Iron-Based Electrocatalysts for Energy Conversion: E ﬀ ect of Ball Milling on Oxygen Reduction Activity

Featured Application: Iron-based electrocatalysts were developed as oxygen-reducing catalysts for microbial fuel cell application. Abstract: In this work, we synthesized new materials based on Fe(II) phthalocyanine (FePc), urea and carbon black pearls (BP), called Fe-N-C, as electrocatalysts for the oxygen reduction reaction (ORR) in neutral solution. The electrocatalysts were prepared by combining ball-milling and pyrolysis treatments, which a ﬀ ected the electrochemical surface area (ECSA) and electrocatalytic activity toward ORR, and stability was evaluated by cyclic voltammetry and chronoamperometry. Ball-milling allowed us to increase the ECSA, and the ORR activity as compared to the Fe-N-C sample obtained without any ball-milling. The e ﬀ ect of a subsequent pyrolysis treatment after ball-milling further improved the electrocatalytic stability of the materials. The set of results indicated that combining ball-milling time and pyrolysis treatments allowed us to obtain Fe-N-C catalysts with high catalytic activity toward ORR and stability which makes them suitable for microbial fuel cell applications. nonpyrolyzed samples. This ﬁnding points toward the beneﬁcial e ﬀ ect of pyrolysis in enhancing catalytic stability, and adequate Jretention values were obtained for FeNC_BM1_P, FeNC_BM3_P and FeNC_BM6_P (50%, 75% and 80%, respectively).


Introduction
Bioelectrochemical Systems (BESs) are classified as one of the most attractive renewable technology for energy conversion [1,2]. Among the various BESs, Microbial Fuel Cells (MFCs) allow simultaneous electrical energy harvesting and wastewater treatment [1,[3][4][5][6][7] by the catalytic action of microorganisms, which oxidize the organic matter in the waste at the anode side of the cell to produce carbon dioxide, protons and electrons. Protons and electrons migrate through an external circuit and recombine with oxygen at the cathode side, producing water [1,4]. However, MFC technology is still in a pioneer stag, due to various critical issues to overcome. One of these issues is the slow kinetics of oxygen reduction reaction (ORR) at the cathode side and the need for a highly efficient catalyst for a fast reduction of oxygen to water involving a four-electron pathway [4,[8][9][10][11]. Hence, the development of effective and low-cost electrocatalysts is of crucial importance to develop highly efficient and durable MFC systems.
In this context, many studies have been addressed to develop alternative catalysts to noble metals (i.e., platinum), which are expensive, rare and sensitive to poisoning [3,8]. Platinum-group-metal-free (PGM-free) catalysts represent a very promising category of new catalysts due to their tuneable composition based on transition metals (Fe, Ni, Co, Mn, Cu) supported on carbon substrates (graphene, graphene oxide, carbon nanotubes, black pearls, carbon black and biochar), which are also doped with heteroatoms (N, P, S, O) [12][13][14][15][16][17][18][19][20] using a Whatman membrane filters nylon (pore size: 0.45 µm, diameter: 47 mm) and a diaphragm vacuum pump (Vacuubrand GMBH). Then, the material was washed with distilled water until neutral pH was obtained, dried in an oven at 70 • C overnight and milled in mortar agate (treated BP was labelled as BPO).
Preparation of electrocatalysts: 0.5 g of BPO, 0.5 g of iron (II) phthalocyanine (FePc, Aldrich) and 2 g of CH 4 N 2 O (Urea, VWR Chemicals) were mixed by hand using a mortar for 30 min, and part of this sample was then pyrolyzed in an Al 2 O 3 -tube furnace (Forni De Marco, Italy) at 900 • C for 1 h under an Ar atmosphere, obtaining two samples labelled as FeNC and FeNC_P, respectively. In addition, a further part of FeNC sample (2 g) was ball-milled in a Zirconia jar (V = 80 mL) with 100 Zirconia balls (5-mm diameter) in a planetary ball-miller (Pulverisette 6, Fritsch GmbH) for either 6 or 12 cycles of 30 min milling and 5-min pause at a rotation rate of 400 rpm, and then pyrolyzed at 900 • C for 1 h under Ar atmosphere, obtaining two samples labelled as FeNC_BM3_P and FeNC_BM6_P. Two further samples were obtained by ball-milling FeNC continuously for 1 h (FeNC_BM1) and then pyrolyzing as described before (FeNC_BM1_P). Figure 1 shows a diagram illustrating the procedure for sample preparation, and Table 1 lists all the samples prepared with the corresponding label.

