the samples were weighed at the end of the immersion phase (W<sub>wet</sub>) and twice at the end of the drying phase, before and after brushing (W<sub>dry</sub> and W<sub>dry, brushed</sub>). All these values were averaged for the samples of the same group. A total of 12 cycles was carried out following this procedure. After 12 cycles, the samples were still in good conditions, probably due to a too limited aggressiveness of the procedure adopted. In fact, the brushing of the efflorescence after each drying phase likely reduced the amount of salt present inside the samples and, moreover, the high air RH present in some drying phases may have prevented the complete drying and the transformation of mirabilite into thenardite, limiting the damage in the subsequent rewetting. Thus, it was decided to carry out further eight cycles, each lasting 24 h and consisting of 4 h of capillary absorption of deionized water, 18 h of oven drying at 50 °C (temperature chosen not to damage the polymeric coating) and 2 h of cooling in room conditions. In this way, the oven drying, with RH varying between 8 and 12%, certainly caused formation of thenardite

at each cycle and the subsequent formation of mirabilite in the wetting phase, leading to the ideal conditions for the occurrence of a more severe damage. The brushing phase and the weight measurements at each cycle remained the same.

In a second type of crystallization test, two prismatic samples for each condition were subjected to continuous capillary absorption of the saline solution with concomitant evaporation, a condition called “wick effect” (Goudie, 1986; Andreotti et al., 2019), which reproduces the typical salt supply that occurs during capillary rise from the soil in a real building. Each sample was placed vertically in a beaker filled with 200 ml of 14 wt% sodium sulphate decahydrate solution (height of immersion: 5 cm). To prevent evaporation from the free surface of the saline solution, a layer of melted paraffin was poured over the solution: after solidification, the paraffin adhered to the sample and the beaker, preventing evaporation. The samples were placed in a climatic chamber at T = 22 ± 0.5 °C and RH = 80 ± 2%. The test ended when the solution was completely absorbed by the samples and evaporated from their surface, which happened after 5 days for G and 7 days for L. The slightly different drying times for the different samples and treatments were probably due also to the morphology and thickness of the layers of efflorescence on the samples surface. After drying, the samples were gently brushed to remove efflorescence and debris and placed in an oven at 60 °C until complete drying, and finally weighed (W<sub>dry, brushed</sub>). Considering that all the prisms were still in good conditions, it was decided to repeat this same test again, on one sample for each type.

The debris obtained after the first continuous absorption test were collected separately for each type of samples, then they were firstly dried in oven at 60 °C for 24 h and then cooled to room temperature in a desiccator. Once dry, they were put in boiling deionized water for 10 min and subsequently filtered, in order to extract the salt ions from the solid part (debris of stone). The saline solutions obtained were then analysed by ion chromatography (Dionex ICS 1000) to obtain the amount of sulphate anions and, by calculation, the percentage weight of solid fraction (ΔW<sub>solid</sub>) and sodium sulphate (Na<sub>2</sub>SO<sub>4</sub>) fraction (ΔW<sub>salt</sub>) referred to the initial weight of the samples.

# RESULTS AND DISCUSSION

## Characteristics of the Stones and Effects of the Treatments

The characteristics of stone cylinders before and after treatments are reported in Table 1, in terms of weight and elastic modulus variation, while the characteristics of stone prisms before and after treatments are reported in Table 2. The weight increases of the samples are almost negligible for all the treatments, due to the low concentration of the solutions applied, which is positive as it suggests that only a thin coating of the two substances formed over the pore surface, avoiding any pore clogging effect. For HAP + CHIT samples, due to the double treatment, the weight variation was slightly greater but, in any case, extremely limited. Interestingly, the weight increases due to chitosan application in the double treatment (HAP + CHIT) was lower with respect to
<table><caption>**TABLE 1** | Characteristics of cylindrical samples: mass variation after treatment in dry condition (ΔW), dynamic elastic modulus before and after treatment (E<sub>d</sub>), variation of dynamic elastic modulus (ΔE<sub>d</sub>).</caption><thead><tr><th rowspan="2">Substrate</th><th rowspan="2">Treatment</th><th colspan="2">ΔW [g]</th><th colspan="2">E<sub>d</sub> [GPa]</th><th rowspan="2">ΔE<sub>d</sub> [GPa]</th></tr><tr><th>After DAP</th><th>After chitosan</th><th>Before</th><th>After</th></tr></thead><tbody><tr><td rowspan="2">L_C</td><td>CHIT</td><td></td><td></td><td>16.25 ± 3.278</td><td>17.01 ± 3.402</td><td>+0.76</td></tr><tr><td>HAP</td><td>+0.23 ± 0.022</td><td>+0.22 ± 0.043</td><td>16.10 ± 2.932</td><td>16.56 ± 3.162</td><td>+0.45</td></tr><tr><td rowspan="2">G_C</td><td>HAP + CHIT</td><td>+0.28 ± 0.059</td><td>+0.05 ± 0.027</td><td>18.27 ± 2.177</td><td>18.99 ± 2.210</td><td>+0.72</td></tr><tr><td>CHIT</td><td></td><td></td><td>13.76 ± 0.449</td><td>13.94 ± 0.457</td><td>+0.17</td></tr><tr><td rowspan="2">L_P</td><td>HAP</td><td>+0.27 ± 0.019</td><td>+0.22 ± 0.055</td><td>13.67 ± 0.244</td><td>13.93 ± 0.083</td><td>+0.27</td></tr><tr><td>HAP + CHIT</td><td>+0.33 ± 0.102</td><td>+0.02 ± 0.015</td><td>13.90 ± 0.268</td><td>14.04 ± 0.158</td><td>+0.13</td></tr></tbody></table>

