Chemicals
Hexahydrates of Ni and copper Cu nitrates (Ni(NO3)2.6H2O and Cu(NO3)2.6H2O) were purchased from Fisher Scientific (USA). Graphene (G) powder (average pore diameter 100 ± 10 Å) and 5wt.% Nafion solutions were purchased from Sigma-Aldrich (MO, USA). Pt on carbon Vulcan (30% Pt/C, E-tek, USA) electrocatalyst was obtained from the Fuel Cell Store (TX, USA). All supplies and chemical materials were of analytical grade purity and were used without further purification. All aqueous solutions were freshly prepared using double distilled water.
Synthesis of NiO–CuO/G electrocatalyst
NiO–CuO/G composites were prepared using a co-precipitation method [29]. In a typical synthesis, the mixed metal salt precursors of Ni(NO3)2.6H2O and Cu (NO3)2.6H2O were dispersed on high surface area G in double-distilled water. Then, pH was adjusted to approximately 10 using the drop-wise addition of 1.0 M NaOH using Benchtop pH meter (Adwa AD1000, Hungary) followed by vigorous stirring for 3 h to avoid agglomeration and to generate a homogeneous dispersion. The resulting black metal-hydroxide precipitate was filtered and washed several times using double-distilled water and adjusted to pH 7. After this, the final product was generated by drying the precipitate at 80 °C (TiTANOX oven, Italy) for 6 h to remove excess water. The dried precipitate was calcinated for 3 h at 300 °C in a muffle furnace (ShinSaeng Scientific, Korea) to form metal oxides. The corresponding mass ratio in the NiO–CuO/G was 30% of NiO–CuO to 70% of Graphene.
Electrochemical measurements using a rotating disk electrode
Electrochemical measurements were performed at room temperature (25 ± 1 °C) using an electrochemical workstation (PST006 Voltamaster, USA) and a rotating disk electrode (RDE; CTV 101 speed control unit, USA). The working electrode (with a geometrical surface area of 0.196 cm2) was either NiO–CuO/G- or Pt/C-deposited thin film on the surface of the glassy carbon electrode (GCE), while Pt wire and Ag/AgCl (Metrohm, Netherlands) were used as counter and reference electrodes, respectively. Before depositing the catalyst layer on the GCE, the GCE was mechanically polished using 0.05 μm alumina slurry on a soft cloth to generate a mirror-like surface. Finally, the GCE was rinsed in double-distilled water and acetone. Following this, 1.0 mg of electrocatalyst was sputtered and mixed with a drop of isopropanol on the GCE. After the isopropanol was dried, a drop of Nafion solution (5%) was pipetted onto the GCE surface to form a homogeneous thin layer. Finally, a second drop of isopropanol was added, and the electrocatalyst film dried at room temperature overnight.
Linear sweep voltammetry (LSV) studies were conducted in a 50-mM phosphate buffer solution (PBS, pH 7.2) at a scan rate of 10 mV s−1 in a potential range of (− 1000 to + 1000 mV vs. Ag/AgCl) (equivalent to − 780 to + 1220 mV vs. standard hydrogen electrode (SHE)) with different rotation speeds (i.e., 0 to 2400 rpm). Before electrochemical measurements were conducted, the electrolyte was sparged with ultra-pure oxygen (O2) for 30 min. For comparison, all electrochemical measurements were performed in nitrogen-saturated PBS. A schematic summary is presented in Figure S1.
Kinetic parameters were analyzed based on the Koutechy-Levich (K-L) equation derived from RDE studies to calculate electron transfer numbers (n) involved in the ORR process [35] as follows:
$$\frac{\mathbf{1}}{{\mathbf{I}}_{\mathbf{d}}}=\frac{\mathbf{1}}{{\mathbf{I}}_{\mathbf{k}}}+\frac{\mathbf{1}}{\mathbf{0.62nFA}{\mathbf{D}}^{{\mathbf{2}}_{/\mathbf{3}}}{\mathbf{c}\boldsymbol{\upnu}}^{-\mathbf{1}\left/\mathbf{6}\right.}{\boldsymbol{\upomega}}^{\mathbf{1}\left/ \mathbf{2}\right.}}$$
(1)
Where Id is the disk current density; Ik is the electrode potential-dependent kinetic current density; ω is the angular momentum (rads−1s1/2); n is the average number of electrons in the catalytic reaction; F is Faraday’s constant (96,485 C mol−1); D and C are the diffusion coefficient of dissolved oxygen (1.9 × 10−5 cm2 s−1) and the concentration of dissolved oxygen in 50 mM PBS (1.117 × 10−6 mol mL−1), respectively; v is the kinetic viscosity of the electrolyte (0.01073 cm2 s−1); and A is the geometric area of the disk electrode (0.196 cm2) [36].