Materials
Treatment of carbon support: Carbon Black pearls 2000 (BP) were purchased by Cabot corporation (MA, US) and treated in refluxing concentrated HNO3 65 wt.% at 90 °C for 16 h. Then, BP was filtered using a Whatman membrane filters nylon (pore size: 0.45 μm, diameter: 47 mm) and a diaphragm vacuum pump (Vacuubrand GMBH). Then, the material was washed with distilled water until neutral pH was obtained, dried in an oven at 70 °C overnight and milled in mortar agate (treated BP was labelled as BPO).
Preparation of electrocatalysts: 0.5 g of BPO, 0.5 g of iron (II) phthalocyanine (FePc, Aldrich) and 2 g of CH4N2O (Urea, VWR Chemicals) were mixed by hand using a mortar for 30 min, and part of this sample was then pyrolyzed in an Al2O3-tube furnace (Forni De Marco, Italy) at 900 °C for 1 h under an Ar atmosphere, obtaining two samples labelled as FeNC and FeNC_P, respectively. In addition, a further part of FeNC sample (2 g) was ball-milled in a Zirconia jar (V = 80 mL) with 100 Zirconia balls (5-mm diameter) in a planetary ball-miller (Pulverisette 6, Fritsch GmbH) for either 6 or 12 cycles of 30 min milling and 5-min pause at a rotation rate of 400 rpm, and then pyrolyzed at 900 °C for 1 h under Ar atmosphere, obtaining two samples labelled as FeNC_BM3_P and FeNC_BM6_P. Two further samples were obtained by ball-milling FeNC continuously for 1 h (FeNC_BM1) and then pyrolyzing as described before (FeNC_BM1_P). Figure 1 shows a diagram illustrating the procedure for sample preparation, and Table 1 lists all the samples prepared with the corresponding label.

Methods
Thermogravimetric analysis (TGA) was performed using a TGA/DSC1 Star System (Mettler Toledo) in N 2 with a heating rate of 5 • C/min. The samples (mass ranging from 9 mg to 24 mg), which was held in platinum sample holder with a cover having one vent central hole.
Catalyst ink preparation: The catalyst ink was prepared by dispersing 0.2 mg of catalyst in 270 µL of DI water and 135 µL of Isopropanol. This dispersion was treated in an ultrasonic bath (Elmasonic P 30 H, Elma Schmidbauer GmbH) for 45 min. Then, 50 µL of Nafion solution (5 wt.% in lower aliphatic alcohols and water, Aldrich) were added to the dispersion and treated in the ultrasonic bath for further 15 min. The ink was dropped (7 µL) onto a working electrode (glassy carbon disk/platinum ring rotating ring disk electrode: RRDE AFE6R2GCPT, area = 0.2375 cm 2 , Pine Research Instrumentation), resulting in a catalyst loading of 0.15 mgcm −2 . Before use, the modified glassy carbon electrode was dried in a convection oven at 40 • C for 4 min.
Fourier Transform Infrared Spectroscopy (FTIR) was carried out by means of a FTIR100 spectrometer (Perkin Elmer) in transmittance mode. Samples were pelleted in 150 mg of KBr using a Specac manual hydraulic press, by applying a pressure of 7 tons for 5 min. The diameter of pellets was 13 mm. X-Ray Diffraction (XRD) was carried out by means of a Philips PW1730 diffractometer with Cu Kα radiation (λ = 1.5406 Å).
Cyclic Voltammetry (CV). The catalytic activity toward ORR of the prepared materials was examined by cyclic voltammetry, using a VMP3 Potentiostat (Bio Logic Science Instrument) controlled by computer through EC-Lab V10.18 software. A conventional tree electrode cell was used. The reference electrode was saturated calomel electrode SCE (Amel 303/SCG/12), the auxiliary electrode was a platinum wire (Amel 805/SPG/12) and the glassy carbon disk RRDE modified with the catalyst layer as a working electrode. All potential values in the manuscript were measured vs. SCE and then converted to the reversible hydrogen electrode (RHE) scale, according to Equation (1): where E 0 (SCE) = 0.241 V. CV experiment was carried out in 125 mL of neutral phosphate buffer solution (PBS), saturated in either nitrogen or oxygen atmosphere (gas purging for at least 20 min to obtain complete oxygen saturation of the media) at room temperature, with 10 mVs −1 scan rate and 1.4 V to −0.4 V vs. RHE potential window. Specific capacitance (Cs) was calculated by integrating capacitive current (CV curve in N 2 atmosphere) over a 1.1 V vs. RHE potential window, according to Equation (2) [49]: where Cs is the specific capacitance (Fg −1 ), A is the integrated area of the CVs, m is mass of electrocatalyst at the electrode surface (g), k is potential scan rate (mVs −1 ) and ∆V is the potential window. The electrochemical surface area (ECSA) was obtained by Equation (3) [50][51][52]: where C DL is the double layer capacitance (Fm −2 ), taken as 30 µFcm −2 , as previously reported [18]. Stability tests were carried out by chronoamperometry. Current density was measured at 0.26 V vs. RHE for 10 h, in O 2 -saturated phosphate-buffered saline (PBS) in static condition. The current density retention (Jretention) was calculated as follows: where J t is the measured current density at time t and J t0 is the initial current density.
