Biohydrogen (H2) is an efficient form of renewable energy generated from various biological organisms. Specifically, primitive plants such as algae which are photosynthetic organisms can produce several commercial products, including biofuels due to their simple form, short life span, efficient photosynthetic capacity, and ability to grow in non-potable water sources. But these algae are often neglected and considered waste. Several studies have revealed the importance and role of algal species in generating biofuels, especially biohydrogen. Considerable research has been conducted in order to understand hydrogen production from algal sources. This review emphasizes the photolysis of water-based hydrogen production in algae apart from the metabolites fermentation process. The influence of physico-chemical factors, including oxygen scavengers, nanoparticles, and hydrogenases, was highlighted in this review to enhance H2 production from algal species. Also, several algal species used for hydrogen production are summarized in detail. Overall, this review intends to summarize the developments in hydrogen production from algal species keeping in view of excellent prospects. This knowledge certainly would provide a good opportunity for the industrial production of hydrogen using algal species, which is one of the most concerned areas in the energy sector.

The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H2). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O2 sensitivity, binding specificity, or H2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS]4 linker group (CbHydA1-PsaE). We demonstrate that the CbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H2 at a rate of 84.9 ± 3.1 µmol H2 mgchl-1 h-1. Further, when prepared and illuminated in the presence of O2, the nanoconstruct retains the ability to generate H2, though at a diminished rate of 2.2 ± 0.5 µmol H2 mgchl-1 h-1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O2-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H2 generating system that can function in the presence of O2.

[NiFe] hydrogenases catalyze the reversible cleavage of molecular hydrogen into protons and electrons. Here, we have studied the impact of temperature and illumination on an oxygen-tolerant and thermostable [NiFe] hydrogenase by IR and EPR spectroscopy. Equilibrium mixtures of two catalytic [NiFe] states, Nia-C and Nia-SR\'\', were found to drastically change with temperature, indicating a thermal exchange of electrons between the [NiFe] active site and iron-sulfur clusters of the enzyme. In addition, IR and EPR experiments performed under illumination revealed an unusual photochemical response of the enzyme. Nia-SR\'\', a fully reduced hydride intermediate of the catalytic cycle, was found to be reversibly photoconverted into another catalytic state, Nia-L. In contrast to the well-known photolysis of the more oxidized hydride intermediate Nia-C, photoconversion of Nia-SR\'\' into Nia-L is an active-site redox reaction that involves light-driven electron transfer towards the enzyme\'s iron-sulfur clusters. Omitting the ground-state intermediate Nia-C, this direct interconversion of these two states represents a potential photochemical shortcut of the catalytic cycle that integrates multiple redox sites of the enzyme. In total, our findings reveal the non-local redistribution of electrons via thermal and photochemical reaction channels and the potential of accelerating or controlling [NiFe] hydrogenases by light.

In an effort to develop the biomimetic chemistry of [FeFe]‑hydrogenases for catalytic hydrogen evolution reaction (HER) in aqueous environment, we herein report the integrations of diiron dithiolate complexes into carbon nanotubes (CNTs) through three different strategies and compare the electrochemical HER performances of the as-resulted 2Fe2S/CNT hybrids in neutral aqueous medium. That is, three new diiron dithiolate complexes [{(μ-SCH2)2N(C6H4CH2C(O)R)}Fe2(CO)6] (R = N-oxylphthalimide (1), NHCH2pyrene (2), and NHCH2Ph (3)) were prepared and could be further grafted covalently to CNTs via an amide bond (this 2Fe2S/CNT hybrid is labeled as H1) as well as immobilized noncovalently to CNTs via π-π stacking interaction (H2) or via simple physisorption (H3). Meanwhile, the molecular structures of 1-3 are determined by elemental analysis and spectroscopic as well as crystallographic techniques, whereas the structures and morphologies of H1-H3 are characterized by various spectroscopies and scanning electronic microscopy. Further, the electrocatalytic HER activity trend of H1 > H2 ≈ H3 is observed in 0.1 M phosphate buffer solution (pH = 7) through different electrochemical measurements, whereas the degradation processes of H1-H3 lead to their electrocatalytic deactivation in the long-term electrolysis as proposed by post operando analysis. Thus, this work is significant to extend the potential application of carbon electrode materials engineered with diiron molecular complexes as heterogeneous HER electrocatalysts for water splitting to hydrogen.

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 μmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.

In the green alga Chlamydomonas reinhardtii, hydrogenase HydA1 converts protons and electrons to H2 at the H-cluster, which includes a [4Fe-4S] cluster linked to a [2Fe] cluster. The yield of H2 is limited by the electron transfer to HydA1, mediated by the iron-sulfur unit of a photosynthetic electron transfer ferredoxin (PetF). In this study, I have investigated by molecular dynamics and the hybrid quantum mechanics/molecular mechanics method two canonical iron-sulfur peptides (PM1 and FBM) that hold potential as PetF replacements. Using a docking approach, I predict that the distance between the two iron-sulfur clusters in FBM/HydA1 is shorter than in PM1/HydA1, ensuring a greater electron transfer rate. This finding is in line with the reported higher H2 production rates for FBM/HydA1. I also show that the redox potential of these peptides, and therefore their electron transfer properties, can be changed by single-residue mutations in the secondary coordination sphere of their cluster. In particular, I have designed a PM1 variant that disrupts the hydrogen-bonding network between water and the cluster, shifting the redox potential negatively compared to PM1. These results will guide experiments aimed at replacing PetF with peptides that can unlock the biotechnological potential of the alga.