<table><caption>**TABLE 2** | Characteristics of prismatic samples: mass variation after treatment in dry condition (ΔW), dynamic elastic modulus before and after treatment (E<sub>d</sub>), variation of dynamic elastic modulus (ΔE<sub>d</sub>).</caption><thead><tr><th rowspan="2">Substrate</th><th rowspan="2">Treatment</th><th colspan="2">ΔW [g]</th><th colspan="2">E<sub>d</sub> [GPa]</th><th rowspan="2">ΔE<sub>d</sub> [GPa]</th></tr><tr><th>After DAP</th><th>After chitosan</th><th>Before</th><th>After</th></tr></thead><tbody><tr><td rowspan="2">L_P</td><td>CHIT</td><td></td><td></td><td>15.54 ± 0.109</td><td>15.86 ± 0.034</td><td>+0.33</td></tr><tr><td>HAP</td><td>+0.55 ± 0.014</td><td>+0.63 ± 0.007</td><td>15.73 ± 0.288</td><td>16.79 ± 0.160</td><td>+1.06</td></tr><tr><td rowspan="2">G_P</td><td>HAP + CHIT</td><td>+0.45 ± 0.127</td><td>+0.15 ± 0.057</td><td>15.70 ± 0.235</td><td>16.71 ± 0.164</td><td>+1.01</td></tr><tr><td>CHIT</td><td></td><td></td><td>13.08 ± 0.035</td><td>13.25 ± 0.118</td><td>+0.17</td></tr><tr><td rowspan="2">G_P</td><td>HAP</td><td>+0.66 ± 0.007</td><td>+0.64 ± 0.014</td><td>13.06 ± 0.056</td><td>13.66 ± 0.052</td><td>+0.60</td></tr><tr><td>HAP + CHIT</td><td>+0.72 ± 0.000</td><td>+0.12 ± 0.014</td><td>13.41 ± 0.084</td><td>13.89 ± 0.246</td><td>+0.48</td></tr></tbody></table>

the CHIT treatment alone, which was found also in a previous study (Andreotti et al., 2019). This aspect is probably related to the mechanism of adsorption of chitosan over different solid substrates (in this case, the layer of CaP phases), which may depend on several factors, including the chemical nature of the substrate, the pH of the solution, etc. (Andreotti et al., 2019), and which was not specifically investigated in the present study. It is also possible that the capillary absorption of the chitosan solution was somehow slowed down in the HAP-treated stone (see Figure 1 below).

All the treatments also caused a systematic increase in the dynamic elastic modulus, although quite limited and difficult to distinguish from the intrinsic variability for this parameter in the two stones analysed. In fact, the elastic modulus of untreated Lecce stone and Globigerina limestone, averaged between all samples used, is 16.47 ± 2.756 GPa and 13.75 ± 0.345 GPa, respectively. The E<sub>d</sub> increase reached a maximum of 0.72 GPa for Lecce stone and 0.27 GPa for Globigerina limestone (Table 1), hence within the respective standard deviations of the untreated value. The standard deviation of the dynamic elastic modulus for Lecce stone is quite high due to the presence of marine fossils within this stone and hence its heterogeneous microstructure. Both the weight increase and the E<sub>d</sub> increase after treatments are generally slightly higher for prisms than for cylinders, owing to the smaller thickness of the first ones, which allowed a greater absorption of the solutions and deposition of HAP and/or chitosan in the stone.

The results of the water absorption test on the cylinders are shown in Figure 1, while the average values of capillary water absorption coefficients (AC) and the total water absorption at 24 h (WA%) are reported in.