Appl. Sci. 2020, 10, 5278 5 of 16 Figure 2a shows TGA curves of FePc, urea, carbon black pearls treated with nitric acid (BPO), and pristine carbon black pearls (BP). Whereas BP were thermally stable over the whole temperature range investigated, BPO had a total weight loss of 33.5% due to the splitting off of oxygen containing surface groups introduced with the treatment with nitric acid [6]. As far as urea is concerned, three main weight losses can be identified. In the first temperature region (room temperature to 230 • C), mass loss can be associated with urea decomposition to ammonium cyanate, cyanic acid (HNCO) and ammonia. As the temperature increased (230 • C-400 • C), HNCO can further react with undecomposed urea to produce different decomposition products that eventually decomposed to carbon dioxide and ammonia up to 400 • C [53].

Results and Discussion
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 16 Figure 2a shows TGA curves of FePc, urea, carbon black pearls treated with nitric acid (BPO), and pristine carbon black pearls (BP). Whereas BP were thermally stable over the whole temperature range investigated, BPO had a total weight loss of 33.5% due to the splitting off of oxygen containing surface groups introduced with the treatment with nitric acid [6]. As far as urea is concerned, three main weight losses can be identified. In the first temperature region (room temperature to 230 °C), mass loss can be associated with urea decomposition to ammonium cyanate, cyanic acid (HNCO) and ammonia. As the temperature increased (230 °C-400 °C), HNCO can further react with undecomposed urea to produce different decomposition products that eventually decomposed to carbon dioxide and ammonia up to 400 °C [53]. In the case of FePc, the first weight loss (188 °C) did not involve a degradation step due to evaporation of adsorbed water. The second weight loss (200-450 °C) was the first degradation stage, leading to phthalocyanine cores being decomposed, followed by a second (550-650 °C) and third (650-800 °C) degradation stage, overall corresponding to breakdown of the three-dimensional polymer structure and following decomposition of the breakdown products [54].

Results and Discussion
Weight loss in the prepared Fe-N-C electrocatalysts reflected weight losses of the precursors, and no substantial changes were observed among FeNC_BM1, FeNC_BM3 and FeNC_BM6 in terms of thermal stability. The first weight loss in Fe-N-C electrocatalysts can be ascribed to a combination of the splitting off of oxygen containing surface groups in BPO, the evaporation of adsorbed water in both BPO and FePc, and the first step of urea decomposition. The second weight loss corresponds to the first thermal degradation step of FePc combined with further decomposition of unreacted urea. After 350 °C, the weight loss in Fe-N-C electrocatalysts was less pronounced and followed the decomposition of the FePc and urea breakdown products. Table 2 lists thermal parameters extrapolated from TGA curves for both precursors and electrocatalysts.
A structural analysis of Fe-N-C samples was carried out by FTIR spectroscopy and XRD. Figure 3a shows the FTIR spectrum of FeNC_BM1, FeNC_BM3 and FeNC_BM6 in a broad range of wavenumber (400-4000 cm −1 ), while Figure 3b shows a portion of the same spectra in a reduced range of wavenumber (500-1800 cm −1 ). As a general comment, the FTIR spectrum of the three catalysts showed all the vibration bands of urea, and two main contributions of FePc at 1335 cm −1 and 727 cm −1 , corresponding to the C=C stretching vibration, and the Carom.-H out-of-plane bending vibration [55,56]. The FTIR spectra of FePc and urea, together with a complete assignment of all In the case of FePc, the first weight loss (188 • C) did not involve a degradation step due to evaporation of adsorbed water. The second weight loss (200-450 • C) was the first degradation stage, leading to phthalocyanine cores being decomposed, followed by a second (550-650 • C) and third (650-800 • C) degradation stage, overall corresponding to breakdown of the three-dimensional polymer structure and following decomposition of the breakdown products [54].
