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Introduction: Desulfovibrio vulgaris Hildenborough is a gram-negative anaerobic bacterium belonging to the sulfate-reducing bacteria that exhibits highly versatile metabolism. By switching from one energy mode to another depending on nutrients availability in the environments„ it plays a central role in shaping ecosystems. Despite intensive efforts to study D. vulgaris energy metabolism at the genomic, biochemical and ecological level, bioenergetics in this microorganism remain far from being fully understood. Alternatively, metabolic modeling is a powerful tool to understand bioenergetics. However, all the current models for D. vulgaris appeared to be not easily adaptable to various environmental conditions. Methods: To lift off these limitations, here we constructed a novel transparent and robust metabolic model to explain D. vulgaris bioenergetics by combining whole-cell proteomic analysis with modeling approaches (Flux Balance Analysis). Results: The iDvu71 model showed over 0.95 correlation with experimental data. Further simulations allowed a detailed description of D. vulgaris metabolism in various conditions of growth. Altogether, the simulations run in this study highlighted the sulfate-to-lactate consumption ratio as a pivotal factor in D. vulgaris energy metabolism. Discussion: In particular, the impact on the hydrogen/formate balance and biomass synthesis is discussed. Overall, this study provides a novel insight into D. vulgaris metabolic flexibility. [ABSTRACT FROM AUTHOR]

Several species of microalgae can convert light energy into molecular hydrogen (H2) by employing enzymes of early phylogenetic origin, [FeFe]-hydrogenases, coupled to the photosynthetic electron transport chain. Bacterial [FeFe]-hydrogenases consist of a conserved domain that harbors the active site cofactor, the H-domain, and an additional domain that binds electron-conducting FeS clusters, the F-domain. In contrast, most algal hydrogenases characterized so far have a structurally reduced, so-termed M1-type architecture, which consists only of the H-domain that interacts directly with photosynthetic ferredoxin PetF as an electron donor. To date, only a few algal species are known to contain bacterial-type [FeFe]-hydrogenases, and no M1-type enzymes have been identified in these species. Here, we show that the chlorophycean alga Uronema belkae possesses both bacterial-type and algal-type [FeFe]-hydrogenases. Both hydrogenase genes are transcribed, and the cells produce H2 under hypoxic conditions. The biochemical analyses show that the two enzymes show features typical for each of the two [FeFe]-hydrogenase types. Most notable in the physiological context is that the bacterial-type hydrogenase does not interact with PetF proteins, suggesting that the two enzymes are integrated differently into the alga's metabolism. [ABSTRACT FROM AUTHOR]

IntroductionDesulfovibrio vulgaris Hildenborough is a gram-negative anaerobic bacterium belonging to the sulfate-reducing bacteria that exhibits highly versatile metabolism. By switching from one energy mode to another depending on nutrients availability in the environments„ it plays a central role in shaping ecosystems. Despite intensive efforts to study D. vulgaris energy metabolism at the genomic, biochemical and ecological level, bioenergetics in this microorganism remain far from being fully understood. Alternatively, metabolic modeling is a powerful tool to understand bioenergetics. However, all the current models for D. vulgaris appeared to be not easily adaptable to various environmental conditions.MethodsTo lift off these limitations, here we constructed a novel transparent and robust metabolic model to explain D. vulgaris bioenergetics by combining whole-cell proteomic analysis with modeling approaches (Flux Balance Analysis).ResultsThe iDvu71 model showed over 0.95 correlation with experimental data. Further simulations allowed a detailed description of D. vulgaris metabolism in various conditions of growth. Altogether, the simulations run in this study highlighted the sulfate-to-lactate consumption ratio as a pivotal factor in D. vulgaris energy metabolism.DiscussionIn particular, the impact on the hydrogen/formate balance and biomass synthesis is discussed. Overall, this study provides a novel insight into D. vulgaris metabolic flexibility.

Several species of microalgae can convert light energy into molecular hydrogen (H2) by employing enzymes of early phylogenetic origin, [FeFe]-hydrogenases, coupled to the photosynthetic electron transport chain. Bacterial [FeFe]-hydrogenases consist of a conserved domain that harbors the active site cofactor, the H-domain, and an additional domain that binds electron-conducting FeS clusters, the F-domain. In contrast, most algal hydrogenases characterized so far have a structurally reduced, so-termed M1-type architecture, which consists only of the H-domain that interacts directly with photosynthetic ferredoxin PetF as an electron donor. To date, only a few algal species are known to contain bacterial-type [FeFe]-hydrogenases, and no M1-type enzymes have been identified in these species. Here, we show that the chlorophycean alga Uronema belkae possesses both bacterial-type and algal-type [FeFe]-hydrogenases. Both hydrogenase genes are transcribed, and the cells produce H2 under hypoxic conditions. The biochemical analyses show that the two enzymes show features typical for each of the two [FeFe]-hydrogenase types. Most notable in the physiological context is that the bacterial-type hydrogenase does not interact with PetF proteins, suggesting that the two enzymes are integrated differently into the alga’s metabolism.

