Adaptation of a methanogen to Fe0 corrosion via direct contact.

Due to unique genomic adaptations, Methanococcus maripaludis Mic1c10 is highly corrosive when in direct contact with Fe0. A critical adaptation involves increased glycosylation of an extracellular [NiFe]-hydrogenase, facilitating its anchoring to cell surface proteins. Corrosive strains adapt to the constructed environment via horizontal gene transfer while retaining ancestral genes important for intraspecies competition and surface attachment. This calls for a reevaluation of how the built environment impacts methane cycling.

CyAbrB2 is a nucleoid-associated protein in Synechocystis controlling hydrogenase expression during fermentation.

The hox operon in Synechocystis sp. PCC 6803, encoding bidirectional hydrogenase responsible for H2 production, is transcriptionally upregulated under microoxic conditions. Although several regulators for hox transcription have been identified, their dynamics and higher-order DNA structure of hox region in microoxic conditions remain elusive. We focused on key regulators for the hox operon: cyAbrB2, a conserved regulator in cyanobacteria, and SigE, an alternative sigma factor. Chromatin immunoprecipitation sequencing revealed that cyAbrB2 binds to the hox promoter region under aerobic conditions, with its binding being flattened in microoxic conditions. Concurrently, SigE exhibited increased localization to the hox promoter under microoxic conditions. Genome-wide analysis revealed that cyAbrB2 binds broadly to AT-rich genome regions and represses gene expression. Moreover, we demonstrated the physical interactions of the hox promoter region with its distal genomic loci. Both the transition to microoxic conditions and the absence of cyAbrB2 influenced the chromosomal interaction. From these results, we propose that cyAbrB2 is a cyanobacterial nucleoid-associated protein (NAP), modulating chromosomal conformation, which blocks RNA polymerase from the hox promoter in aerobic conditions. We further infer that cyAbrB2, with altered localization pattern upon microoxic conditions, modifies chromosomal conformation in microoxic conditions, which allows SigE-containing RNA polymerase to access the hox promoter. The coordinated actions of this NAP and the alternative sigma factor are crucial for the proper hox expression in microoxic conditions. Our results highlight the impact of cyanobacterial chromosome conformation and NAPs on transcription, which have been insufficiently investigated.

Molecular detection and characterization of Trichomonas gallinae isolated from ornamental birds in Tehran, Iran.

Trichomonas gallinae is a widespread protozoan parasite that primarily affects birds, causing a disease known as avian trichomonosis. The present study aimed to investigate the prevalence and genetic diversity of T. gallinae, a parasite causing avian trichomoniasis in feral pigeons, budgerigars, and finches in Tehran, Iran. The 5.8S ribosomal RNA locus, along with the internal transcribed spacer 2 (ITS2) region, has been extensively utilized for genotype identification and for determining inter- and intra-specific diversity. More recently, the Fe-hydrogenase (Fe-Hyd) gene has been suggested as an additional genetic marker to enhance the accuracy of strain subtyping discrimination. In the present study, a total of 12% (12/100) birds examined were infected with T. gallinae using microscopy and PCR methods. Infection was found in seven of 30 (23.3%) feral pigeons, three of 40 (7.5%) budgerigars, and two of 30 (6.66%) finches. Analysis of the ITS2 region of T. gallinae isolates revealed two highly similar sequences. The first sequence (GenBank: OQ689964-OQ689970) was found in five feral pigeons and two budgerigars, whereas the second sequence (GenBank: OQ689971-OQ689975) was identified in two feral pigeons, one budgerigar, and two finches. Phylogenetic analysis confirmed the presence of two distinct clusters (cluster I and cluster II) within the trichomonads based on the ITS2 region. However, further analysis using Fe-Hyd revealed greater diversity, with three subtypes identified (A1, A2, and C1). One isolate identified in the present study (GenBank accession number: OQ694508.1) belonged to subtype A1. Combining ITS2 and Fe-Hyd markers holds promise for a more comprehensive understanding of the population structure of T. gallinae and the potential role of ITS2 in host adaptation.

Secondary structure changes as the potential H2 sensing mechanism of group D [FeFe]-hydrogenases.