Weight loss in the prepared Fe-N-C electrocatalysts reflected weight losses of the precursors, and no substantial changes were observed among FeNC_BM1, FeNC_BM3 and FeNC_BM6 in terms of thermal stability. The first weight loss in Fe-N-C electrocatalysts can be ascribed to a combination of the splitting off of oxygen containing surface groups in BPO, the evaporation of adsorbed water in both BPO and FePc, and the first step of urea decomposition. The second weight loss corresponds to the first thermal degradation step of FePc combined with further decomposition of unreacted urea. After 350 • C, the weight loss in Fe-N-C electrocatalysts was less pronounced and followed the decomposition of the FePc and urea breakdown products. Table 2 lists thermal parameters extrapolated from TGA curves for both precursors and electrocatalysts. A structural analysis of Fe-N-C samples was carried out by FTIR spectroscopy and XRD. Figure 3a shows the FTIR spectrum of FeNC_BM1, FeNC_BM3 and FeNC_BM6 in a broad range of wavenumber (400-4000 cm −1 ), while Figure 3b shows a portion of the same spectra in a reduced range of wavenumber (500-1800 cm −1 ). As a general comment, the FTIR spectrum of the three catalysts showed all the vibration bands of urea, and two main contributions of FePc at 1335 cm −1 and 727 cm −1 , corresponding to the C=C stretching vibration, and the C arom. -H out-of-plane bending vibration [55,56]. The FTIR spectra of FePc and urea, together with a complete assignment of all vibration bands, is reported in Figure S1 and Table S1, respectively, while the assignment of vibration band in the FeNC catalysts is reported in Table 3.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 16 vibration bands, is reported in Figure S1 and Table S1, respectively, while the assignment of vibration band in the FeNC catalysts is reported in Table 3.   On the other hand, a band due to symmetric vibrations of the isoindole fragments was clearly visible for FeNC_BM1 around 1120 cm −1 , while it became a shoulder for FeNC_BM3 and FeNC_BM6. This finding suggests a higher vibrational activity of the isoindole groups on FeNC-BM1, indicating that increasing ball-milling time enhanced the interaction between FePc macrocycle and the carbon support. Figure 4a shows XRD patterns of FeNC_BM1, FeNC_BM3 and FeNC_BM6. The XRD patterns of FePc, urea and BPO are shown in Figure S2. In the XRD pattern of the three catalysts, the major  On the other hand, a band due to symmetric vibrations of the isoindole fragments was clearly visible for FeNC_BM1 around 1120 cm −1 , while it became a shoulder for FeNC_BM3 and FeNC_BM6. This finding suggests a higher vibrational activity of the isoindole groups on FeNC-BM1, indicating that increasing ball-milling time enhanced the interaction between FePc macrocycle and the carbon support. Figure 4a shows XRD patterns of FeNC_BM1, FeNC_BM3 and FeNC_BM6. The XRD patterns of FePc, urea and BPO are shown in Figure S2. In the XRD pattern of the three catalysts, the major contribution of urea can be observed, while the more intense peak of graphitic carbon (from BPO support around 25 2θ • ) and the peaks of FePc were hidden due to the greater crystalline contribution of urea.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 16 support around 25 2θ°) and the peaks of FePc were hidden due to the greater crystalline contribution of urea. However, as shown in Figure S3, the contribution of graphitic carbon was greater for FeNC-BM6, especially compared to FeNC_BM1. The contribution in the amorphous fraction was comprised in the 2θ range in which FePc exhibited a greater number of peaks (i.e., 15-85 2θ°). Therefore, we consider that the increase in ball-milling time led to an enhanced incorporation of FePc in BPO carbon support, in good agreement with FTIR results. Figure 4b shows the diffractograms of the catalysts obtained after heat treatment. The diffraction peak around 25 2θ degrees was observed for all materials, indicating a strong contribution of graphitic carbon to the XRD pattern of all catalysts, which is different from what observed for nonpyrolyzed catalysts. This was due to a precursor decomposition and breakdown during pyrolysis, as pointed out by TGA. Diffraction peaks for metal iron and iron oxide were also evident [57], and their intensity increased as the ball milling-time increased. This was particularly evident in the case of the diffraction peaks at 45 2θ° and 82 2θ° for the FeNC_BM6_P sample, suggesting an increase in iron content in the samples with the ball-milling time, in accordance with FTIR results.