Aerobic hydrogen-oxidizing bacteria (HOB) is a group of active, abundant, and diverse microorganisms with representatives in nearly all phyla, distributed in soil exposed to atmospheric or elevated hydrogen. In this review, first, we discuss the fundamental physiology, isolation, and identification techniques for HOB. On this basis, the hydrogenase genetic organization and metabolic strategy of Cupriavidus necator, Mycobacterium smegmatis as representative HOB are summarized. Availability of hydrogen, oxygen, nutrients, and environmental variables such as temperature and moisture are key ecological factors influencing H2 oxidation activity, hydrogenase groups, and microbial composition. Finally, we systematically illustrate the ecological roles of HOB and the interactions between HOB and other soil microbiota, particularly rhizobia in agricultural soils and Cyanobacteria in deserts. Intensive studies should focus on the cell functioning regulated by hydrogenases and on the full play of ecological roles by competitive HOB consortiums in soil niches, which is expected to facilitate the biogeochemical process of carbon dioxide fixation and energy utilization. [ABSTRACT FROM AUTHOR]

The misuse and continues discharge of antibiotics can cause serious pollution, which is urgent to take steps to remit the environment pollution. In this study, anaerobic bacteria isolated from the aeration tank of a local sewage treatment plant were employed to investigate hydrogen production and tetracycline (TC) degradation during anaerobic fermentation. Results indicate that low concentrations of TC enhanced hydrogen production, increasing from 366 mL to a maximum of 480 mL. This increase is attributed to stimulated hydrolysis and acidogenesis, coupled with significant inhibition of homoacetogenesis. Furthermore, the removal of TC, facilitated by adsorption and biodegradation, exceeded 90 %. During the fermentation process, twenty-one by-products were identified, leading to the proposal of four potential degradation pathways. Analysis of the microbial community revealed shifts in diversity and a decrease in the abundance of hydrogen-producing bacteria, whereas bacteria harboring tetracycline resistance genes became more prevalent. This study provides a possibility to treat tetracycline-contaminated wastewater and to produce clean energy simultaneously by anaerobic fermentation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, the effect of modulating fulvic acid (FA) concentrations (0, 25 and 50 mg/L) on nitrogen removal in a bioelectrochemical hydrogen autotrophic denitrification system (BHDS) was investigated. Results showed that FA increased the nitrate (NO 3 - -N) removal rate of the BHDSs from 37.8 to 46.2 and 45.2 mg N/(L·d) with a current intensity of 40 mA. The metagenomic analysis revealed that R2 (25 mg/L) was predominantly populated by autotrophic denitrifying microorganisms, which enhanced denitrification performance by facilitating electron transfer. Conversely, R3 (50 mg/L) exhibited an increase in genes related to the heterotrophic process, which improved the denitrification performance through the collaborative action of both autotrophic and heterotrophic denitrification pathways. Besides, the study also identified a potential for nitrogen removal in Serpentinimonas, which have been rarely studied. The interesting set of findings provide valuable reference for optimizing BHDS for nitrogen removal and promoting specific denitrifying genera within the system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Anaerobic electrochemical digestion (AED) is an effective system for recovering biogas from organic wastes. However, the effects of different anode potentials on anaerobic activated sludge remain unclear. This study confirmed that biofilms exhibited the best electroactivity at -0.2 V (vs. Ag/AgCl) compared to -0.4 V and 0 V. Gas was further regulated, with the highest hydrogen content (47 ± 7 %) observed at -0.2 V. The 0 V system produced the largest amount of methane (70 ± 8 %) and exhibited the greatest presence of hydrogen-utilizing microorganisms. The gas yield at -0.4 V was the lowest, with no hydrogen detected. Excess bioelectrohydrogen at -0.2 V and 0 V caused the co-enrichment of Methanobacterium and Acetoanaerobium, establishing a thermodynamically feasible current-acetate-hydrogen electron cycle to improve electrogenesis. These results provide insights into the regulatory strategies of MEC technology during anaerobic digestion, which play a decisive role in determining the composition of biogas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Biomethanation converts carbon dioxide (CO 2 ) emissions into renewable natural gas (RNG) using mixed microbial cultures enriched with hydrogenotrophic archaea. This study examines the performance of a single methanogenic archaeon converting biogas with added hydrogen (H 2 ) into methane (CH 4 ) using a trickle-bed bioreactor with enhanced gas-liquid mass transport. The process in continuous operation followed the theoretical reaction of hydrogenotrophic methanogenesis (CO 2  + 4 H 2  → CH 4  + 2 H 2 O), producing RNG with over 99 % CH 4 and more than 0.9 H 2 conversion efficiency. The Monod constants of H 2 uptake were experimentally determined using kinetic modelling. Also, a dimensionless parameter was used to quantify the ratio between the H 2 mass transfer rate and the maximum attainable H 2 consumption rate. Single-culture biomethanation averts the formation of secondary metabolites and bicarbonate buffer interferences, resulting in lower demands for H 2 than mixed-culture biomethanation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