**Supplementary Table S1.** The MIP curves of the samples with and without the treatments are reported in Figure 2.

After 24 h of capillary absorption, the samples were not completely saturated yet, as the slope of the last part of the curves in Figure 1 is still slightly increasing. This is due to the presence of pores of different sizes within the material, as shown by the MIP analyses in Figure 2. The bigger pores are the first to absorb water and be saturated, later acting as reservoir for the thinner pores that could take a much longer time to be saturated. Nonetheless, in the untreated condition the capillary absorption was almost complete after 24 h. The total water absorption at 24 h and the total porosity are similar for the two stones, with slightly higher porosity for Globigerina limestone and slightly higher variability of results for Lecce stone, the latter due to the more heterogeneous microstructure of this lithotype. The AC coefficient of Globigerina limestone is higher than that of Lecce stone, consistently with its slightly higher porosity and larger pore mean radius found by MIP.

After treatments, all the samples exhibited similar total porosity and pore size distribution (Figure 2 and Supplementary Table S1), and the differences are attributable to the intrinsic variability of the stones. Despite this, the treatments modified the capillary water absorption rate of the stones. In Lecce stone, all the treatments slowed down the water capillary absorption compared to UT samples, especially in the case of HAP and HAP + CHIT. In Globigerina limestone, slight variations in AC were registered after the CHIT and the HAP treatments, while the combined treatment HAP + CHIT caused a marked reduction in AC. The modification of the capillary absorption rate could suggest a pore clogging effect, but the variation in the pore size distribution is negligible, as shown
<figure><img src="image_179.png" /><figcaption>**FIGURE 1** | Capillary water absorption curves for the untreated (UT) and treated cylindrical samples for (A) Lecce stone and (B) Globigerina limestone. Values are averages for at least two samples.</figcaption></figure>

in the results of MIP test (Figure 2), hence no significant pore occlusion occurred. The reduction of AC could be due to changes in the roughness of the pores surface after treatments and/or modifications in the contact angle with water. In the case of HAP-treated stone, changes in sorptivity comparable to those found in the present paper have been found in previous studies (Sassoni et al., 2011; Sassoni et al., 2016a), as a result of the moderate alterations in the static contact angle (Sassoni et al., 2016a; Sassoni et al., 2020) and in pore size distribution (Sassoni et al., 2011; Sassoni et al., 2016a).

Based on SEM images taken on the external surface of untreated and treated samples of Globigerina (Supplementary Figure S1), it is possible to observe that neither the single treatments (HAP or CHIT) nor the combined treatment (HAP + CHIT) led to formation of a clearly distinguishable coating over the stone surface. Accordingly, EDS analysis did not reveal any element that could be attributed to newly formed coatings (such as P in the HAP and HAP + CHIT coatings). Given the high porosity of the stone, the DAP and chitosan solutions were quickly absorbed into the pore network (thus leading to the changes in pore size distribution and water sorptivity discussed above), while no accumulation on the treated surface took place.

<figure><img src="image_180.png" /><figcaption>**FIGURE 2** | Pore size distribution of various type of stone samples, obtained with MIP test, compared to the relative UT samples, for (A) Lecce stone and (B) Globigerina limestone.</figcaption></figure>

The lack of coating accumulation on the surface was likely helped by rinsing with water at the end of the treatment application. The samples were rinsed with water to remove the excess of unreacted DAP (with chitosan possibly deposited on top) that accumulated on the sample surface, so as to prevent formation of a surface crust and pore clogging, with consequent alterations in the stone transport properties. Similar results were obtained also in the case of Lecce stone (SEM images are omitted for brevity's sake).

In any case, the present results suggest that:

- the AC modifications due to the three kinds of treatments are different in the two stones. In particular, the impact of the treatments is smaller in L than in G, probably due to the much quicker capillary absorption of the latter, which overcomes the possible microstructure modifications caused by the treatments;

- the same chitosan solution tested here, when applied as a coating over a glass slide, exhibited a static contact angle of about 65°, i.e. much higher than stone, although still in the hydrophilic field (<90°). This may explain the slower capillary absorption of L_CHIT and L_HAP + CHIT compared with the untreated Lecce stone. In the case of Globigerina limestone, having a much faster capillary absorption, the AC coefficient of the stone was almost unaltered by CHIT treatment, while it
A

<figure><img src="image_181.png" /></figure>

B

<figure><img src="image_182.png" /></figure>

**FIGURE 3** | Significant UT cylinders at the 1st, 2nd, 4th, 6th, 10th, 12th, 16th and 20th cycles of the salt crystallization test for: (A) Lecce stone and (B) Globigerina limestone. The photos were taken after the drying phase, before brushing.

decreased for the double treatment (G_HAP + CHIT), although less than in Lecce stone.