[FeFe]-hydrogenases function as both H2 catalysts and sensors. While catalysis is well investigated, details regarding the H2 sensing mechanism are limited. Here, we relate protein structure changes to H2 sensing, similar to light-driven bio-sensors. Our results highlight how identical cofactors incorporated in alternative protein scaffolds serve different functions in nature.

Ethanol-type anaerobic digestion enhanced methanogenic performance by stimulating direct interspecies electron transfer and interspecies hydrogen transfer.

Ethanol pre-fermentation of food waste effectively alleviates acidification; however, its effects on interspecies electron transfer remain unknown. This study configured the feed according to COD ratios of ethanol: sodium acetate: sodium propionate: sodium butyrate of 5:2:1.5:1.5 (ethanol-type anaerobic digestion) and 0:5:2.5:2.5 (control), and conducted semi-continuous anaerobic digestion (AD) experiments. The results showed that ethanol-type AD increased maximum tolerable organic loading rate (OLR) to 6.0 gCOD/L/d, and increased the methane production by 1.2-14.8 times compared to the control at OLRs of 1.0-5.0 gCOD/L/d. The abundance of the pilA gene, which was associated with direct interspecies electron transfer (DIET), increased by 5.6 times during ethanol-type AD. Hydrogenase genes related to interspecies hydrogen transfer (IHT), including hydA-B, hoxH-Y, hnd, ech, and ehb, were upregulated during ethanol-type AD. Ethanol-type AD improved methanogenic performance and enhanced microbial metabolism by stimulating DIET and IHT.

Diverse non-canonical electron bifurcating [FeFe]-hydrogenases of separate evolutionary origins in Hydrogenedentota.

Hydrogenedentota, a globally distributed bacterial phylum-level lineage, is poorly understood. Here, we established a comprehensive genomic catalog of Hydrogenedentota, including a total of seven clades (or families) with 179 genomes, and explored the metabolic potential and evolutionary history of these organisms. We show that a single genome, especially those belonging to Clade 6, often encodes multiple hydrogenases with genomes in Clade 2, which rarely encode hydrogenases being the exception. Notably, most members of Hydrogenedentota contain a group A3 [FeFe]-hydrogenase (BfuABC) with a non-canonical electron bifurcation mechanism, in addition to substrate-level phosphorylation and electron transport-linked phosphorylation pathways, in energy conservation. Furthermore, we show that BfuABC from Hydrogenedentota fall into five sub-types. Phylogenetic analysis reveals five independent routes for the evolution of BfuABC homologs in Hydrogenedentota. We speculate that the five sub-types of BfuABC might be acquired from Bacillota (synonym Firmicutes) through separate horizontal gene transfer events. These data shed light on the diversity and evolution of bifurcating [FeFe]-hydrogenases and provide insight into the strategy of Hydrogenedentota to adapt to survival in various habitats. IMPORTANCE: The phylum Hydrogenedentota is widely distributed in various environments. However, their physiology, ecology, and evolutionary history remain unknown, primarily due to the limited availability of the genomes and the lack of cultured representatives of the phylum. Our results have increased the knowledge of the genetic and metabolic diversity of these organisms and shed light on their diverse energy conservation strategies, especially those involving electron bifurcation with a non-canonical mechanism, which are likely responsible for their wide distribution. Besides, the organization and phylogenetic relationships of gene clusters coding for BfuABC in Hydrogenedentota provide valuable clues to the evolutionary history of group A3 electron bifurcating [FeFe]-hydrogenases.

A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO2 and N2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Light-induced H2 generation in a photosystem I-O2-tolerant [FeFe] hydrogenase nanoconstruct.

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.

Ultrathin Film Antimony-Doped Tin Oxide Prevents [NiFe] Hydrogenase Inactivation at High Electrode Potentials.