A high rate upflow anaerobic polyfoam-based bioreactor (UAPB) was developed for lab-scale in-situ biogas upgrading by H 2 injection. The reactor, with a volume of 440 mL, was fed with synthetic wastewater at an organic loading rate (OLR) of 3.5 g COD/L·day and a hydraulic retention time (HRT) of 7.33 h. The use of a porous diffuser, alongside high gas recirculation, led to a higher H 2 liquid mass transfer, and subsequently to a better uptake for high CH 4 content of 56% (starting from 26%). Our attempts to optimize both operational parameters (H 2 flow rate and gas recirculation ratio, which is the total flow rate of recirculated gas over the total outlet of gas flow rate) were not initially successful, however, at a very high recirculation ratio (32) and flow rate (54 mL/h), a significant improvement of the hydrogen consumption was achieved. These operational conditions have in turn driven the methanogenic community toward the dominance of Methanosaetaceae, which out-competed Methanosarcinaceae. Nevertheless, highly stable methane production rates of 1.4-1.9 L CH 4 /L reactor .day were observed despite the methanogenic turnover. During the different applied operational conditions, the bacterial community was especially impacted, resulting in substantial shifts of taxonomic groups. Notably, Aeromonadaceae was the only bacterial group positively correlated with increasing hydrogen consumption rates. The capacity of Aeromonadaceae to extracellularly donate electrons suggests that direct interspecies electron transfer (DIET) enhanced biogas upgrading. Overall, the proposed innovative biological in-situ biogas upgrading technology using the UAPB configuration shows promising results for stable, simple, and effective biological biogas upgrading., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

In this study, the optimal hydrogen (H 2 ) production conditions of the high-efficiency H 2 -producing mutant strain Ethanoligenens harbinense YR-3 (carbon-nitrogen ratio 5.5, phosphate buffer 80 mM, initial pH 6.0, biotin 1.4 mg/L) are obtained by intermittent experiments. The maximum specific H 2 production rate of YR-3 (2.85 mol H 2 /mol glucose) was 1.4 times that of the wild strain ZGX4 (2.04 mol H 2 /mol glucose). The liquid-phase products are mainly ethanol and acetic acid, indicating that the metabolic pathway has not changed. Two-dimensional electrophoresis and mass spectrometry were used to compare and analyze the protein map differences between YR-3 and ZGX4. The results show that 1,6-fructose diphosphate aldolase and the flavoprotein in hydrogenase are highly expressed. This study will provide a theoretical basis for the genetic modification of high-efficiency H 2 -producing strains and the improvement of H 2 production capacity., (Copyright © 2024. Published by Elsevier B.V.)

The extensive use of plastic products in food packaging and daily life makes them inevitably enter the treatment process of food waste (FW). Plasticizer as a new pollutant is threatening the dark fermentation of FW. Our study showed that bisphenol A (BPA) at > 250 mg/L had a significant inhibition on hydrogen production from FW by thermophilic dark fermentation. The endogenous ATP content and lactate dehydrogenase (LDH) release showed that high level of BPA not only inhibited the growth of hydrogen-producing consortium, but also led to cell death. In addition, BPA mainly affects the hydrogen-producing consortium by reducing cell membrane fluidity, damaging cell membrane integrity and reducing cell membrane potential, resulting in cell death. This study provides some new insights into the mechanism of the effect of BPA on hydrogen production from FW by thermophilic dark fermentation, and lays the foundation on the utilization of FW., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The septic tank is the most commonly used decentralized wastewater treatment systems for household wastewater treatment in on-site applications. The removal rate of various pollutants is lower in different septic tank configurations. The integration of a microbial electrolysis cells (MEC) into septic tank or biofilm-based reactors can be a green and sustainable technology for household wastewater treatment and energy production. In this study, a 50-L septic tank was converted into a 50-L MEC coupled with biofilm-based reactor for simultaneous household wastewater treatment and hydrogen production. The biofilm-based reactor was integrated by an anaerobic packed-bed biofilm reactor (APBBR) and an aerobic moving bed biofilm reactor (aeMBBR). The MEC/APBBR/aeMBBR was evaluated at different organic loading rates (OLRs) by applying voltage of 0.7 and 1.0 V. Result showed that the increase of OLRs from 0.2 to 0.44 kg COD/m 3 d did not affect organic matter removals. Nutrient and solids removal decreased with increasing OLR up to 0.44 kg COD/m 3 d. Global removal of chemical oxygen demand (COD), biochemical oxygen demand (BOD), total nitrogen (TN), ammoniacal nitrogen (NH 4 + ), total phosphorus (TP) and total suspended solids (TSS) removal ranged from 81 to 84%, 84 to 85%, 53 to 68%, 88 to 98%, 11 to 30% and 76 to 88% respectively, was obtained in this study. The current density generated in the MEC from 0 to 0.41 A/m 2 contributed to an increase in hydrogen production and pollutants removal. The maximum volumetric hydrogen production rate obtained in the MEC was 0.007 L/L . d (0.072 L/d). The integration of the MEC into biofilm-based reactors applying a voltage of 1.0 V generated different bioelectrochemical nitrogen and phosphorus transformations within the MEC, allowing a simultaneous denitrification-nitrification process with phosphorus removal., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A hydrogen (H2)-based membrane biofilm reactor (H2-MBfR) can reduce electron acceptors nitrate (NO3-), selenate (SeO42-), selenite (HSeO3-), and sulfate (SO42-), which are in wastewaters from coal mining and combustion. This work presents a model to describe a H2-driven microbial community comprised of hydrogenotrophic and heterotrophic bacteria that respire NO3-, SeO42-, HSeO3-, and SO42-. The model provides mechanistic insights into the interactions between autotrophic and heterotrophic bacteria in a microbial community that is founded on H2-based autotrophy. Simulations were carried out for a range of relevant solids retention times (SRT; 0.1-20 days) and with adequate H2-delivery capacity to reduce all electron acceptors. Bacterial activity began at an ∼0.6-day SRT, when hydrogenotrophic denitrifiers began to accumulate. Selenate-reducing and selenite-reducing hydrogenotrophs became established next, at SRTs of ∼1.2 and 2 days, respectively. Full NO3-, SeO42-, and HSeO3- reductions were complete by an SRT of ∼5 days. SO42- reduction began at an SRT of ∼10 days and was complete by ∼15 days. The desired goal of reducing NO3-, SeO42-, and HSeO3-, but not SO42-, was achievable within an SRT window of 5-10 days. Autotrophic hydrogenotrophs dominated the active biomass, but nonactive solids were a major portion of the solids, especially for an SRT ≥ 5 days., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Biodegradability testing in soil helps to identify safe synthetic organic chemicals but is still obscured by the formation of soil-bound 'non-extractable' residues (NERs). Present-day methodologies using radiocarbon or stable ( 13 C, 15 N) isotope labeling cannot easily differentiate soil-bound parent chemicals or transformation products (xenoNERs) from harmless soil-bound biomolecules of microbial degraders (bioNERs). Hypothesizing a minimal retention of hydrogen in biomolecules, we here apply stable hydrogen isotope - deuterium (D) - labeling to unravel the origin of NERs. Soil biodegradation tests with D- and 13 C-labeled 2,4-D, glyphosate and sulfamethoxazole reveal consistently lower proportions of applied D than 13 C in total NERs and in amino acids, a quantitative biomarker for bioNERs. Soil-bound D thus mostly represents xenoNERs and not bioNERs, enabling an efficient quantification of xenoNERs by just measuring the total bound D. D or tritium (T) labeling could thus improve the value of biodegradability testing results for diverse organic chemicals forming soil-bound residues., (© 2024. The Author(s).)

