Hydrogenase as the basis for green hydrogen production and utilization

Hydrogenase is a paradigm of highly efficient biocatalyst for H_(2) production and utilization evolved in nature. A dilemma is that despite the high activity and efficiency expected for hydrogenases as promising catalysts for the hydrogen economy, the poor oxygen tolerance and low yield of hydrogenases largely hinder their practical application. In these years, the enigmas surrounding hydrogenases regarding their structures, oxygen tolerance, mechanisms for catalysis, redox intermediates, and proton-coupled electron transfer schemes have been gradually elucidated;the schemes, which can well couple hydrogenases with other highly efficient(in)organic and biological catalysts to build novel reactors and drive valuable reactions, make it possible for hydrogenases to find their niches. To see how scientists put efforts to tackle this issue and design novel reactors in the fields where hydrogenases play crucial roles, in this review,recent advances were summarized, including different strategies for protecting enzyme molecules from oxygen, enzyme-based assembling systems for H_(2) evolution in the photoelectronic catalysis, enzymatic biofuel cells for H_(2) utilization and storage and the efficient electricity-hydrogen-carbohydrate cycle for high-purity hydrogen and biofuel automobiles. Limitations and future perspectives of hydrogenasebased applications in H_(2) production and utilization with great impact are discussed. In addition, this review also provides a new perspective on the use of biohydrogen in healthcare beyond energy.

ISOLATION, PURIFICATION AND CHARACTERIZATION OF THE HYDROGEN EVOLUTION PROMOTING FACTOR OF HYDROGENASE OF SPIRULINA PLATENSIS

A component （s-factor） with obvious promoting effect on hydrogen evolution of hydrogenase has beenisolated and extracted from a Cell=free preparation of Spirulina platensis.The effect of the s-factor in the re-action system is similar to that of Na2S2O4, but is coupled with ligh. The s-factor has the maximumabsorption peak at 620 nm in the oxidized state, at 590 nm in the reduced state. The partially purifieds-factor showed two bands by SDS-PAGE and is distinctly different from phycocyanin,which has nochange of oxidized state and reduced state absorption spectra, and also has no promoting effect onhydrogenase of Spirulina platensis under the light.

Photoinduced hydrogen evolution in an artificial system containing photosystem I, hydrogenase, methy1 viologen and mercaptoacetic acid

Hydrogen evolution was detected in an artificial system composed of light-harvesting unit of purified photosystem I, catalyst of hydrogenase, methyl viologen and electron donor under radiation. Absorption spectral features confirmed that electron transfer from electron donors to proton was via a photoinduced reductive process of methyl viologen.

Optimization of detecting hydrogenase activity for acidogenic fermentation of activated sludge

In order to evaluate the hydrogen-producing efficiency of anaerobic activated sludge in Anaerobic Baffled Reactor(ABR)fermentation processes,the optimal conditions for hydrogen producing hydrogenase method on methyl viologen(MV)assay was used to detect the hydrogen production activity of the activated sludge.The most favorable parameters such as 0.6 mL sodium acetate buffer(pH 5.0),100 μL lysozyme,0.2 mL sodium dibromoethane(9.0 mmol/L)and 0.7 mmol/L iron added into 1 mL activated sludge(2.66～26.64 gMLVSS/L)were found.Furthermore,reaction temperature and culture time were detected as 40 ℃ and 30 min respectively.Sodium thiosulfate and sodium sulfides were taken as the reducing agent while trichloroacetic acid as terminator.Under the MV optimal conditions,micro-toxic Dimethyl sulfoxide(DMSO)get higher security and better accuracy.The sensitivity of the detection methods(DMSO as electron carrier)was increased by more than 30%.The results show that the optimal conditions can be applied to measure hydrogenase activity correlating with its specific hydrogen production rate in a hydrogen-producing anaerobic activated sludge system.