For hydrogenases to serve as effective electrocatalysts in hydrogen biotechnological devices, such as enzymatic fuel cells, it is imperative to design electrodes that facilitate stable and functional enzyme immobilization, efficient substrate accessibility, and effective interfacial electron transfer. Recent years have seen considerable advancements in this area, particularly concerning hydrogenases. However, a significant limitation remains: the inactivation of hydrogenases at high oxidative potentials across most developed electrodes. Addressing this issue necessitates a thorough understanding of the interactions between the enzyme and the electrode surface. In this study, we employ ATR-IR spectroscopy combined with electrochemistry in situ to investigate the interaction mechanisms, electrocatalytic behavior, and stability of the oxygen-tolerant membrane-bound [NiFe] hydrogenase from Cupriavidus necator (MBH), which features a His-tag on its small subunit C-terminus. Antimony-doped tin oxide (ATO) thin films were selected as electrodes due to their protein compatibility, suitable potential window, conductivity, and transparency, making them an ideal platform for spectroelectrochemical measurements. Our comprehensive examination of the physiological and electrochemical processes of [NiFe] MBH on ATO thin film electrodes demonstrates that by tuning the electron transport properties of the ATO thin film, we can prevent MBH inactivation at extended oxidative potentials while maintaining direct electron transfer between the enzyme and the electrode.

Gene-centered metagenome analysis of Vulcano Island soil (Aeolian archipelago, Italy) reveals diverse microbial key players in methane, hydrogen and sulfur cycles.

The Aeolian archipelago is known worldwide for its volcanic activity and hydrothermal emissions, of mainly carbon dioxide and hydrogen sulfide. Hydrogen, methane, and carbon monoxide are minor components of these emissions which together can feed large quantities of bacteria and archaea that do contribute to the removal of these notorious greenhouse gases. Here we analyzed the metagenome of samples taken from the Levante bay on Vulcano Island, Italy. Using a gene-centric approach, the hydrothermal vent community appeared to be dominated by Proteobacteria, and Sulfurimonas was the most abundant genus. Metabolic reconstructions highlight a prominent role of formaldehyde oxidation and the reverse TCA cycle in carbon fixation. [NiFe]-hydrogenases seemed to constitute the preferred strategy to oxidize H2, indicating that besides H2S, H2 could be an essential electron donor in this system. Moreover, the sulfur cycle analysis showed a high abundance and diversity of sulfate reduction genes underpinning the H2S production. This study covers the diversity and metabolic potential of the microbial soil community in Levante bay and adds to our understanding of the biogeochemistry of volcanic ecosystems.

Covalent versus noncovalent attachments of [FeFe]­hydrogenase models onto carbon nanotubes for aqueous hydrogen evolution reaction.

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.

Oxygen sensitivity of [FeFe]-hydrogenase: a comparative study of active site mimics inside vs. outside the enzyme.

[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.

Engineering bionanoreactor in bacteria for efficient hydrogen production.

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.

Co-expression analysis reveals distinct alliances around two carbon fixation pathways in hydrothermal vent symbionts.

Most autotrophic organisms possess a single carbon fixation pathway. The chemoautotrophic symbionts of the hydrothermal vent tubeworm Riftia pachyptila, however, possess two functional pathways: the Calvin-Benson-Bassham (CBB) and the reductive tricarboxylic acid (rTCA) cycles. How these two pathways are coordinated is unknown. Here we measured net carbon fixation rates, transcriptional/metabolic responses and transcriptional co-expression patterns of Riftia pachyptila endosymbionts by incubating tubeworms collected from the East Pacific Rise at environmental pressures, temperature and geochemistry. Results showed that rTCA and CBB transcriptional patterns varied in response to different geochemical regimes and that each pathway is allied to specific metabolic processes; the rTCA is allied to hydrogenases and dissimilatory nitrate reduction, whereas the CBB is allied to sulfide oxidation and assimilatory nitrate reduction, suggesting distinctive yet complementary roles in metabolic function. Furthermore, our network analysis implicates the rTCA and a group 1e hydrogenase as key players in the physiological response to limitation of sulfide and oxygen. Net carbon fixation rates were also exemplary, and accordingly, we propose that co-activity of CBB and rTCA may be an adaptation for maintaining high carbon fixation rates, conferring a fitness advantage in dynamic vent environments.

Oxygen-resistant [FeFe]hydrogenases: new biocatalysis tools for clean energy and cascade reactions.