MFC configuration
All MFC studies were conducted using single-chamber, air-cathode MFCs (6 cm long, 4.6 cm in diameter, total working volume = 100 mL) as described elsewhere [37] and illustrated in Figure S2. Anodes were three-dimensional carbon felt glued to the top of an externally connected anode port with effective dimensions of 2.5 × 2.5 × 0.6 cm and a projected surface area of 18.50 cm2. They were positioned on the other side of the cell (Parallel to the cathode) at a distance of ~ 5 cm from the cathode. Gas diffusion carbon cloth electrodes were used as cathode electrodes (6 × 6 cm each; surface area = 16.63 cm2) with a catalyst loading of 0.30 mg/cm2. Titanium wire were used as current collectors for both electrodes.
Cathode electrodes preparation
Cathode electrodes were prepared as described elsewhere [38]. The catalyst was maintained on the water-facing side of a cathode at a mass loading of 0.3 mg cm−2. Before coating, catalyst slurry was prepared by mixing NiO–CuO/G composites with a 5% Nafion solution. The mixture was ultra-sonicated at 60 °C for 30 min and uniformly dispersed onto the carbon cloth surface electrode (mesoporous gas diffusion; Fuel Cell Store, TX, USA). To reach the load of the electrode (0.3 mg cm−2), multiple catalyst ink layers were deposited on top of each other. Electrodes were dried at room temperature for 24 h before MFC studies. For comparison, 30 wt.% of a Pt catalyst on carbon Vulcan (surface area = ~ 220 m2 g−1; E-Tek, USA) was used as a cathode at a catalyst mass loading of 0.3 mg cm−2, using the same procedure as previously described.
MFC operation and analysis
MFCs were inoculated with aerobic activated sludge from a local municipal wastewater treatment plant (Benha, Egypt) and operated under a fed-batch mode for 60 days to allow biofilms to grow on anode surfaces [39]. MFCs were fed with artificial wastewater containing sodium acetate (2.0 g L−1) as the sole organic substrate in 50 mM phosphate buffer solution (BPS) (chemical oxygen demand (COD) concentration = 1472 ± 17 mg/L) supplemented with a 12.5 mL mineral solution and a 12.5 mL vitamin solution. The 50 mM PBS solution contained: NaHCO3: 2.5 g/L, NH4Cl: 0.2 g/L, KH2PO4: 13.6 g/L, KCl: 0.33 g/L, NaCl: 0.3 g/L, K2HPO4: 17.4 g/L, CaCl2.2H2O: 0.15 g/L, MgCl2: 3.15 g/L, and a yeast extract: 1 g/L. All MFC studies were conducted in triplicate to calculate average values.
MFCs were operated in a fed-batch mode at room temperature. They were monitored using a data acquisition system (LabJack U6-PRO, USA) connected to a personal computer. An external resistance of 1000 Ώ was used, unless otherwise stated. Current (mA m−2) and power densities (mW m−2) were calculated according to Ohm’s law as previously described [5, 37]. Polarization and power curves were plotted using a single-cycle technique by recording the pseudo-steady-state voltage across different external resistances, ranging from 175 KΩ to 50 Ω [40]. Internal resistance (Rint) was determined using linear regression corresponding to the Ohmic zone on the linear section of the polarization curve [41].