Comparative evaluation of different cell disruption methods for the release of hydrogenase from H2 -producing bacterium E. harbinenase YUAN-3T

A comparative evaluation of three different cell-disruption methods for the release of hydrogenase from H 2-producing bacterium E.harbinenase YUAN-3 T was investigated.The cell disruption techniques evaluated in this study were ultrasonication,high-speed homogenization and bead milling.Ultrasonication process was found to be the most effective method in terms of cell disruption.As for the specific activity of hydrogenase,there is no significant difference among the three kinds of methods.An orthogonal experiment L 9 (3 4) was designed to optimize the procedures of ultrasonication for cell disruption.The optimized ultrasonication disruption conditions were the treatment at 250 W,20 kHz,30 s /15 s and 0.30 g bacteria cell (dry weight) in 15 mL suspension buffer.As a result,the optimized conditions allow the hydrogenase to maintain the active form with the yield of 93.95 mg protein /g cell and the final activity of 0.252 μmol /min /mg protein.In this work,we have developed and optimized an ultrasonication protocol for YUAN-3 T cells,which is adapted to laboratoryscale release of hydrogenase proteins.

Synthesis and Crystal Structure of a Model for the Active Sites of Fe-only Hydrogenases:[Fe_2(SCH_2)_2N(3-PhCF_3)(CO)_6]_2

A model compound for the active sites of Fe-only hydrogenases, [Fe2（SCH2）2N（3- PhCF3）（CO）6]2, has been synthesized and structurally characterized by single-crystal X-ray diffraction. It crystallizes in tetragonal, space group P43, with α = 12.6324（3）, b = 12.6324（3）, c = 24.0453（12） A, V = 3837.1（2） A^3, Z= 4, Fe4S4N2C30O12F6H16, Mr= 1062.09, Dc= 1.839 g/cm^3, μ（MoKα） = 1.791 mm^-1, F（000） = 2112, T= 293（2） K, Flack = 0.034（9）, R = 0.0282 and wR = 0.0685 for 8148 observed reflections with I 〉 20（I）. In the title compound, each Fet atom is coordinated by three terminal carbonyl C atoms （Fe-C： 1.783（3）- 1.816（3） A）, two bridging S atoms （Fe-S： 2.2609（7）-2.2757（8） A） and another Fe atom （Fe-Fe 2.5011（5） A）, adopting a distorted octahedral geometry with trans angles ranging from 152.45（11） to 157.77（10）°.

Five-heterocyclic-biphosphine-substituted Fe-only Hydrogenase Mimic： Synthesis,Characterization and Properties

A new five-heterocyclic-biphosphine-substituted Fe-only hydrogenase mimic,[（μ-pdt）Fe_2（CO）_5]_2（PTP）（1）,has been synthesized at room temperature. 1·H_2O crystallizes in triclinic system,space group P1,with a = 11.5897（4）,b = 13.6156（4）,c = 18.0333（6） ？,α = 76.306（3）,β = 72.742（3）,γ = 68.939（3）°,V = 2508.84（14） ？~3,Dc = 1.570 g/cm3,Z = 2,M_r = 1186.37,F（000） = 1204,the final R = 0.0748,and wR = 0.2012. In the tetranuclear complex 1·H_2O,each [2Fe_2S] butterfly unit is attached to one P atom of the diphosphine bridge and exhibits a square-pyramidal geometry. Complex 1 was characterized by elemental analysis,IR spectra,UV-vis absorption spectra,~1H-NMR and ^（31）P-NMR. The cyclic voltammetry behavior of compound 1 was investigated as well.

浮动海上有机农场——Hydrogenase

浮动有机农场Hydrogenase外形看起来如同丝网编织的精美花边,是一个真正的净化站.位于4个碳回收塔内的绿色海藻将回收船舶带来的碳废物,可以直接用于这座"飞艇"形的有机农场的生物制氢,相当于传统飞机跑道边的加油站.