It is also possible that the slowing down of the capillary absorption rate found in the test is transitory, maybe related to some washout of the chitosan layer and/or of some unreacted DAP. In fact, in the previous study, the chitosan coating applied over calcite was found to be washed out by water until reaching the adsorbed layer, which in turn was not sensitive to water flow (Andreotti et al., 2019). Unfortunately, it was not possible to repeat the test on the same cylindrical samples, as they were broken and used for other analyses, hence this aspect will be investigated in future studies.

## Effects of the Salt Crystallization Tests on Cylinders

The appearance of the untreated cylindrical samples during the 20 crystallization cycles (the first 12 carried out with drying at room temperature and the other 8 with oven drying at 50 °C) is reported in **Figure 3**, for some selected cycles and for one representative sample. At the end of the first cycle, no deterioration was found, as expected, because no thenardite to mirabilite transformation had taken place yet. It can be clearly observed that oven drying increased the aggressiveness of the crystallization cycles (see the photos of the 16th and 20th cycles in **Figure 3**), as expected. However, the untreated cylindrical samples were still in relatively good conditions even at the end of
<figure><img src="image_183.png" /></figure>

<figure><img src="image_184.png" /></figure>

**FIGURE 4** | Weight variation of the cylindrical samples after drying and brushing, at each cycle of the salt crystallization test for: (A) Lecce stone (UT and with different treatments) and (B) Globigerina limestone (UT and with different treatments).

the 20th crystallization cycle. The limited severity of the adopted testing conditions seems to be due to two main reasons:

- the saline solution was used in the first wetting cycle only, while deionized water was used in the following cycles. This

means that a major amount of the salt that was introduced in the first absorption cycle was removed when efflorescence and debris were brushed away at the end of the first cycle and this amount was not re-introduced in the samples in the subsequent cycles. This is confirmed by the definitely more

<figure><img src="image_185.png" /></figure>

**FIGURE 5** | Change in the final dry weight, after brushing, compared to the initial weight of the cylindrical samples.
<figure><img src="image_186.png" /><figcaption>**FIGURE 6** | Lecce stone samples and Globigerina limestone samples during the cycles (dried condition, before brushing), where the difference in the amount of efflorescence formed in the different type of samples can be seen. The pictures of the cycles with oven drying are not reported, because the "cementification" of debris during drying made impossible to distinguish the layer of efflorescence.</figcaption></figure>
<figure><img src="image_187.png" /></figure>

<figure><img src="image_188.png" /><figcaption>**FIGURE 7** | Pore size distribution of stone samples, obtained with MIP test, after the salt crystallization test (and desalination by poulting) for: (A) Lecce stone and (B) Globigerina limestone.</figcaption></figure>

abundant salt crystals formation observed at the end of the second cycle (Figure 3), which was not found in the following ones;—the stone fragments that tended to detach during the wetting phase were apparently “cemented” by salt during the subsequent drying phase and this greatly limited the weight loss of the samples, making it difficult to quantify the actual degree of deterioration.

The weight variations of the samples after each drying + brushing phase (W<sub>dry, brushed</sub>) are reported in **Figure 4**, while the variation of the final dry weight compared to the initial one (ΔW<sub>final</sub>) is reported in **Figure 5**.

The graphs in **Figure 4** clearly indicate that the first part of the test, with drying at room temperature, led to a strong weight increase, with no apparent damage in the stones. This can be ascribed to the fact that the samples did not completely dry in laboratory conditions prior to the next wetting cycle, thus also preventing the transformation from mirabilite to thenardite and limiting the damage. Moreover, the peaks in the first part of the test suggest that the variations in the air RH at each drying cycle caused a variable degree of drying before the next wetting phase. Only in the second part, when oven drying was used, there was a continuous weight loss, below the initial weight of the samples. Concerning the effect of the treatments, it can be noted that the

curves of the UT_SALT samples are the lowest in **Figure 4**, with a maximum weight loss (ΔW<sub>final</sub>) equal to -2.36 ± 0.21% for L and -2.89 ± 0.13% for G (Figure 5). All the treatments improved the performance of the stones, the benefit increasing in the following order: UT ≈ CHIT < HAP < HAP + CHIT. The fact that the HAP treatment alone has a positive effect on the salt resistance of the stone can be ascribed to several factors, which act simultaneously: 1) an increase in the stone tensile strength (as suggested by the increase in the elastic modulus and as seen in the literature (Naidu et al., 2011; Sassoni et al., 2011; Sassoni et al., 2016a; Sassoni et al., 2016b; Sassoni, 2018)), with a consequent improvement in the salt resistance (Sassoni et al., 2016b); 2) the fact that a greater roughness of the pore surfaces allows for a better capillary flow towards the outside once this roughness is wet, promoting efflorescence (Scherer and Wheeler, 2008; Andreotti et al., 2019); 3) the preventive action towards calcite dissolution (Graziani et al., 2015; Sassoni et al., 2016a; Sassoni et al., 2016b; Andreotti et al., 2019). All these positive effects seem to add to those of chitosan in the samples with the double treatment, where the presence of the polymer helps reduce the stone degradation, by increasing the formation of external efflorescence compared to the UT samples (Figure 6). In fact, the HAP + CHIT_SALT samples exhibited a systematically lower weight loss at every cycle than all the other samples, which
<figure><img src="image_189.png" /></figure>