To evaluate catalytic activity toward ORR of the prepared catalysts, cyclic voltammetry was carried out in either nitrogen-or oxygen-saturated phosphate buffer. No peaks were detected in N2saturated PBS for both FeNC and FeNC_P (Figure 5), and the resulting capacitive current was much higher for FeNC_P as compared to FeN, because an expected effect of pyrolysis in increasing surface area of the catalyst. When the electrolyte was saturated with oxygen, a well-defined reduction peak around 0.6 V vs. RHE can be seen for both samples demonstrating the occurrence of ORR at the surface of Fe-N-C catalysts. The ORR peak was shifted toward more positive voltage values for FeNC_P, pointing at the beneficial effect of pyrolysis in enhancing ORR. ORR peak current density also increased, indicating an increased density of active sites after pyrolysis. However, as shown in Figure S3, the contribution of graphitic carbon was greater for FeNC-BM6, especially compared to FeNC_BM1. The contribution in the amorphous fraction was comprised in the 2θ range in which FePc exhibited a greater number of peaks (i.e., 15-85 2θ • ). Therefore, we consider that the increase in ball-milling time led to an enhanced incorporation of FePc in BPO carbon support, in good agreement with FTIR results. Figure 4b shows the diffractograms of the catalysts obtained after heat treatment. The diffraction peak around 25 2θ degrees was observed for all materials, indicating a strong contribution of graphitic carbon to the XRD pattern of all catalysts, which is different from what observed for nonpyrolyzed catalysts. This was due to a precursor decomposition and breakdown during pyrolysis, as pointed out by TGA. Diffraction peaks for metal iron and iron oxide were also evident [57], and their intensity increased as the ball milling-time increased. This was particularly evident in the case of the diffraction peaks at 45 2θ • and 82 2θ • for the FeNC_BM6_P sample, suggesting an increase in iron content in the samples with the ball-milling time, in accordance with FTIR results.
To evaluate catalytic activity toward ORR of the prepared catalysts, cyclic voltammetry was carried out in either nitrogen-or oxygen-saturated phosphate buffer. No peaks were detected in N 2 -saturated PBS for both FeNC and FeNC_P (Figure 5), and the resulting capacitive current was much higher for FeNC_P as compared to FeN, because an expected effect of pyrolysis in increasing surface area of the catalyst. When the electrolyte was saturated with oxygen, a well-defined reduction peak around 0.6 V vs. RHE can be seen for both samples demonstrating the occurrence of ORR at the surface of Fe-N-C catalysts. The ORR peak was shifted toward more positive voltage values for FeNC_P, pointing at the beneficial effect of pyrolysis in enhancing ORR. ORR peak current density also increased, indicating an increased density of active sites after pyrolysis. The same characterization was carried out for all the prepared electrocatalysts: The samples prepared with different ball-milling times (FeNC_BM1, FeNC_BM3 and FeNC_BM6) and the samples obtained after a further pyrolysis treatment of the above-mentioned samples (FeNC_BM1_P, FeNC_BM3_P and FeNC_BM6_P). The corresponding CV curves are shown in Figure 6. CV analysis indicated that both ball-milling and pyrolysis affect capacitive current density values, ORR peak potential and peak current density. Electrochemical surface area (ECSA) was then calculated by integrating capacitive current, and the results are shown in Table 4, together with The same characterization was carried out for all the prepared electrocatalysts: The samples prepared with different ball-milling times (FeNC_BM1, FeNC_BM3 and FeNC_BM6) and the samples obtained after a further pyrolysis treatment of the above-mentioned samples (FeNC_BM1_P, FeNC_BM3_P and FeNC_BM6_P). The corresponding CV curves are shown in Figure 6. The same characterization was carried out for all the prepared electrocatalysts: The samples prepared with different ball-milling times (FeNC_BM1, FeNC_BM3 and FeNC_BM6) and the samples obtained after a further pyrolysis treatment of the above-mentioned samples (FeNC_BM1_P, FeNC_BM3_P and FeNC_BM6_P). The corresponding CV curves are shown in Figure 6. CV analysis indicated that both ball-milling and pyrolysis affect capacitive current density values, ORR peak potential and peak current density. Electrochemical surface area (ECSA) was then calculated by integrating capacitive current, and the results are shown in Table 4, together with CV analysis indicated that both ball-milling and pyrolysis affect capacitive current density values, ORR peak potential and peak current density. Electrochemical surface area (ECSA) was then Appl. Sci. 2020, 10, 5278 9 of 16 calculated by integrating capacitive current, and the results are shown in Table 4, together with further electrochemical parameters extrapolated from CV analysis, such as ORR peak potential (Epr) and peak current density (Jpr). Table 4. Electrochemical surface area (ECSA) and electrochemical parameters (oxygen reduction reaction (ORR) peak potential, E pr , peak current density, J pr , and current density retention (@ 0.26 V vs. RHE) after 10 h, Jretention) of the prepared electrocatalysts.