A functional hydrogenase mimic that catalyzes robust H_(2) evolution spontaneously in aqueous environment

Although great progress has been made in improving hydrogen production,highly efficient catalysts,which are able to produce hydrogen in a fast and steady way at ambient temperature and pressure,are still in large demand.Here,we report a[NiCo]-based hydrogenase mimic,NiCo_(2)O_(4) nanozyme,that can catalyze robust hydrogen evolution spontaneously in water without external energy input at room temperature.This hydrogenase nanozyme facilitates water splitting reaction by forming a three-center Ni-OH-Co bond analogous to the[NiFe]-hydrogenase reaction by using aluminum as electron donor,and realizes hydrogen evolution with a high production rate of 915 L·h^(-1) per gram of nanozymes,which is hundreds of times higher than most of the natural hydrogenase or hydrogenase mimics.Furthermore,the NiCo_(2)O_(4) nanozyme can robustly disrupt the adhesive oxidized layer of aluminum and enable the full consumption of electrons from aluminum.In contrast to the often-expensive synthetic catalysts that rely on rare elements and consume high energy,we envision that this NiCo_(2)O_(4) nanozyme can potentially provide an upgrade for current hydrogen evolution,accelerate the development of scale-up hydrogen production,and generate a clean energy future.

Immobilizing [FeFe]-hydrogenase mimics to metal–organic frameworks for enhanced hydrogen production

Production of hydrogen from water by solar-energy conversion has long been considered a promising way to solve the climate change and energy crisis(1)However,some critical issues at this stage,such as catalysts for hydrogen evolution with high efficiency and low cost,definitely hinder the practical application of photocatalytic hydrogen production from water.[Fe Fe]-hydrogenase,an excellent natural biological enzyme catalyst bearing unique organometallic clusters with noble-metal-free element,is most efficient in reducing protons to hydrogen and demonstrates remarkable turnover frequencies(TOF 6000–9000 s1 per active site)[2].

An[Fe-Fe]-Hydrogenase Mimic Immobilized on MCM-41 for the Photochemical Production of Hydrogen in Pure Water

A composite proton-reducing catalyst Hy@MCM-41 was created by using a hydrophobic[Fe-Fe]-hydrogenase mimic(Hy)incorporated into the K^(+)-exchanged molecular sieve MCM-41.Hy acts as the active site to generate H_(2) by reducing H^(+),and MCM-41 provide a large surface area to maintain the dispersion of Hy and to prevent aggrega-tion and precipitation.Hy@MCM-41 was successfully applied to the light-induced hydrogen production in pure water with an iridium compound[Ir(ppy)_(2)bpy]Cl and triethylamine(TEA)as the photosensitizer and the sacrificial electron donor,respectively,exhibiting good stability and catalytic activity.The present study provides a general strategy for the application of water-insoluble catalysts in pure water by using mesoporous molecular sieves.

Synthesis, structure and electrocatalytic H2-evoluting activity of a dinickel model complex related to the active site of [NiFe]-hydrogenases

Structural and functional biomimicking of the active site of [NiFe]-hydrogenases can provide helpful hints for designing bioinspired catalysts to replace the expensive noble metal catalysts for H2 generation and uptake.Treatment of dianion [Ni(phma)]2-[H4 phma=N,N’-1,2-phenylenebis(2-mercaptoacetamide)] with [NiCl2(dppp)](dppp=bis(diphenylphosphino)propane) yielded a dinickel product[Ni(phma)(μ-S,S’)Ni(dppp)](1) as the model complex relevant to the active site of [NiFe]-H2 ases.The structure of complex 1 has been characterized by single-crystal X-ray analysis.From cyclic voltammetry and controlled potential electrolysis studies,complex 1 was found to be a moderate electrocatalyst for the H2-evoluting reaction using ClCH2COOH as the proton source.

Challenge of new biological energy resources——Arguments on structure of active site of NiFe hydrogenase

Hydrogenases are enzymes that can reversibly split molecular hydrogen. Study on the structure of the active site and the mechanism of catalysis has drawn great attention because the results may be useful for the design of cheap biomimetic hydrogen catalysts for fuel cells, or as model for the photoproduction of H\-2. At one time the active site was generally considered to be composed of mononuclear nickel complex with ligands from the polypeptide. A breakthrough in the understanding of the structure of Hases occurred with the resolution crystal structure of D. gigas Hases in 1995. The unexpected result challenged the previously reported spectroscopic studies and caused some academic arguments. Some methods and results used for insight into Hases have to be reconsidered. Different viewpoints concerning the structure of active site of Hases in different periods and some remaining questions will be presented.