indicates better resistance to salt damage: at the end of the cycles, the HAP + CHIT treatment made the weight loss decrease by 84% in Lecce stone and by 69% in Globigerina limestone, with respect to the untreated samples. The polymeric treatment alone induced a behavior quite similar to the untreated samples, hence it seems that the inhibiting action of chitosan alone is not sufficient, or, most likely, the polymer is not well distributed and anchored to the pore surface. As a matter of fact, chitosan seems to promote the formation of efflorescence rather than subflorescence, as confirmed by the fact that the final weight loss of the samples is lower than that of the untreated samples. However, it is also possible that the inhibiting effect of chitosan causes a greater supersaturation of the saline solution inside the pores, which causes local stress concentrations in those areas of the pores surface that are not covered by the chitosan coating. In the case of HAP + CHIT, this problem was not found, as the CaP layer improved the coverage and the adhesion of chitosan.

The MIP analysis was carried out on the samples subjected to the saline crystallization test, after desalination of the fragments by poultering, and the results are reported in **Figure 7** and **Supplementary Table S2**, in comparison with UT samples (not subjected to any salt crystallization). The differences in the curves for the various treatments and with respect to UT samples are very limited and seem ascribable to the intrinsic variability of the material, which is lower for G and higher for L. In fact, it is noteworthy that, being MIP a destructive test, it was

not possible to perform it on exactly the same cylinders before and after the crystallization test. The limited porosity variation is probably related also to the low aggressiveness of the crystallization cycle procedure, which led to a limited deterioration in all the samples.

The results of the tensile splitting test carried out on the stone cylinders after the salt crystallization cycles, drying and brushing, are reported in **Figure 8**. In the case of Globigerina limestone, it can be clearly observed that, for the untreated condition, salt crystallization cycles caused a marked reduction in tensile strength. In the case of the treated specimens, a lower reduction in tensile strength was experienced, the benefit increasing in the order UT ≈ CHIT < HAP < HAP + CHIT. This trend confirms what already found in terms of weight loss (Figure 5). In the case of Lecce stone, the results of the tensile splitting test exhibit a less evident trend (Figure 8A), but again the HAP + CHIT treatment was able to provide the greatest benefit. In particular, it should be considered that the consolidating treatment of hydroxyapatite increases the strength of the material, which is subsequently decreased by degradation due to salt crystallization test. Therefore, the tensile strength of the L_HAP + CHIT sample, which is higher than the L_UT sample by 9.55%, represents the residual strength after consolidation and subsequent salt degradation. The fact that, for this treatment condition, the residual strength is the highest is in agreement with the lowest weight loss measured at the end of the salt crystallization test for this condition. In fact, the reduced material loss is expected to have helped maintain the high tensile strength even after salt degradation. However, when evaluating the results of the tensile splitting test, it should be kept in mind that only two replicates for each condition were available for each condition, because all the samples were obtained from the same quarry slab (for greater representativeness and homogeneity of the material) and some replicates had been used for other analyses. Additional tests on a higher number of samples will be necessary to confirm the observed trends.

## Tests on Prisms

As reported above, the prismatic samples, at the end of the first absorption test, exhibited a considerable amount of efflorescence on the surface, as shown in **Figure 9**, but after brushing no really significant deterioration of the stone substrates was observed. The repetition of the test (on a single sample per type) caused significant degradation, with formation of efflorescence and detachment of a layer from the sample surface, as reported in **Figure 10**. The weight variation of the prisms after the first and the second continuous absorption tests, after drying and brushing (W<sub>dry, brushed</sub>), is reported in **Supplementary Figure S2**, while the corresponding changes of final dry weight compared to the initial one (ΔW<sub>final</sub>), are reported in **Figure 11**. These values refer to the single prismatic sample of each condition that was used for both tests. **Supplementary Table S3** also report a summary of the weight loss reduction caused by the treatments in the two stones in comparison with untreated references, in the two salt crystallization tests. The values in **Figures 9–11** and **Supplementary Table S3** allow to derive some remarks:
<figure><img src="image_190.png" /><figcaption>**FIGURE 9** | Prismatic samples at the end of the first continuous absorption test, before brushing.</figcaption></figure>