The "knallgas" bacterium Cupriavidus necator is attracting interest due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO 2 via the Calvin-Benson-Bassham (CBB) cycle, and grows on organic acids and sugars. Its tripartite genome is notable for its size and duplications of key genes (CBB cycle, hydrogenases, and nitrate reductases). Little is known about which of these isoenzymes and their cofactors are actually utilized for growth on different substrates. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on succinate, fructose, hydrogen (H 2 /CO 2 ), and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that (i) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. (ii) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. (iii) For hydrogenases, both soluble and membrane-bound enzymes were utilized for lithoautotrophic growth. (iv) Of the six terminal respiratory complexes in C. necator H16, only some are utilized, and utilization depends on the energy source. (v) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necator 's genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium and inspire future engineering strategies.IMPORTANCEThe soil bacterium Cupriavidus necator can grow on gas mixtures of CO 2 , H 2 , and O 2 . It also consumes formic acid as carbon and energy source and various other substrates. This metabolic flexibility comes at a price, for example, a comparatively large genome (6.6 Mb) and a significant background expression of lowly utilized genes. In this study, we mutated every non-essential gene in C. necator using barcoded transposons in order to determine their effect on fitness. We grew the mutant library in various trophic conditions including hydrogen and formate as the sole energy source. Fitness analysis revealed which of the various energy-generating iso-enzymes are actually utilized in which condition. For example, only a few of the six terminal respiratory complexes are used, and utilization depends on the substrate. We also show that the protein cost for the various lowly utilized enzymes represents a significant growth disadvantage in specific conditions, offering a route to rational engineering of the genome. All fitness data are available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/., Competing Interests: The authors declare no conflict of interest.

[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, Ni a -C and Ni a -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. Ni a -SR'', a fully reduced hydride intermediate of the catalytic cycle, was found to be reversibly photoconverted into another catalytic state, Ni a -L. In contrast to the well-known photolysis of the more oxidized hydride intermediate Ni a -C, photoconversion of Ni a -SR'' into Ni a -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 Ni a -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., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Pyrogenic carbon is considered an enhancer to H 2 -yielding dark fermentation (DF), but little is known about how it regulates extracellular electron transfer (EET) and influences transmembrane respiratory chains and intracellular metabolisms. This study addressed these knowledge gaps and demonstrated that wood waste pyrogenic carbon (biochar) could significantly improve the DF performance; e.g., addition of pyrogenic carbon produced by pyrolysis at 800 °C (PC800) increased H 2 yield by 369.7%. Biochemical quantification, electrochemical analysis, and electron respiratory chain inhibition tests revealed that PC800 promoted the extracellular flavin-based electron transfer process and further activated the acceleration of the transmembrane electron transfer. Comparative metagenome/metatranscriptome analyses indicated that the flavin-containing Rnf complex was the potential transmembrane respiratory enzyme associated with PC800-mediated EET. Based on NADH/NAD + circulation, the promoted Rnf complex could stimulate the functions of the electron bifurcating Etf/Bcd complex and startup of glycolysis. The promoted Etf/Bcd could further contribute to balance the NADH/NAD + level for glycolytic reactions and meanwhile provide reduced ferredoxin for group A1 [FeFe]-hydrogenases. This proton-energy-linked mechanism could achieve coupling production of ATP and H 2 . This study verified the important roles of pyrogenic carbon in mediating EET and transmembrane/intracellular pathways and revealed the crucial roles of electron bifurcation in DF for hydrogen production.