Sample
ECSA (m 2 g −1 ) E pr (V) J pr (mAcm −2 ) Jretention (%) As a general comment, ECSA values changed after either ball-milling time and subsequent pyrolysis, and variations in Epr and Jpr also took place. To identify a trend in ECSA, Epr and Jp after ball-milling and pyrolysis, those parameters were plotted in Figure 7 for nonpyrolyzed ( Figure 7a) and pyrolyzed catalysts (Figure 7b).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 9 of 16 further electrochemical parameters extrapolated from CV analysis, such as ORR peak potential (Epr) and peak current density (Jpr). As a general comment, ECSA values changed after either ball-milling time and subsequent pyrolysis, and variations in Epr and Jpr also took place. To identify a trend in ECSA, Epr and Jp after ball-milling and pyrolysis, those parameters were plotted in Figure 7 for nonpyrolyzed ( Figure 7a) and pyrolyzed catalysts (Figure 7b). Trend of electrochemical surface area (ECSA), ORR peak potential (Epr) and peak current density (Jpr) of the prepared FeNC-based electrocatalyst before and after ball-milling (a) and pyrolysis (b) (symbols: experimental data; dash lines: guide for eyes).
In the case of nonpyrolyzed sample, ECSA increased as the ball-milling time increased up to 3 h (80 m 2 g −1 for FeNC_BM3). After that, ECSA decreased for the sample prepared with 6 h of ball-milling time (46 m 2 g −1 for FeNC_BM6). This finding indicates that increasing ball-milling is effective in increasing surface area of the electrocatalysts up to 3 h, but longer ball-milling time may reverse the effect as previously reported [44,58]. Ball milling is indeed a very well-known method to increase materials surface area and porosity, contributing to breakdown of larger solid clusters into smaller ones, and optimizing ball-milling time is crucial to achieve desirable surface area and morphology [39]. ORR peak current density (ORR) followed the same trend as ECSA due to an increased density Figure 7. Trend of electrochemical surface area (ECSA), ORR peak potential (Epr) and peak current density (Jpr) of the prepared FeNC-based electrocatalyst before and after ball-milling (a) and pyrolysis (b) (symbols: experimental data; dash lines: guide for eyes).
In the case of nonpyrolyzed sample, ECSA increased as the ball-milling time increased up to 3 h (80 m 2 g −1 for FeNC_BM3). After that, ECSA decreased for the sample prepared with 6 h of ball-milling time (46 m 2 g −1 for FeNC_BM6). This finding indicates that increasing ball-milling is effective in increasing surface area of the electrocatalysts up to 3 h, but longer ball-milling time may reverse the effect as previously reported [44,58]. Ball milling is indeed a very well-known method to increase materials surface area and porosity, contributing to breakdown of larger solid clusters into smaller ones, and optimizing ball-milling time is crucial to achieve desirable surface area and morphology [39].
ORR peak current density (ORR) followed the same trend as ECSA due to an increased density of active sites for higher ECSA samples. By contrast, Epr values increased as the ball-milling time increased, FeNC_BM6 sample having the more positive Epr value (Epr = 0.73 V vs. RHE).
The samples were further subjected to a pyrolysis treatment to overcome a possible heterogeneity in porosity and modification in surface chemistry induced by ball-milling, which may have contributed to decrease the active site utilization [59]. The pyrolysis step after ball-milling led to an increase in ECSA only for the sample prepared with no ball-milling (146 m 2 g −1 for FeNC_P vs. 19 m 2 g −1 for FeNC) and the sample obtained with 1 h of ball-milling time (69 m 2 g −1 for FeNC_BM1_P vs. 54 m 2 g −1 for FeNC_BM1). By contrast, the pyrolysis treatment led to a decrease in ECSA for ball-milling time higher than 1 h. This can be ascribed to an agglomeration of solid clusters and pore growth between them, as previously reported by Martinaiou et al., who demonstrated that ESCA of Fe-N-C materials is reduced by a pyrolysis step after ball milling due to changes in porosity [60]. Epr and Jpr values were affected by pyrolysis with a similar trend as compared to that described for ECSA and illustrated in Figure 7.