Peptide self‐assembly as a strategy for facile immobilization of redox enzymes on carbon electrodes

Redox-enzyme‐mediated electrochemical processes such as hydrogen production,nitrogen fixation,and CO_(2) reduction are at the forefront of the green chemistry revolution.To scale up,the inefficient two‐dimensional(2D)immobilization of redox enzymes on working electrodes must be replaced by an efficient dense 3D system.Fabrication of 3D electrodes was demonstrated by embedding enzymes in polymer matrices.However,several requirements,such as simple immobilization,prolonged stability,and resistance to enzyme leakage,still need to be addressed.The study presented here aims to overcome these gaps by immobilizing enzymes in a supramolecular hydrogel formed by the self‐assembly of the peptide hydrogelator fluorenylmethyloxycarbonyldiphenylalanine.Harnessing the self‐assembly process avoids the need for tedious and potentially harmful chemistry,allowing the rapid loading of enzymes on a 3D electrode under mild conditions.Using the[FeFe]hydrogenase enzyme,high enzyme loads,prolonged resistance against electrophoresis,and highly efficient hydrogen production are demonstrated.Further,this enzyme retention is shown to arise from its interaction with the peptide nanofibrils.Finally,this method is successfully used to retain other redox enzymes,paving the way for a variety of enzyme‐mediated electrochemical applications.

Plant growth promoting H₂-oxidizing bacteria as seed inoculants for cereal crops

The long-term success of hydrogenase uptake negative legume-rhizobia associations, in spite of their apparent inefficiency, may be explained by the positive effects of H2 release to soil. A primary benefit of H2 release to soil is the stimulation of H2-oxidizing, plant growth promoting rhizobacteria (PGPR) [1]. Two such previously isolated strains were tested as seed inoculants for barley and spring wheat;there were significant differences between treatments and controls in tiller and grain head production, supported by data from greenhouse trials. T-RFLP analysis of barley soil samples, supported by DNA sequencing data, successfully distinguished both species inoculated. Successful re-isolation indicates that these isolates can reproduce themselves in soils and can be used as effective inoculants with peat as the standard carrier. This study showed that we are able to achieve some of the beneficial effects of crop rotation without the need to implement actual crop rotation.

Microbial Water Source for the Desert Plants

Finding water resources for the desert plants is one of the important research areas since it enables saving water resources.Bromus inermis plant was noticed to keep 5%of moisture in its rhizosphere zone;therefore,this study aimed to identify this source of water.Thirteen endophytic bacteria were isolated from the root of B.inermis and identified.Their specific respiration rate was determined.Alcaligenes faecalis showed the highest specific respiration rate.It is a facultative chemoautotrophic hydrogen-fixing bacterium that utilizes the hydrogen gas as energy source and the water is produced as an end product.The source of hydrogen gas for this bacterium is not only from air and soil gases but also from the hydrogen-producing bacteria such as Enterobacter spp.,which was among the isolated bacteria.The hydrogenases synthesizing genes(HoxC,HypA and HypB)were detected in most of the isolated bacteria and roots of four wild plants,out from 18 wild plant samples,epically the grains of the wild wheat plant.This result suggests that the hydrogen-fixing and hydrogen-producing bacteria transfer from the root through the plant to inhabit the grain/seeds.This can help the grain/seeds to germinate in drought environment.