Purple non-sulfur bacteria (PNSB) are a diverse group of bacteria studied for various possible applications. They are commonly surveyed in bioenergy research as they produce biohydrogen, a candidate for clean alternative energy. This study aimed to assess the biohydrogen production ability and genetically characterize a high biohydrogen-producing PNSB (MAY2) isolated from Los Baños, Laguna, Philippines via whole genome sequencing (WGS). MAY2, when grown in mixed volatile fatty acids, produced biogas with 38% hydrogen. WGS results revealed that the isolate is positively classified under the genus Rhodobacter johrii. Also, 82 genetic hallmarks for biohydrogen production were found in the isolated genome which are involved in the production of key enzymes and proteins relevant to the photofermentative and hydrogen regulation pathways. Its nitrogenase gene cluster is stringently regulated by two genes, nifA and rofN, whose function and expression are easily affected by several environmental factors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Methanogenic communities of hypersaline microbial mats of Guerrero Negro, Baja California Sur, Mexico, have been recognized to be dominated by methylotrophic methanogens. However, recent studies of environmental samples have evidenced the presence of hydrogenotrophic and methyl-reducing methanogenic members, although at low relative abundances. Physical and geochemical conditions that stimulate the development of these groups in hypersaline environments, remains elusive. Thus, in this study the taxonomic diversity of methanogenic archaea of two sites of Exportadora de Sal S.A was assessed by mcrA gene high throughput sequencing from microcosm experiments with different substrates (both competitive and non-competitive). Results confirmed the dominance of the order Methanosarcinales in all treatments, but an increase in the abundance of Methanomassiliiccocales was also observed, mainly in the treatment without substrate addition. Moreover, incubations supplemented with hydrogen and carbon dioxide, as well as the mixture of hydrogen, carbon dioxide and trimethylamine, managed to stimulate the richness and abundance of other than Methanosarcinales methanogenic archaea. Several OTUs that were not assigned to known methanogens resulted phylogenetically distributed into at least nine orders. Environmental samples revealed a wide diversity of methanogenic archaea of low relative abundance that had not been previously reported for this environment, suggesting that the importance and diversity of methanogens in hypersaline ecosystems may have been overlooked. This work also provided insights into how different taxonomic groups responded to the evaluated incubation conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ramírez-Arenas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A novel mesophilic bacterium, strain SS33 T , was isolated from a deep-sea hydrothermal vent chimney at Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean. The cells of strain SS33 T were motile short rods with a single polar flagellum. The growth of strain SS33 T was observed at the temperature range between 33 and 55 °C (optimum growth at 45 °C), at the pH range between 5.0 and 7.1 (optimum growth at pH 6.0) and in the presence of between 2.0 and 4.5% (w/v) NaCl [optimum growth at 3.5% (w/v)]. Strain SS33 T was a facultative anaerobic chemolithoautotroph using molecular hydrogen and elemental sulphur as the sole electron donor. Nitrate, nitrous oxide, sulphate, elemental sulphur and molecular oxygen were capable of serving as the sole electron acceptor. Phylogenetic analysis based on 16S rRNA gene sequences placed strain SS33 T in the genus Hydrogenimonas belonging to the class Epsilonproteobacteria . The closely related species of strain SS33 T were Hydrogenimonas urashimensis SSM-Sur55 T (95.96%), Hydrogenimonas thermophila EP1-55-1% T (95.75%) and Hydrogenimonas cancrithermarum ISO32 T (95.24%). According to the taxonomic and physiological characteristics, it is proposed that strain SS33 T was classified into a novel species of genus Hydrogenimonas , Hydrogenimonas leucolamina sp. nov., with SS33 T (=JCM 39184 T =KCTC 25253 T ) as the type strain. Furthermore, the genome comparison of Epsilonproteobacteria revealed that their [NiFe] hydrogenase genes belonging to Group 1b could be divided into two phylogenetic lineages and suggested that the reverse gyrase gene has been lost after division to the genus Hydrogenimonas .

Lignocellulosic biomass offers substantial potential as an ideal feedstock for dark fermentative hydrogen production due to its sustainability and cost-effectiveness. The current study examined the influence of furfural on fermentative hydrogen production using lignocellulosic hydrolysate in the presence of furfural. Synthetic lignocellulosic hydrolysate, consisting primarily of 76% xylose, 10% glucose, 9% arabinose, and a mixture of other sugars such as galactose and mannose (85% pentose sugars and 15% hexose sugars), was employed as the substrate. Various substrate concentrations ranging from 2 to 32 g/L were tested, along with furfural concentrations of 0, 1, and 2 g/L. The investigation aimed to assess the effects of initial substrate concentration, initial furfural concentration, furfural-to-biomass ratio (F/B), and furfural-to-substrate ratio (F/S) on biohydrogen production yields. The maximum specific substrate utilization rates at different substrate concentrations were effectively characterized using Haldane's substrate inhibition model. Among the tested concentrations, the 16 g/L emerged as the optimal substrate concentration. The initial furfural concentration was identified as the most significant parameter impacting biohydrogen production, with complete inhibition observed at a furfural concentration of 2 g/L. Higher F/S ratios at substrate concentrations ranging from 2 to 16 g/L resulted in reduced maximum specific hydrogen production rates (MSHPR) and hydrogen yields. Substrate inhibition was observed at 24 g/L and 32 g/L. Lactate was the predominant metabolite in all batches containing 2-g/L furfural, as well as in batches with 1-g/L furfural and substrate concentrations of 24 and 32 g/L. Furfural at a concentration of 1 g/L was not inhibitory in any of the batches. Overall, the mixed cultures in this study could efficiently produce hydrogen from lignocellulosic hydrolysates and degrade furfural, providing new insights into fermentative hydrogen-producing bacteria with furfural tolerance., (© 2024. Crown.)