- The first absorption test led to an increase in the weight of all samples, due to the accumulation of salt in the stone, but the damage was very limited. The deposition of salts inside both

stones was higher for UT samples, indicating that all the treatments favored the formation of efflorescence rather than subflorescence.
<figure><img src="image_191.png" /><figcaption>**FIGURE 10** | Prismatic samples at the end of the second continuous absorption test, before brushing.</figcaption></figure>

- After the first test, the samples were oven-dried (expectedly causing the formation of thenardite) and the second test started, hence the salt already present in the pores transformed into mirabilite, causing severe deterioration. The final weight loss at the end of the second test was quite limited in L and evident in G. It is interesting to note that the “wick test” carried out on prisms produced a very different damage in the two stones, while their deterioration was similar after the salt crystallization cycles on cylinders (Figure 5),

although again slightly higher in G than in L. This can be ascribed to the different mechanism of salt damage in the two tests, but also to the smaller thickness of prisms compared to cylinders, making the impact of weight loss larger in the former ones.

- After the second test, all the treatments caused a lower deterioration (smaller weight loss) in the stone samples, the improvement increasing in the following order: CHIT < HAP < HAP + CHIT, again confirming the trend already
<figure><img src="image_192.png" /><figcaption>**FIGURE 11** | Change in the final dry weight of the prisms after the second absorption test, after brushing, compared to the initial weight for the samples.</figcaption></figure>

<figure><img src="image_193.png" /></figure>

<figure><img src="image_194.png" /><figcaption>**FIGURE 12** | Weight percentages of salt fraction and solid fraction in debris, referred to the initial weight of the prismatic samples, obtained after brushing at the end of the first absorption test, for: (A) Lecce stone and (B) Globigerina limestone.</figcaption></figure>
<figure><img src="image_195.png" /><figcaption>**FIGURE 13** | Dynamic elastic modulus of the prismatic samples at each stage of the research (the dotted line indicates the initial value of the UT reference), for: (A) Lecce stone and (B) Globigerina limestone. The dotted line indicates the initial value of the UT reference (15.49 GPa for L and 13.11 GPa for G).</figcaption></figure>

observed. The combined HAP + CHIT treatment led to a remarkable improvement with respect to the untreated conditions, as the weight loss decreased by 84% in Lecce stone and by 68% in Globigerina limestone, which were actually the same percentages of reduction found in the cylinders. Unlike the crystallization cycles on cylinders, in this case also the chitosan treatment alone led to a significant improvement (Supplementary Table S3). This was ascribed to the fact that the chitosan alone was not as well anchored to the pore surface as in the case of the HAP pre-treatment, so it was probably partially removed (alongside calcite) during the multiple wetting cycles carried out in the cylinders, while it was not so altered during the two cycles of continuous absorption carried out in the prisms.

The debris brushed at the end of the first absorption test were analysed and the results are reported in **Figure 12**, as weight percentages of solid fraction (ΔW<sub>solid</sub>) and sodium sulphate (Na<sub>2</sub>SO<sub>4</sub>) fraction (ΔW<sub>salt</sub>) with respect to the initial weight of the samples. The results in terms of stone loss confirm the limited deterioration achieved after the first complete absorption of the saline solution. The presence of chitosan in this type of test, both alone and in combination with hydroxyapatite, caused an increase in the saline fraction, therefore in the amount of efflorescence formed, as can be seen also in **Figure 9**. Moreover, in HAP + CHIT samples, there was also a lower quantity of solid fraction, indicating that the combined treatment

has a beneficial effect, promoting efflorescence and limiting damage in the stone. This confirms what was also found in the crystallization test on the cylinders. The CHIT samples, on the other hand, despite a greater quantity of efflorescence than the untreated ones, did not cause any reduction in the solid fraction lost (or even a slight increase), indicating that the polymer alone was not so effective.

Although the weight variation of the samples during the salt crystallization tests can generally provide a useful indication about the deterioration state of the sample, it must be noted that this variation is the sum of a positive term (the salts deposited in the stone pores) and a negative term (the stone debris lost due to deterioration). For this reason, to better evaluate the deterioration of the stone, the dynamic elastic modulus was measured before treatment, after treatment, after the salt crystallization test (after brushing) and also after progressive desalination by poulticing. The values obtained at each phase are reported in **Figure 13**. An increase in the dynamic elastic modulus may indicate an effective consolidation treatment and/or some salt accumulation in the pores, while a reduction indicates internal damage in the material due to salt crystallization (cracking). It is noteworthy that the sensitivity of ultrasonic velocity measurement to micro-cracks development in the stone is very high, as these micro-cracks strongly affect the path followed by ultrasonic wave.