The body of ECSA, Epr and Jp results indicated that ball-milling time in the range of 1-3 h allowed for the production of electrocatalysts with high ECSA, high density of ORR active sites (Jp), and good catalytic activity toward ORR (Epr). Longer ball-milling time (6 h) led to a catalyst (FeNC_BM6) with lower ECSA and Jp, but the lowest ORR overpotential (as can be seen from Epr value) still pointed at a good electrocatalytic activity toward ORR. The good catalytic activity of FeNC_BM6 can be explained by considering the enhanced FePc/carbon support interaction, as indicated by FTIR and XRD data. These findings confirm that ORR activity in the FeNC_catalysts arises from a combination of high surface area and the high content of FeN 4 active sites well connect with the carbon support.
After pyrolysis, ECSA and Jp of FeBM3_P and FeBM6_P decreased, and Epr was shifted toward less positive values, indicating that pyrolysis is not effective in improving ORR performance of FeNC_BM3 and FeNC_BM6. This can be explained by the role of pyrolysis combined with long ball-milling time in increasing metal iron and Fe 3 O 4 /Fe 2 O 3 content in the resulting materials. In fact, it has been previously proved that iron oxide has lower catalytic activity than nitrogen coordinated iron (FeN 4 ) sites. Metal iron has no ORR activity, also causing a reduction in the available area of FeN 4 active sites [61].
As far as FeNC_BM1 sample is concerned, the pyrolysis induced an increase in ECSA and Jp, while Epr remained almost constant.
Along with ORR performance, electrocatalytic stability under operative conditions is also of paramount importance for determining the applicability of an electrocatalysts. Hence, the elect of ball-milling and pyrolysis on electrocatalytic stability was evaluated by chronoamperometry in O 2 -saturated PBS. Figure 8 shows current density retention (Jretention) as a function of time.
In the case of nonpyrolyzed samples (Figure 8a), current density decreased over time, and eventually reached a plateau after 8 h. On the other hand, current density retention increased in the order FeNC < FeNC_BM1 < FeNC_BM3 > FeNC_BM6, indicating that ball-milling time was very effective in enhancing catalytic stability of Fe_N_C catalysts. Table 3 lists Jretention values for all samples: 6 h of ball-milling allowed retention up to 65% of initial current density after 10 h of operation. In the case of nonpyrolyzed samples (Figure 8b), current density retention was much higher than that of the corresponding nonpyrolyzed samples. This finding points toward the beneficial effect of pyrolysis in enhancing catalytic stability, and adequate Jretention values were obtained for FeNC_BM1_P, FeNC_BM3_P and FeNC_BM6_P (50%, 75% and 80%, respectively).

Contextualization into Existing Literature
Fe-N-C catalysts can be considered as one of the most promising catalyst families to replace platinum at the cathode side of energy conversion devices such as fuel cells. Previous reports, which have focused on alkaline anion exchange membrane fuel cells, deeply investigated the ORR activity of Fe-N-C catalysts in alkaline environments, and found that optimizing the preparation procedure boosted catalytic activity and durability. Fe-N-C catalyst obtained by supporting FePc on graphene showed a ORR peak potential in the 0.7-0.84 V vs. RHE in an alkaline environment [22,62,63]. Oliveira et al. investigated ORR behaviour in an alkaline electrolyte of FeN4 macrocycles supported over carbon nanotubes and carbon spheres via a wet impregnation strategy. Those catalysts achieved a reduction potential of 0.82 V and 0.85 V vs. RHE, with an ECSA of 45 m 2 g −1 and 57 m 2 g −1 , respectively [12]. Optimizing surface area and porosity improves the mass transport, and consequently, ORR performance [64]. Atanassov and coworkers demonstrated that a ball-mill treatment followed by pyrolysis induced a decrease in relative amount of both mesopores and macropores homogenizing morphology of Fe-N-C catalysts. In an alkaline environment, these materials directly catalysed oxygen reduction to water, with a reduction potential of 0.93 V vs. RHE [59].
In this context, the FeNC_BM6 catalyst discussed in this work showed an ORR peak potential as high as 0.73 V vs. RHE in neutral media. This value is very close to the values obtained for similar catalysts in alkaline environments, demonstrating that FeNC_BM6 is very active for ORR studies in pH environments, such as neutral, along with its electrochemical stability.