In this study, a directional electrode separator (DES) was designed and incorporated into a single-chamber bioelectrochemical system (BES) to reduce migration and reoxidation of hydrogen. This issue arises when H 2 , generated at the cathode, travels to the anode where anodic biofilms use H 2 . To test the feasibility of our design, a 3D-printed BES reactor equipped with a DES was inoculated with anaerobic digestor granules and operated under fed-batch conditions using fermented corn stover effluent. The DES equipped reactor achieved significantly higher current densities (∼53 A/m²) compared to a conventional single-chamber BES without a separator (∼16 A/m²), showing a 3.3 times improvement. Control abiotic electrochemical experiments revealed that the DES exhibited significantly higher proton conductivity (456±127 µS/mm) compared to a proton exchange membrane (67±21 µS/mm) with a statistical significance of P=0.03. The DES also effectively reduced H 2 migration to the anode by 21-fold relative to the control. Overall, incorporating a DES in a single-chamber BES enhanced anodic current density by reducing H 2 migration to the anode., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study evaluated the dark-fermentative hydrogen (H 2 ) production potential of isolated and identified Shigella flexneri SPD1 from various pure (glucose, fructose, sucrose, lactose, and galactose) and biowastes (coconut coir, cotton fiber, groundnut shells, rice-, and wheat-straws)-derived sugars. Among sugars, S. flexneri SPD1 exhibited high H 2 production of up to 3.20 mol/mole of hexose using glucose (5.0 g/L). The pre-treatment of various biowastes using green solvents (choline chloride and lactic acid mixture) and enzymatic hydrolysis resulted in the generation of up to 36.0 g/L of sugars. The maximum H 2 production is achieved up to 2.92 mol/mol of hexose using cotton-hydrolysate. Further, the upscaling of bioprocess up to 5 L of capacity resulted in a maximum yield of up to 3.06 mol/mol of hexose. These findings suggested that S. flexneri SPD1, a novel H 2 -producer, can be employed to develop a circular economy-based approach to produce clean energy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Screening and isolating acid-tolerant bacteria capable of efficient hydrogen production can mitigate the inhibitory effects on microbial activity caused by rapid pH drops during fermentation. In this study, we isolated an acid-tolerant and highly efficient hydrogen-producing bacterium, named Clostridium sp. BLY-1, from acidic soil. Compared to the model strain Clostridium pasteurianum DSM 525, BLY-1 demonstrates a faster growth rate and superior hydrogen production capabilities. At an initial pH of 4.0, BLY-1's hydrogen production is 7.5 times greater than that of DSM 525, and under optimal conditions (pH=5.0), BLY-1's hydrogen production rate is 42.13% higher than DSM 525. Genomic analysis revealed that BLY-1 possesses a complete CiaRH two-component system and several stress-resistance components absent in DSM 525, which enhance its growth and hydrogen production in acidic environments. These findings provide a novel avenue for boosting the hydrogen production capabilities of Clostridium strains, offering new resources for advancing the green hydrogen industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study presents a bidirectional flow tidal bioreactor designed to enhance H 2 -driven CO 2 biomethanation. The bioreactor alternated biofilms between immersion in nutrient solution and exposure to H 2 /CO 2 , creating alternating dry and wet states. This tidal operation minimized liquid film thickness during dry periods and ensured uniform nutrient distribution during wet periods. Bidirectional H 2 /CO 2 supply was used to reduce biofilm thickness heterogeneity across the reactor height. CO 2 biomethanation remained stable with an empty bed residence time of 9.7 min, achieving a methane (CH 4 ) formation rate of 26.8 Nm 3 CH 4 /(m 3 ·d). The product gas contained 95.0 ± 2.5 % CH 4 , with a H 2 /CO 2 conversion efficiency of 90.8 %. Tidal operation mitigated the buildup of dissolved and suspended organics, such as organic acids and detached biofilms. Dominant bacteria in biofilms included fermentative species like Petrimonas and H 2 -utilizing homoacetogens like Sporomusa. Enriched hydrogenotrophic methanogens, particularly Methanobacterium, were observed. Overall, this study highlights the bioreactor's effectiveness in improving CO 2 biomethanation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Measurements of stable isotope ratios in organic compounds are widely used tools for plant ecophysiological studies. However, the complexity of the processes involved in shaping hydrogen isotope values (δ 2 H) in plant carbohydrates has limited its broader application. To investigate the underlying biochemical processes responsible for 2 H fractionation among water, sugars, and cellulose in leaves, we studied the three main CO 2 fixation pathways (C 3 , C 4 , and CAM) and their response to changes in temperature and vapor pressure deficit (VPD). We show significant differences in autotrophic 2 H fractionation (ε A ) from water to sugar among the pathways and their response to changes in air temperature and VPD. The strong 2 H depleting ε A in C 3 plants is likely driven by the photosynthetic H + production within the thylakoids, a reaction that is spatially separated in C 4 and strongly reduced in CAM plants, leading to the absence of 2 H depletion in the latter two types. By contrast, we found that the heterotrophic 2 H-fractionation (ε H ) from sugar to cellulose was very similar among the three pathways and is likely driven by the plant's metabolism, rather than by isotopic exchange with leaf water. Our study offers new insights into the biochemical drivers of 2 H fractionation in plant carbohydrates., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Sulfidogenesis is a promising technology for the selective recovery of chalcophile bulk metals (e.g. Cu, Zn, and Co) from metal-contaminated waters such as acid mine drainage (AMD) and metallurgy waste streams. The use of elemental sulfur (S 0 ) instead of sulfate (SO 4 2- ) as electron acceptor reduces electron donor requirements four-fold, lowering process costs, and expanding the range of operating conditions to a more acidic pH. We previously reported autotrophic S 0 reduction using an industrial mesophilic granular sludge as inoculum under thermoacidophilic conditions. Here, we examined the effect of pH on the S 0 reduction performance of the same inoculum, in a gas-lift reactor run at 30°C under neutral (pH 6.9) and acidic (pH 3.8) conditions, continuously fed with mineral media and H 2 and CO 2 . Steady-state volumetric sulfide production rates (VSPR) dropped 2.5-fold upon transition to acidic pH, from 1.79 ± 0.18 g S 2- ·L -1 ·d -1 to 0.71 ± 0.07 g S 2- ·L -1 ·d -1 . Microbial community composition was analyzed using 16S rRNA gene amplicon sequencing. At neutral pH (6.9), the high relative abundance of the S 0 -reducing genus Sulfurospirillum, previously known only for heterotrophic members, combined with the presence of Acetobacterium and detection of acetate, suggests an important role for heterotrophic S 0 reduction facilitated by acetogenesis. Conversely, at acidic pH (3.9), S 0 reduction appeared autotrophic, as indicated by the high relative abundance of Desulfurella., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Previous studies have indicated that hydrogen-rich water (HW) treatment can delay fruit ripening and senescence. However, little is known about the HW-delaying pulp breakdown. In this study, eight physiological characteristics revealed that HW treatment delayed both pericarp browning and pulp breakdown of litchi fruit. To gain a comprehensive understanding of the changes in litchi pulp, a combination of multiple metabolomics and gene expression analyses was conducted, assessing 67 primary metabolites, 103 volatiles, 31 amino acids, and 13 crucial metabolite-related genes. Results showed that HW treatment promoted starch degradation, decelerated cell wall degradation and glycolysis, and maintained the flavor and quality of litchi fruit. Furthermore, HW treatment stimulated the production of volatile alcohols, aldehydes, ketones, olefins, and amino acids, which might play a vital role in HW-delaying pulp breakdown. This study sheds light on the mechanism by which HW delayed pulp breakdown by investigating small molecule metabolites and metabolic pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Molecular hydrogen is beneficial for fruits quality improvement. However, the mechanism involved, especially cellular metabolic responses, has not been well established. Here, the integrated widely targeted metabolomics analysis (UPLC-MS/MS) and biochemical evidence revealed that hydrogen-based irrigation could orchestrate, either directly or indirectly, an array of physiological responses in blueberry (Vaccinium spp.) during harvesting stage, especially for the delayed senescence in harvested stage (4 °C for 12 d). The hubs to these changes are wide-ranging metabolic reprogramming and antioxidant machinery. A total of 1208 distinct annotated metabolites were identified, and the characterization of differential accumulated metabolites (DAMs) revealed that the reprogramming, particularly, involves phenolic acids and flavonoids accumulation. These changes were positively matched with the transcriptional profiles of representative genes for their synthesis during the growth stage. Together, our findings open a new window for development of hydrogen-based agriculture that increases the shelf-life of fruits in a smart and sustainable manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Yan Zeng, Xu Cheng, and Pathier Didier are employees and hold ownership interests (including patents) in Air Liquide (China) R&D Co., Ltd., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