As desirable, after treatments, all the samples showed an increase in the elastic modulus, obviously greater for the
consolidated ones (HAP and HAP + CHIT). After the salt crystallization test, the maximum $E_d$ value was found, as the salt crystals inside the stone samples partially filled the pores and provided a faster ultrasonic transmission. In each of the three desalination phases, the salt ions were transported into the poultice and removed from the samples. Consistently, the elastic modulus progressively decreased after each poultice application, as the pores (previously filled by salts) were progressively emptied. In the case of Globigerina limestone, after desalination for the third time, the UT, CHIT and HAP samples exhibited a lower $E_d$ value than the initial condition, which suggests crack formation during the salt crystallization test. On the contrary, in the case of the HAP + CHIT treatment, the final $E_d$ was still higher than the initial condition (the value being actually very close to that right after treatment), which suggests limited damaging during the salt crystallization test. In the case of Lecce stone, after desalination for 3 times all the specimens exhibited an $E_d$ value still higher than the initial one, again the value of the HAP + CHIT sample being the highest. Although the permanence of some residual salts inside the samples even after the third desalination cannot be completed excluded, still the observed trend confirms the potential of the HAP + CHIT treatment to prevent salt damage.

## CONCLUSION

In the present paper, three treatments were tested, aiming at preventing salt crystallization damage in porous limestone: an hydroxyapatite-based consolidant (expected to strengthen the stone), a chitosan solution (expected to inhibit salt crystallization and favor the formation of efflorescence rather than harmful subflorescence) and a combination of these two treatments (where hydroxyapatite is expected to also improve the anchoring of chitosan). Lecce stone and Globigerina limestone were used, being representative of highly porous lithotypes widely used in heritage buildings and severely affected by salt damage onsite.

All the treatments were characterized by low concentration and hence they caused only a slight increase in the weight of the samples, indicating a small amount of products deposited. As a consequence, the treatments basically did not alter the porosity of the stone, demonstrating an excellent compatibility, although they somehow altered the capillary water absorption rate (especially for the combined treatment), although this latter aspect is still under investigation. The possibility that the alteration of the absorption rate given by the treatments is transitory is supported by the results of the crystallization tests. In fact, the prisms, despite absorbing saline solution at slightly different rates, all dried out in similar times (6-7 days); moreover, the weights of the cylinders at the end of the wetting phase of each cycle did not show substantial differences. Therefore, it seems that this slowdown was only transitory and did not have an important impact on the results. This aspect, however, still requires full elucidation and, in order to better understand the transitory or permanent nature of the phenomenon, further tests are necessary. All the treatments also caused a slight increase in the dynamic elastic modulus of the stones, suggesting a positive effect in terms of mechanical properties.

Two accelerated laboratory tests were carried out to investigate the effectiveness of the treatments. The first one was carried out on stone cylinders and involved salt crystallization cycles in a sodium sulphate solution (wetting and drying cycles). These cycles were designed by modifying the procedure of the EN 12370 standard, in order to make the entire test less aggressive than that in the standard. This first kind of salt crystallization test allowed to make the following remarks:

- drying in room conditions (adopted in the first 12 cycles) likely prevented the full transformation thenardite ↔ mirabilite during the cycles, hence it was too mild and made it necessary to adopt oven drying at 50 °C for the following eight cycles. Although a clear weight loss was induced in the samples by this second drying method, an improvement of the procedure used for the cycles still seems necessary, to find a compromise between the too aggressive method suggested by the EN standard and the too mild one adopted in this research;

- all the treatments promoted the formation of efflorescence compared to the untreated stone samples. In the presence of chitosan, this was ascribed to the inhibiting effect of the polymer, while in the case of the HAP treatment alone this seems due to the extremely rough surface of the calcium phosphate phases formed over the pores surface, promoting the water flow towards the stone surface (Scherer and Wheeler, 2008);

- all the treatments led to an improvement in the stone resistance to salt crystallization, measured in terms of sample weight loss. The improvement increased according to the order CHIT < HAP < HAP + CHIT, the combined treatment providing an outstanding improvement (weight loss decrease by 84% in Lecce stone and by 69% in Globigerina limestone, with respect to the untreated samples);

- the combined HAP + CHIT treatment also gave the best results in terms of tensile strength of the stone after the salt crystallization cycles, basically leading to a residual strength after the cycles equal to (or even slightly higher than) the unweathered original stones.