Conclusions
Ball-milling and pyrolysis strongly affect electrochemical surface area (ECSA), catalytic activity, and stability of Fe-N-C electrocatalysts prepared using Iron(II) phthalocyanine, urea and carbon black pearls as precursors. Ball-milling increased the ECSA, ORR peak potential (Epr) and peak current density (Jpr) as compared to the FeNC sample obtained without any ball-milling treatments. Different ball-milling times (1 h, 3 h, and 6 h) were explored, and ORR performance of all the three catalysts prepared (FeNC_BM1, FeNC_BM3_FeNC_BM6) displayed a good catalytic activity toward ORR. In particular, 3 h and 6 h of ball-milling time allowed for the production of samples with the highest ECSA, Jp and Epr values due to its beneficial effect in enhancing the interaction between FePc

Contextualization into Existing Literature
Fe-N-C catalysts can be considered as one of the most promising catalyst families to replace platinum at the cathode side of energy conversion devices such as fuel cells. Previous reports, which have focused on alkaline anion exchange membrane fuel cells, deeply investigated the ORR activity of Fe-N-C catalysts in alkaline environments, and found that optimizing the preparation procedure boosted catalytic activity and durability. Fe-N-C catalyst obtained by supporting FePc on graphene showed a ORR peak potential in the 0.7-0.84 V vs. RHE in an alkaline environment [22,62,63]. Oliveira et al. investigated ORR behaviour in an alkaline electrolyte of FeN 4 macrocycles supported over carbon nanotubes and carbon spheres via a wet impregnation strategy. Those catalysts achieved a reduction potential of 0.82 V and 0.85 V vs. RHE, with an ECSA of 45 m 2 g −1 and 57 m 2 g −1 , respectively [12]. Optimizing surface area and porosity improves the mass transport, and consequently, ORR performance [64]. Atanassov and coworkers demonstrated that a ball-mill treatment followed by pyrolysis induced a decrease in relative amount of both mesopores and macropores homogenizing morphology of Fe-N-C catalysts. In an alkaline environment, these materials directly catalysed oxygen reduction to water, with a reduction potential of 0.93 V vs. RHE [59].
In this context, the FeNC_BM6 catalyst discussed in this work showed an ORR peak potential as high as 0.73 V vs. RHE in neutral media. This value is very close to the values obtained for similar catalysts in alkaline environments, demonstrating that FeNC_BM6 is very active for ORR studies in pH environments, such as neutral, along with its electrochemical stability.

Conclusions
Ball-milling and pyrolysis strongly affect electrochemical surface area (ECSA), catalytic activity, and stability of Fe-N-C electrocatalysts prepared using Iron(II) phthalocyanine, urea and carbon black pearls as precursors. Ball-milling increased the ECSA, ORR peak potential (Epr) and peak current density (Jpr) as compared to the FeNC sample obtained without any ball-milling treatments. Different ball-milling times (1 h, 3 h, and 6 h) were explored, and ORR performance of all the three catalysts prepared (FeNC_BM1, FeNC_BM3_FeNC_BM6) displayed a good catalytic activity toward ORR. In particular, 3 h and 6 h of ball-milling time allowed for the production of samples with the highest ECSA, Jp and Epr values due to its beneficial effect in enhancing the interaction between FePc and carbon support. Ball-milling also affected catalytic stability of the Fe-N-C catalysts, as increasing ball-milling time led to an increase of current density retention up to 65% for the FeNC_BM6 sample. The effect of a subsequent pyrolysis treatment after ball-milling on ORR catalytic activity and stability was also investigated. We found that pyrolysis after ball-milling had a detrimental effect on ECSA, Jp and Epr, with such a negative effect being more relevant for the samples obtained with higher ball-milling time (FeNC_BM3_P and FeNC_BM6_P). In fact, pyrolysis combined with long ball-milling time caused the increase in metal iron and iron oxide in the samples, which are less effective as ORR active sites as compared to nitrogen-coordinated iron. On the other hand, pyrolysis further improved electrocatalytic stability of the materials, allowing retention up to 80% of initial current density for FeNC_BM3_P and FeNC_BM3_P samples.
Overall, considering the requirements of high ORR catalytic activity and good stability under operative conditions, 6 h of ball-milling without any further pyrolysis was effective to prepare a Fe-N-C material with a good compromise between ORR activity and stability (FeNC_BM6). Also, 1 h of ball-milling followed by pyrolysis was effective in producing a Fe-C-materials with high ORR activity and stability (FeNC_BM1_P). Hence, FeNC_BM6 and FeNC_BM1_P can be good candidates for use at the cathode side of microbial fuel cells operating in neutral pH.