[FeFe]-hydrogenases function as both H 2 catalysts and sensors. While catalysis is well investigated, details regarding the H 2 sensing mechanism are limited. Here, we relate protein structure changes to H 2 sensing, similar to light-driven bio-sensors. Our results highlight how identical cofactors incorporated in alternative protein scaffolds serve different functions in nature.

Ruminal microbes catabolise feed carbohydrates mainly into SCFA, methane (CH 4 ), and carbon dioxide (CO 2 ), with predictable relationships between fermentation end products and net microbial increase. We used a closed in vitro batch culture system, incubating grass and maize silages, and measured total gas production at 8 and 24 h, as well as the truly degraded substrate, the net production of SCFA, CH 4 , and microbial biomass at 24 h, and investigated the impact of silage type and inoculum microbial mass on fermentation direction. Net microbial yield was negatively correlated with total gas at 8 h (P < 0•001), but not at 24 h (P = 0•052), and negatively correlated with CH 4 production (P < 0•001). Higher initial inoculum microbial mass was related to a lower net microbial yield (P < 0•001) but a higher CH 4 production (P < 0•001). A significant difference between grass silage and maize silage was detected within the context of these relationships (P < 0•050). The metabolic hydrogen (2H) recovery was 102.8 ± 12.3 % for grass silages and 118.8 ± 13.3% for maize silages. Overall, grass silages favoured more substrate conversion to microbial biomass and less to fermentation end products than maize silage. Lower inoculum microbial mass facilitated more microbial growth and, because of the 2H sink by microbial synthesis, decreased CH 4 production., Competing Interests: The authors declare no conflict of interest., (© Published by Cambridge University Press on behalf of The Nutrition Society 2024.)