The use of enzymes to generate hydrogen, instead of using rare metal catalysts, is an exciting area of study in modern biochemistry and biotechnology, as well as biocatalysis driven by sustainable hydrogen. Thus far, the oxygen sensitivity of the fastest hydrogen-producing/exploiting enzymes, [FeFe]hydrogenases, has hindered their practical application, thereby restricting innovations mainly to their [NiFe]-based, albeit slower, counterparts. Recent exploration of the biodiversity of clostridial hydrogen-producing enzymes has yielded the isolation of representatives from a relatively understudied group. These enzymes possess an inherent defense mechanism against oxygen-induced damage. This discovery unveils fresh opportunities for applications such as electrode interfacing, biofuel cells, immobilization, and entrapment for enhanced stability in practical uses. Furthermore, it suggests potential combinations with cascade reactions for CO2 conversion or cofactor regeneration, like NADPH, facilitating product separation in biotechnological processes. This work provides an overview of this new class of biocatalysts, incorporating unpublished protein engineering strategies to further investigate the dynamic mechanism of oxygen protection and to address crucial details remaining elusive such as still unidentified switching hot-spots and their effects. Variants with improved kcat as well as chimeric versions with promising features to attain gain-of-function variants and applications in various biotechnological processes are also presented.

Mechanistic Principles of Hydrogen Evolution in the Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+ exchange across the membrane. Despite recent structural advances, the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L toward the [NiFe] site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the [NiFe] active site of Mbh to drive PCET reactions and ion transport.

Hydrogen production by a fully de novo enzyme.

Molecular catalysts based on abundant elements that function in neutral water represent an essential component of sustainable hydrogen production. Artificial hydrogenases based on protein-inorganic hybrids have emerged as an intriguing class of catalysts for this purpose. We have prepared a novel artificial hydrogenase based on cobaloxime bound to a de novo three alpha-helical protein, α3C, via a pyridyl-based unnatural amino acid. The functionalized de novo protein was characterised by UV-visible, CD, and EPR spectroscopy, as well as MALDI spectrometry, which confirmed the presence and ligation of cobaloxime to the protein. The new de novo enzyme produced hydrogen under electrochemical, photochemical and reductive chemical conditions in neutral water solution. A change in hydrogen evolution capability of the de novo enzyme compared with native cobaloxime was observed, with turnover numbers around 80% of that of cobaloxime, and hydrogen evolution rates of 40% of that of cobaloxime. We discuss these findings in the context of existing literature, how our study contributes important information about the functionality of cobaloximes as hydrogen evolving catalysts in protein environments, and the feasibility of using de novo proteins for development into artificial metalloenzymes. Small de novo proteins as enzyme scaffolds have the potential to function as upscalable bioinspired catalysts thanks to their efficient atom economy, and the findings presented here show that these types of novel enzymes are a possible product.

Redirecting electron flow in Acetobacterium woodii enables growth on CO and improves growth on formate.

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.

Minimal and hybrid hydrogenases are active from archaea.

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

Characterization and potentiality of plant-derived silver nanoparticles for enhancement of biomass and hydrogen production in Chlorella sp. under nitrogen deprived condition.

Energy is a crucial entity for the development and it has various alternative forms of energy sources. Recently, the synthesis of nanoparticles using benign biocatalyst has attracted increased attention. In this study, silver nanoparticles were synthesized and characterized using Azadirachta indica plant-derived phytochemical as the reducing agent. Biomass of the microalga Chlorella sp. cultivated in BG11 medium increased after exposure to low concentrations of up to 0.48 mg L-1 AgNPs. In addition, algal cells treated with 0.24 mg L-1 AgNPs and cultivated in BG110 medium which contained no nitrogen source showed the highest hydrogen yield of 10.8 mmol L-1, whereas the untreated cells under the same conditions showed very low hydrogen yield of 0.003 mmol L-1. The enhanced hydrogen production observed in the treated cells was consistent with an increase in hydrogenase activity. Treatment of BG110 grown cells with low concentration of green synthesized AgNPs at 0.24 mg L-1 enhanced hydrogenase activity with a 5-fold increase of enzyme activity compared to untreated BG110 grown cells. In addition, to improve photolytic water splitting efficiency for hydrogen production, cells treated with AgNPs at 0.24 mg L-1 showed highest oxygen evolution signifying improvement in photosynthesis. The silver nanoparticles synthesized using phytochemicals derived from plant enhanced both microalgal biomass and hydrogen production with an added advantage of CO2 reduction which could be achieved due to an increase in biomass. Hence, treating microalgae with nanoparticles provided a promising strategy to reduce the atmospheric carbon dioxide as well as increasing production of hydrogen as clean energy.