Photocatalytic Hydrogen Production using Polymeric Carbon Nitride with a Hydrogenase and a Bioinspired Synthetic Ni Catalyst

Solar-light-driven H2 production in water with a [NiFeSe]-hydrogenase (H2ase) and a bioinspired synthetic nickel catalyst (NiP) in combination with a heptazine carbon nitride polymer, melon (CNx), is reported. The semibiological and purely synthetic systems show catalytic activity during solar light irradiation with turnover numbers (TONs) of more than 50 000 mol H2 (mol H2ase)−1 and approximately 155 mol H2 (mol NiP)−1 in redox-mediator-free aqueous solution at pH 6 and 4.5, respectively. Both systems maintained a reduced photoactivity under UV-free solar light irradiation (λ>420 nm).


Experimental Section
Materials. Chemicals were purchased from commercial suppliers and used without further purification. The [NiFeSe]-hydrogenase from Desulfomicrobium baculatum (Dmb [NiFeSe]-H 2 ase) was purified under air by a previously published method. [1] The pure enzyme was dialyzed against 20 mM Tris/HCl, pH 7.6. The enzyme integrity was verified by measuring its specific activity with an aliquot of the H 2 ase under H 2 in the presence of 1 mM methylviologen (MV) for 30 min at 30 °C. H 2 oxidation activity was determined spectrophotometrically at 604 nm by following the color change of oxidized MV in a hydrogen-saturated solution, after adding enzyme. The preparation has a specific activity of 2115 µmol H 2 min -1 mg -1 , [2] and the stock enzyme solution was diluted with an aqueous TEOA solution (0.1 M, pH 7) before photocatalytic experiments in an anaerobic glovebox. CN x (melon) [3] and NiP [4] were synthesized as previously described. Reagents for the analytical part of the work were of the highest available purity.

Photocatalysis Experiments.
A standard photocatalytic experimental set-up was used as follows: Melon (CN x ) was added to a borosilicate glass tube containing a magnetic stir bar (total volume 7.74 mL). An aqueous solution of electron donor (usually 0.1 M) was then added along with the catalyst being used (NiP), sealed with a small septum. The suspension was then sonicated for 20 min under air. The suspension was then purged for 20 min with 2% CH 4 in N 2 . In the case of hydrogenase experiments, the enzyme was added after purging and then the vial was purged with 2% CH 4 in N 2 for an additional 5 min.
The vials were then placed in a water-jacketed rack and irradiated with 1 Sun using a Whalogen lamp (Newport Oriel Solar Light Simulator, 1000 W, 100 mW cm -2 ) with an air mass 1.5 global (AM 1.5G) filter in the absence or presence of a 420 nm UV-broad band filter (UQG Optics), with stirring. Headspace gases were sampled using Hamilton air-tight syringes by injecting 20 µL into the gas chromatograph (Agilent 7890A Series GC equipped with a 5 Å molecular sieve column; oven held at 45 °C) at regular intervals. H 2 produced was quantified by comparison to the CH 4 internal standard and each measurement was carried out in triplicate.
Centrifugation Experiments. Photocatalytic experiments were set up under standard conditions and were irradiated with 1 Sun for 2 h and H 2 production monitored by GC. The suspension was then transferred (in air) to centrifuge tubes and spun down for 5 min (5000 rpm). The supernatant was decanted and the pellet washed with distilled water (3 mL). The sample was centrifuged again for 5 min (5000 rpm) and supernatant decanted. The melon pellet was resuspended in 0.1 M EDTA (NiP, pH 4.5; H 2 ase pH 6) and vortexed for 2 min.
The suspension was returned to the borosilicate glass vials, sealed and purged with 2%

Photoluminescence Experiments.
A vial containing 2 mg of CN x in 0.1 M EDTA (pH 6) was sonicated for 20 min. The photoluminescence emission spectrum of the suspension was measured on a Agilent Technologies Cary Fluorescence Spectrophotometer with excitation at 365 nm. The cuvette was sonicated for 1 min to prevent aggregation and settling of the CN x between measurements, then either MV (26 and 50 pmol) or H 2 ase (33 and 50 pmol) was added and the cuvette shaken prior to measurements.

Treatment of Data.
All analytical measurements were performed in triplicate. The data were treated as follows: for a sample of n observations x i , the unweighted mean value x 0 and the standard deviation σ were calculated using the equations A minimum σ of 10% was assumed for all experiments even where triplicate runs gave a weighted mean of less than 10%. The solar light source and the gas chromatograph were calibrated regularly to ensure reproducibility.

S3
Calculation of External Quantum Efficiency (EQE). Hydrogen generation was driven by blue light (λ = 460 nm) of intensity I = 3.5 mW cm -2 and UV light (λ = 365 nm) of intensity I = 47 mW cm -2 at 25 °C from an LED light source controlled by a CompactStat, IVIUM potentiostat. The EQE can be calculated with the following formula: Where n H2 is the moles of H 2 photo-generated, N A is the Avogardo constant, h is the Planck constant, c is the speed of light, t irr is the irradiation time, A is the irradiated area of the photoreactor.
Characterization Details of CN x . Diffuse reflectance UV-Vis spectra were collected on a Cary 5000 spectrometer (referenced to PTFE or barium sulfate) and the spectra in percentage reflectance were converted using the Kubelka Munk function. X-ray diffraction patterns were collected using a STOE Stadi P diffractometer (Cu K α1 ) in transmission mode. ATR-IR spectra were collected with a PerkinElmer UATR TWO spectrometer equipped with a diamond crystal. Surface areas were calculated using Brunauer-Emmett-Teller (BET) theory from the adsorption isotherms of the samples. Samples were heated for 6-12 h overnight at 100 °C to a vacuum of 10 -7 mbar. Isotherms were collected on a Quantachrome Autosorb iQ gas sorption analyzer using argon as the sorbent at 87.45 K.
Multipoint BET calculations were carried with the BET Assistant in the ASiQwin software, using data points from the argon adsorption isotherm at or below the maximum in V·(1 P/P 0 ) in accordance with the ISO recommendations. All characterization data was compared to previously reported data. [3] Scanning electron microscopy (SEM) was performed on a Vega TS 5130MM (Tescan) microscope. Sample was deposited onto a carbon tab (Leco) and sputtered with gold for imaging. Zeta potential was measured using a Malvern Zetasizer Nano ZS. Sample was dispersed with sonication in NaCl solution (10 mM) of different pH (adjusted with HCl or NaOH) and allowed to stand prior to measurements in disposable cuvettes (Malvern). Measurements were conducted as four replicates; average results were quoted using the standard deviation as the error. *Vials were placed 10 cm closer to the light source thus may have been irradiated with a higher intensity of light than that used in standard experiments, thus a control with no filter was also measured. These measurements were performed in duplicate.  Table S4. Photocatalytic H 2 production using CN x (5 mg) in aqueous EDTA (0.1 M, pH 4.5) solution with NiP (20 nmol) under standard conditions with the addition of neutral density filters (50% absorbance, 80% absorbance and no filter).  Figure S1. FT-IR spectrum of CN x . Figure S2. XRD pattern of CN x . S10 Figure S3. SEM image of CN x . Figure S4. Determination of the isoelectric point of CN x using a zeta potential vs pH plot. The isoelectric point was found to be at around 3.3, which is in broad agreement with the value of 4.1 measured in a previous publication. [6]