Syntheses, Characterization and X-ray Crystal Structures of Diiron Ethanedithiolate Complexes Fe_2(S_2C_2H_4 )(CO)_5 (2-Ph_2PC_6H_4NH_2 ) and Fe_2 (S_2C_2H_4 )(CO)_5 (2-Ph_2PC_6H_4CH_2NH_2)

Two new diiron ethanedithiolate complexes Fe2（S2C2H4）（CO）5（2-Ph2PC6H4NH2） （1） and Fez（S2CzH4）（CO）5（2-PhzPC6H4CHzNH2） （2） as active site models of [FeFe] hydrogenases have been prepared by the treatment of （,u-SCHzCH2S-μ）Fe2（CO）6 with 2-PhzPC6H4NH2 or 2-PhzPC6H4CH2NH2 in the presence of the decarbonylating agent Me3NO＇2H20. As new complexes, both 1 and 2 were fully characterized by elemental analysis, IR and^1H （13C, 31p） NMR spectroscopies. In addition, the molecular structure of complex 1 was established by X-ray crystallography. The crystal of Fe2（S2C2H4）（CO）5（2-PhzPC6H4NH2） （1） crystallizes in orthorhombic, spacegroup Pna21 with a = 20.9461（17）, b = 13.7615（11）, c = 9.3133（7）A, V= 2684.6（4） A3, Z = 4, C25Hz0FezNOsPS2, Mr = 621.21, Dc = 1.537 g/cm^3, F（000） = 1264. The final R = 0.0197 and wR = 0.0495 for 4605 observed reflections with I 〉 2a（/） and R = 0.0206 and wR = 0.0501 for all data.

Genomic Analysis of <i>Anabaena variabilis</i>Mutants PK17 and PK84 That Are Characterised by High Production of Molecular Hydrogen

The use of cyanobacteria for producing molecular hydrogen is one of the desirable tasks of photobiotechnology. Some years ago, we isolated several chemically induced mutants of the cyanobacterium Anabaena variabilis ATCC 29413 that exhibited a high level of H2-production;but the genetic nature of these mutants remained unresolved. To reveal mutations that could be responsible for enhancement of H2-production in two independent mutants, PK17 and PK84, the pyrosequencing of their entire genomes was performed. The results were analyzed on the basis of comparison with the complete genome sequence of the reference strain Anabaena variabilis ATCC 29413. The genomes of mutants PK17 and RK84 contain 107 and 104 point deviations from the reference genome, respectively. The most probable reason for the increase of H2-production in mutant PK17 is the mutation identified in the gene hupL encoding the large subunit of uptake hydrogenase. A high level of H2-production in mutant PK84 could be the result of a mutation in a conserved part of the gene hypF, which participates in the post-translation maturation of hydrogenase complexes.

Oxide-supported metal catalysts for anaerobic NAD+regeneration with concurrent hydrogen production

We report SiO_(2)-supported monometallic Pt,Pd,Au,Ni,Cu and Co catalysts for proton-driven NAD+regeneration,co-producing H_(2).All metals are fully selective to NAD+where the order of turnover frequencies(Pt>Pd>Cu>Au,Ni and Co)coincides with those otherwise observed in electrochemical hydrogen evolution reactions.This has revealed that NADH is capable of converting the metal sites into a“cathode”without an external potential and the NADH to NAD+reaction involves transferring electron and hydrogen atom separately.Electron-deficient Ptδ+(on CeO_(2))enhances TOF and the heterogeneous Pt/CeO_(2) catalyst is recyclable without losing any activity/selectivity.

Prospects for engineering Ralstonia eutropha and Zymomonas mobilis for the autotrophic production of 2,3-butanediol from CO_(2)and H_(2)

The decarbonization of the chemical industry and a shift toward circular economies because of high global CO_(2) emissions make CO_(2) an attractive feedstock for manufacturing chemicals.Moreover,H_(2) is a low-cost and carbon-free reductant because technologies such as solar-driven electrolysis and supercritical water(scH_(2)O) gasification enable sustainable production of molecular hydrogen(H_(2)).We review the recent advances in engineering Ralsto-nia eutropha,the representative species of“Knallgas”bacteria,for utilizing CO_(2) and H_(2) to autotrophically produce 2,3-butanediol(2,3-BDO).This assessment is focused on state-of-the-art approaches for splitting H_(2) to supply en-ergy in the form of ATP and NADH to power cellular reactions and employing the Calvin-Benson-Bassham cycle for CO_(2) fixation.Major challenges and opportunities for application and future perspectives are discussed in the context of developing other promising CO_(2) and H_(2)-utilizing microorganisms,exemplified by Zymomonas mobilis.