[FeFe]-hydrogenase is nature\'s most efficient proton reducing and H2-oxidizing enzyme. However, biotechnological applications are hampered by the O2 sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe2(adt)(CO)6-x(CN)x]x- and [Fe2(pdt)(CO)6-x(CN)x]x- (x = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy. Additionally, we describe a straightforward synthetic recipe for the active site precursor complex Fe2(adt)(CO)6. Our data indicate that the aminodithiolate (adt) complex, which is the synthetic precursor of the natural active site cofactor, is most oxygen sensitive. This observation highlights the significance of proton transfer in aerobic deactivation, and supported by DFT calculations facilitates an identification of the responsible reactive oxygen species (ROS). Moreover, we show that the ligand environment of the iron ions critically influences the reactivity with O2 and ROS like superoxide and H2O2 as the oxygen sensitivity increases with the exchange of ligands from CO to CN-. The trends in aerobic deactivation observed for the model complexes are in line with the respective enzyme variants. Based on experimental and computational data, a model for the initial reaction of [FeFe]-hydrogenase with O2 is developed. Our study underscores the relevance of model systems in understanding biocatalysis and validates their potential as important tools for elucidating the chemistry of oxygen-induced deactivation of [FeFe]-hydrogenase.

ConspectusNature\'s prototypical hydrogen-forming catalysts─hydrogenases─have attracted much attention because they catalyze hydrogen evolution at near zero overpotential and ambient conditions. Beyond any possible applications in the energy sphere, the hydrogenases feature complicated active sites, which implies novel biosynthetic pathways. In terms of the variety of cofactors, the [FeFe]-hydrogenase is among the most complex.For more than a decade, we have worked on the biosynthesis of the active site of [FeFe] hydrogenases. This site, the H-cluster, is a six-iron ensemble consisting of a [4Fe-4S]H cluster linked to a [2Fe]H cluster that is coordinated to CO, cyanide, and a unique organic azadithiolate ligand. Many years ago, three enzymes, namely, HydG, HydE, and HydF, were shown to be required for the biosynthesis and the in vitro maturation of [FeFe] hydrogenases. The structures of the maturases were determined crystallographically, but still little progress was made on the biosynthetic pathway. As described in this Account, the elucidation of the biosynthetic pathway began in earnest with the identification of a molecular iron-cysteinate complex produced within HydG.In this Account, we present our most recent progress toward the molecular mechanism of [2Fe]H biosynthesis using a collaborative approach involving cell-free biosynthesis, isotope and element-sensitive spectroscopies, as well as inorganic synthesis of purported biosynthetic intermediates. Our study starts from the radical SAM enzyme HydG that lyses tyrosine into CO and cyanide and forms an Fe(CO)2(CN)-containing species. Crystallographic identification of a unique auxiliary 5Fe-4S cluster in HydG leads to a proposed catalytic cycle in which a free cysteine-chelated \"dangler\" Fe serves as the platform for the stepwise formation of a [4Fe-4S][Fe(CO)(CN)(cysteinate)] intermediate, which releases the [Fe(CO)2(CN)(cysteinate)] product, Complex B. Since Complex B is unstable, we applied synthetic organometallic chemistry to make an analogue, syn-B, and showed that it fully replaces HydG in the in vitro maturation of the H-cluster. Syn-B serves as the substrate for the next radical SAM enzyme HydE, where the low-spin Fe(II) center is activated by 5\'-dAdo• to form an adenosylated Fe(I) intermediate. We propose that this Fe(I) species strips the carbon backbone and dimerizes in HydE to form a [Fe2(SH)2(CO)4(CN)2]2- product. This mechanistic scenario is supported by the use of a synthetic version of this dimer complex, syn-dimer, which allows for the formation of active hydrogenase with only the HydF maturase. Further application of this semisynthesis strategy shows that an [Fe2(SCH2NH2)2(CO)4(CN)2]2- complex can activate the apo hydrogenase, marking it as the last biosynthetic intermediate en route to the H-cluster. This combined enzymatic and semisynthetic approach greatly accelerates our understanding of H-cluster biosynthesis. We anticipate additional mechanistic details regarding H-cluster biosynthesis to be gleaned, and this methodology may be further applied in the study of other complex metallocofactors.

Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to products such as acetate and biofuels. The model acetogen Acetobacterium woodii can grow on CO2, formate or methanol, but not on carbon monoxide, an important industrial waste product. Since hydrogenases are targets of CO inhibition, here, we genetically delete the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. We show that the ∆hydBA/hydA2 mutant indeed grows on CO and produces acetate, but only after a long adaptation period. SNP analyzes of CO-adapted cells reveal a mutation in the HycB2 subunit of the HydA2/HydB2/HydB3/Fdh-containing hydrogen-dependent CO2 reductase (HDCR). We observe an increase in ferredoxin-dependent CO2 reduction and vice versa by the HDCR in the absence of the HydA2 module and speculate that this is caused by the mutation in HycB2. In addition, the CO-adapted ∆hydBA/hydA2 mutant growing on formate has a final biomass twice of that of the wild type.

Pulsed light illumination stands out as a noteworthy technique for photosynthetic H2 production, playing a crucial role in eliminating O2 and activating hydrogenase enzymes. However, further improvements are essential to make H2 photoproduction suitable for future commercial applications. In our study, we observed a distinct enhancement in pulsed light-induced H2 photoproduction in the unicellular green alga Chlamydomonas reinhardtii when treated with the optimal concentration of the mild O2 scavenger Na2SO3. This improvement was a result of reduced O2 content, increased hydrogenase enzyme activity, and suppressed H2-uptake activity. Furthermore, our findings indicate that exposing Na2SO3-treated C. reinhardtii to optimal light waveform continues to significantly boost pulsed light-induced H2 photoproduction, attributed to the alleviation of impaired photosystem II activity. Altogether, the combined application of optimal sulfite concentration and light waveform effectively enhances pulsed light-induced photosynthetic H2 production in the green alga C. reinhardtii.