2. Experimental section

2.1 Materials

Enzymatic hydrolysis lignin (EHL) isolated from the corncob bio-refinery residue through alkali dissolving, acid precipitation, water washing, drying, ball milling and sieving, was provided by Shandong Longli Biotechnology Co., Ltd. Iron chloride hexahydrate (FeCl3·6H2O, AR 99%), cobalt nitrate hexahydrate (Co(NO3)2·6H2O, AR 99%), urea (CO(NH2)2, AR 99%), ammonia (NH3·H2O, AR 25-28%), ethanol (C2H5OH, AR 99.7%), commercial iridium carbon (20 wt%) were purchased from Shanghai Aladdin Chemical Co., Ltd., monochloro acetic acid (C2H3ClO2, AR 98%) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., and acetone (C3H6O, AR 98%), hydrochloric acid (HCl, 36.0-37.0%), nitric acid (HNO3, 65.0-68.0%), sodium hydroxide (NaOH, AR 96%), potassium hydroxide (KOH, AR 85%) were produced by Guangzhou Chemical Reagent Factory. Nafion solution (D520, 5 wt%) was supplied from DuPont. Carbon papers (TGP-H-060, 20 cm×20 cm, 0.19 mm) were produced by Toray Corporation (Japan).

2.2 Preparation of lignin-based CoFe supramolecules

Carboxylated enzymatic hydrolyzed lignin (EHL-COOH) was prepared by the aqueous method according to our previous report 51. Then 1.0 g EHL-COOH was dissolved in 100 mL pure water, while FeCl3·6H2O and Co(NO3)2·6H2O (nFe/nCo = 8:0, 7:1, 6:2, 4:4, 2:6, 1:7, 0:8) were added into 50 mL pure water under vigorous stirring. The mixture was added drop by drop into the aqueous solution of EHL-COOH and stirred for 10 min. The pH of above solution was adjusted to 8 by NH3·H2O and HCl. After aging for 12 h, the precipitate was centrifuged, washed with ultra-pure water, dried at 80℃ for 24 h, and ground to obtain the lignin-based CoFe supramolecules (EHL-COOH-CoFe).

2.3 Preparation of lignin-derived carbon CoFe-CoxN@NC

Firstly, 1.0 g of EHL-COOH-CoFe was ground and mixed with a certain amount of urea, then was heated at a rate of 5 ℃·min-1and pyrolyzed at high temperature for 2 h under the argon atmosphere. The products were washed with 1 mol·L−1 HCl and ultra-pure water, respectively, and dried at 80℃ for 12 h to obtain the bimetal-based functionalized nitrogen-doped lignin-derived carbon materials, denoted as CoFe-CoxN@NC. The effects of metal ratio, pyrolysis temperature, the ratio of metal to lignin, and nitrogen content on the structure and electrocatalytic performance of CoFe-CoxN@NC were systematically investigated. 1.0 g of EHL-COOH was mixed and ground with 1.0 g of urea, then pyrolyzed at 700 ℃ to obtain the metal free nitrogen-doped lignin-derived carbon materials (NC) as comparative sample.

2.4 Structural characterization of CoFe-CoxN@NC

The crystal structure of the electrocatalyst was characterized by Rigaku Miniflex-600 XRD instrument, which used a Cu-Kα radiated X-ray source with a scanning range of\(\mathrm{2}\mathrm{\theta}\mathrm{\ }\mathrm{=}\mathrm{\ }\mathrm{5}\mathrm{-80}\)and a scanning rate of 10 °·min−1. The morphology and elemental composition were characterized by American FEI Tecnai G2 F20 TEM, and its operating voltage was 200 kV. The degree of order and graphitization were characterized by LabRAM HR Evolution Raman spectrometer, with an excitation wavelength of 532 nm. Nitrogen adsorption/desorption isotherms were measured at 77 K with ASAP 2460 surface area and porosity analyzer. The elemental composition and valence state were characterized by Thermo Scientific K-Alpha + X-ray photoelectron spectrometer (XPS). All the peaks were calibrated according to the standard position of C 1s peak (284.8 eV). The percentage of doped metal in the sample was calculated by Varian 720 inductively coupled plasma OES spectrometer (ICP-OES). The band gap width of carbon materials was determined by PE Lambda 950 UV-Vis Diffuse Reflectance Spectrometer. The valence band and conduction band of carbon materials were tested by UV photoelectron spectrometer (Thermo Fischer Escalab 250 Xi).

2.5 Electrochemical measurements

All electrochemical measurements were performed in a three-electrode system using the Gamry Interface 1010 electrochemical workstation. Graphite rod and Hg/ HgO were used as the counter electrode and reference electrode, respectively. The working electrode was prepared as follows, 4 mg of carbon powder was added into 200 μL of 0.25% nafion-ethanol solution. After ultrasonic dispersion of the powder, 50 μL of the slurry was dropped onto the treated carbon paper, and then the carbon paper was held by electrode clamp as the working electrode. The loading capacity of the catalyst was 4 mg·cm−2.
The electrochemical performances were investigated by using linear sweep voltammetry (LSV) and cyclic voltammetry (CV) techniques at room temperature. 1 mol·L−1 KOH solution was used as electrolyte, and the scanning rate recorded by all the polarization curves was 1 mV·s−1. Before LSV testing, the working electrode with loading catalyst was firstly activated by several CV cycles. All the measured data of the polarization curve were corrected by IR, and all the potentials were calculated according to the reversible hydrogen electrode (RHE). The expression is as follows:
\(\mathrm{E}\mathrm{\ }\left(\mathrm{\text{vs.}}\mathrm{\ }\mathrm{\text{RHE}}\right)\mathrm{\ =\ E}\mathrm{\ }\left(\mathrm{\text{vs.}}\mathrm{\ }\mathrm{Hg/HgO}\right)\mathrm{\ +\ 0.098\ +\ 0.0591\ \times\ }\mathrm{\text{pH}}\).

2.6 Density functional theory (DFT) calculations

DFT calculations were carried out using the Vienna ab Initio Simulation Package (VASP) 52. The Perdew-Burke-Ernzerhof (PBE) model in the form of the generalized gradient approximation (GGA) was used to describe the exchange-correlation potential. For all optimization calculations, the energy and force convergence criteria were set to 10-5 eV and 0.05 ev·Å-1, respectively. Total energy was calculated using the Projector Augmented-Wave (PAW) method with a plane-wave cutoff energy of 400 eV. A gamma-centered K-point of 3×3×1 was chosen to describe the Brillouin zone.