The second test used to investigate the salt crystallization resistance was based on the continuous capillary absorption of a fixed amount of sodium sulphate solution by prismatic stone samples, up to their complete drying ("wick test"). Two complete cycles of absorption were carried out, allowing to derive the following conclusions:

- like in the previous test, all the treatments promoted the formation of efflorescence compared to the untreated stones. This was assessed not only by visual observation, but also by weighing the amount of salts detached/brushed from the samples at the end of the test;

- all the treatments led to an improvement in the stone resistance to salt crystallization. The weight loss decreased according to the same order as in the previous test, the combined treatment providing basically the same outstanding improvement as in the test on cylinders;

- after the test and the desalination of the prismatic samples by poulticing, the dynamic elastic modulus was measured in the
samples. The combined HAP + CHIT treatment confirmed its very good performance, leading to the highest values (even higher than the original unweathered stone samples in the case of Globigerina limestone).

The comparison between the combined HAP + CHIT treatment and other modifiers tested over last years and mentioned in the Introduction is not an easy task, as differences in the materials used or in the test carried out lead to results that are difficult to compare. In (Cassar et al., 2008), prismatic samples of different quality types of Globigerina limestone, untreated and treated with a phosphorylated inhibitor, were subjected to weathering by a sodium sulphate solution, in a test similar to the “wick test” used in this research. The inhibitor led to an improvement in the saline solution capillary flow through the porous materials, helping crystallization occur on the surface of the stone and not within the pores. Moreover, the treatment led to a reduction in debris loss during the salt crystallization test by about 47% with respect to the untreated samples while, in similar conditions, HAP + CHIT reduced the weight loss by 69%. In (Sammut et al., 2014), cubic stone samples of Globigerina limestone, treated with citrate and phosphocitrate with different treatment methods (capillary rise, brushing and poultice), were subjected to salt crystallization cycles. Although the treatment gave positive effects on the rate of transport of the saline solution, data on the stone loss during crystallization cycles are not reported, as the differences obtained in various conditions were too small to draw conclusions. Other examples, were different substrates or types of tests were used, are reported in (Rodriguez-Navarro et al., 2002; Selwitz and Doehne, 2002; Lubelli and van Hees, 2007; Rivas et al., 2010; Ruiz-Agudo et al., 2010; Smith et al., 2010; Gupta et al., 2012; Ruiz-Agudo et al., 2012; Granneman et al., 2019; Bracciale et al., 2020). Concerning the cost of the investigated treatment, the experimental campaign is at an early stage, hence this aspect has not been specifically evaluated yet. However, considering that the most expensive chemical used is chitosan and that its concentration in solution is very low (0.05 wt%), the cost/liter of the chitosan treatment is expected to be comparable with other commonly used conservation treatments. Moreover, chitosan is presently exploited in a wide variety of applications and its use is expected to strongly increase in the future, suggesting that its cost might decrease thanks to the set-up of improved technologies for its production.

The combined treatment, composed by a first application of phosphate consolidant and a second application of a chitosan solution, gave extremely promising results in terms of reduction of salt deterioration in both the porous limestones investigated in this study, with an improvement in the reduction of salt deterioration in line or even greater than other crystallization modifiers. The calcium phosphate phases formed in the first step of the treatment have been shown to improve the tensile strength of stone and to favor the formation of efflorescence rather than subflorescence, but they are also thought to improve the anchoring of the chitosan layer and to prevent the leaching of the calcite substrate, which might lead to the leaching of the chitosan layer too.

The water-based hydroxyapatite consolidant is known to be compatible with stone and friendly for the environment and the conservators, and chitosan is a non-toxic and environmentally friendly biopolymer, hence the combined treatment can be seen as a fully “green” solution for conservation.

The performance of the tested treatments was investigated in this paper through short-term laboratory tests on stone specimens, with encouraging results, but further tests will be necessary to assess the long-term effectiveness and the actual duration of the new treatments. In particular, the fact that chitosan is expected to biodegrade with time is considered a positive feature, which avoids that aged and useless polymeric residues remain in the stone and which can be coped with by periodically re-applying the treatment. However, the duration of this polymer in onsite conditions needs to be investigated, in order to assess whether it is compatible with applications in the conservation field. The effectiveness of the new treatments in different heritage substrates is a further aspect needing investigation and requiring further studies.

## DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

## AUTHOR CONTRIBUTIONS

All authors have read and agree to the published version of the manuscript. Author Contributions: Conceptualization, EF; methodology, EF, ES and MB; experimental investigation: MB and ES; data analysis and interpretation, EF, MB and ES; writing—original draft preparation, MB and EF; writing—review and editing, EF, ES and MB; supervision, EF.

## ACKNOWLEDGMENTS

All the members of the RILEM Technical Committee “271-ASC: Accelerated laboratory test for the assessment of the durability of materials with respect to salt crystallization” (Chair: Barbara Lubelli, Deputy Chair: Inge Rörig-Dalgaard) are gratefully acknowledged for the interesting and fruitful discussion about the aggressiveness of standard salt crystallization tests.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmats.2021.583112/full#supplementary-material.