Influent and effluent COD concentrations were analyzed according to APHA standard methods for water and wastewater examination [42]. Organic concentrations were calculated as COD removal efficiency (COD R%), which was calculated using the following equation:
$$\mathrm{COD}\ \mathrm{R}\%=\frac{{\mathrm{COD}}_{\mathrm{initial}}-{\mathrm{COD}}_{\mathrm{final}}}{{\mathrm{COD}}_{\mathrm{initial}}}\ \mathrm{x}\ 100$$
(2)
Where CODinitial is the COD concentration in the influent (mg COD/L), and CODfinal is the COD concentration in the final effluent at the end of MFC batch cycles (mg COD/L).
The Coulombic efficiency (CE) was calculated by normalizing the measured current with respect to the theoretical current based on consumed COD as follows:
$${\mathrm C}_{\mathrm E}\left(\%\right)=\frac{{\mathrm C}_{\mathrm P}}{{\mathrm C}_{\mathrm T}}\;\mathrm x\;100$$
(3)
Where CT is the theoretical coulombs and was estimated according to the following formula: CT = (F × N × W × V)/M), where F is Faraday’s constant (96,485 C mol−1), N is number of moles of electrons (8 mol mol−1), W is the daily COD load removed (g L−1), M is the molecular weight of acetate (59 g mol−1), and V is the medium volume (100 mL) [43]. CP is the Coulombs equivalent to the actual current produced during one batch cycle.
Physical characterization of NiO–CuO/G and anodic biofilms
X-ray diffraction (XRD) patterns
In order to determine the physical characteristics of NiO–CuO/G (such as lattice composition and distinctive crystallite size), XRD was performed using an XRD–RIGAKU-D/MAX-PC 2500 X-ray diffractometer fitted with Ni-filtered Cu Kα as the radiation source (λ = 0.154056 nm) at a tube current of 40 mA with a 40-kV voltage. The 2θ angular regions were detected at a scan rate of 10° min−1 from 10° to 80°. XRD data analyses were conducted using the Materials Studio (Accelrys, USA) software suite Reflex module.
Scanning electron microscopy(SEM) analysis of electrocatalysts and biofilms
Anodic biofilm growth characterization on bioanode electrode surfaces was visualized at the end of batch studies using SEM (JEOL JAX-840A, Japan). The anode was fixed in 2.5% (w/v) glutaraldehyde for 4 h. Following fixation, samples were washed three times in DI water and dehydrated in ascending ethanol gradient steps (30% to 100% with 10 min for each step) to avoid artifact drying. Finally, samples were sputtered with gold and imaged using SEM at 20 kV. Energy dispersive X-rays (EDX) were mapped using SEM instrumentation.
Transmission electron microscopy(TEM) of electrocatalysts and biofilms
The JEOL-JEM 2010 TEM, Japan was used to determine NiO–CuO/G microstructures and particles sizes and to understand the internal morphological characteristics of the isolated bacterial strains. TEM analytical procedures for isolated anodic bacterial strains were conducted under sterile conditions at room temperature (23 °C) by inoculating 100 μL bacterial cultures into a 5-mL sterilized nutrient broth and incubating them at 37 °C for 18–20 h before TEM analysis. Then, samples were fixed in 2.5% glutaraldehyde (w/v) at 4 °C for 10 min. Before TEM imaging, harvested bacterial cells were deposited on the TEM grid, stained with 2% uranyl acetate for 3–5 s on a carbon-coated mesh Cu grid, and air-dried. The Gatan program was used for data processing and particle size measurement [44].
Biochemical identification of isolated anodic bacterial communities
The isolated anodic bacterial communities were identified using Vitek 2 manufacturer’s instructions (Biomeriux VITEK-2 Compact Reference Manual-Ref-414532) [45]. A sufficient quantity of anodic biofilm was transferred using a sterile swab into a polystyrene test tube containing 3 mL sterile saline, and mixed in a suspension well. Turbidity was adjusted to the equivalent of 0.5–0.63 McFarl and turbidity units using a turbidity meter (VITEK®2 DensiCHEK™, France). The biofilm suspension was incubated in a vacuum chamber station with data collected at different time intervals to measure suspension turbidity and/or by-products from donor substrate metabolism. Finally, raw data were processed using a special algorithm to eliminate false readings.