Antibiotic fermentation residue, which is generated from the microbial antibiotic production process, has been a troublesome waste faced by the pharmaceutical industry. Dark fermentation is a potential technology to treat antibiotic fermentation residue in terms of renewable H 2 generation and waste management. However, the inherent antibiotic in antibiotic fermentation residue may inhibit its dark fermentation performance, and current understanding on this topic is limited. This investigation examined the impact of the inherent antibiotic on the dark H 2 fermentation of Cephalosporin C (CEPC) fermentation residue, and explored the mechanisms from the perspectives of bacterial communities and functional genes. It was found that CEP-C in the antibiotic fermentation residue significantly inhibited the H 2 production, with the H 2 yield decreasing from 17.2 mL/g-VS added to 12.5 and 9.6 mL/g-VS added at CEP-C concentrations of 100 and 200 mg/L, respectively. CEP-C also prolonged the H 2 -producing lag period. Microbiological analysis indicated that CEP-C remarkably decreased the abundances of high-yielding H 2 -producing bacteria, as well as downregulated the genes involved in hydrogen generation from the"pyruvate pathway" and"NADH pathway", essentially leading to the decline of H 2 productivity. The present work gains insights into how cephalosporin antibiotics influence the dark H 2 fermentation, and provide guidance for mitigating the inhibitory effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

For a long time, intercropping and rotation of leguminous with non-leguminous crops is widely used to reduce the application of nitrogen fertilizer and increase yield in agroecosystems. At present, most researchers considered that this management measure is helpful for reducing fertilizer consumption and increasing its efficiency, as it can improve nutrient supply of legumestonon-legumes, the spatial nutrient utilization efficiency by enhancing soil spatial heterogeneity, and improve soil structure and disease resistance. However, current theories cannot fully explain the positive effect of crop rotation and inter-cropping systems involving legumes. A large amount of hydrogen (H 2 ) can be produced as an obligatory by-product of nitrogenase responsible for nitrogen (N 2 ) fixation in the root nodules of leguminous plants. Despite of substantial amounts of H 2 enriched in the rhizosphere of legumes, only a minor proportion of H 2 is found to leak to soil surface. Increasing evidence showed that most H 2 released in soil is immediately depleted in the surrounding of N 2 -fixing nodules by H 2 -oxidizing bacteria (HOB) thriving in soil. HOB can use H 2 as an electron donor to assimilate and fix CO 2 through redox reactions to synthesize cellular substances and consequently promote plant growth. To date, however, little is known about the biological mechanism and ecological process behind the "hydrogen fertilizer effect". Therefore, we review the H 2 -induced plant growth-promoting effects and its microbiological mechanisms. Our aims were to explore a new way for enhancing agroecosystem production, and to provide scientific basis for future utilization of H 2 in agricultural production practices.

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., Competing Interests: The authors declare no conflict of interest.

Enhancing the availability of representative isolates from hydrothermal vents (HTVs) is imperative for comprehending the microbial processes that propel the vent ecosystem. In recent years, Campylobacteria have emerged as the predominant and ubiquitous taxon across both shallow and deep-sea vent systems. Nevertheless, only a few isolates have been cultured, primarily originating from deep-sea HTVs. Presently, no cultivable isolates of Campylobacteria are accessible in shallow water vent systems (<200 m), which exhibit markedly distinct environmental conditions from their deep-sea counterparts. In this study, we enriched a novel isolate (genus Sulfurospirillum , Campylobacteria) from shallow-water HTVs of Kueishan Island. Genomic and physiological analysis revealed that this novel Campylobacteria species grows on a variety of substrate and carbon/energy sources. The pan-genome and phenotypic comparisons with 12 previously isolated Sulfurospirillum species from different environments supported the identification of functional features in Sulfurospirillum genomes crucial for adaptation to vent environments, such as sulfur oxidation, carbon fixation, biofilm formation, and benzoate/toluene degradation, as well as diverse genes related with signal transportation. To conclude, the metabolic characteristics of this novel Campylobacteria augment our understanding of Campylobacteria spanning from deep-sea to shallow-water vent systems.IMPORTANCECampylobacteria emerge as the dominant and ubiquitous taxa within vent systems, playing important roles in the vent ecosystems. However, isolated representatives of Campylobacteria have been mainly from the deep-sea hydrothermal fields, leaving a significant knowledge gap regarding the functions, activities, and adaptation strategies of the vent microorganisms in shallow-water hydrothermal vents (HTVs). This study bridges this gap by providing insights into the phenomics and genomic diversity of genus Sulfurospirillum (order Campylobacterales, class Campylobacteria) based on data derived from a novel isolate obtained from shallow-water HTVs. Our mesophilic isolate of Sulfurospirillum not only augments the genus diversity of Campylobacteria pure cultures derived from vent systems but also serves as the inaugural reference isolate for Campylobacteria in shallow-water environments., Competing Interests: The authors declare no conflict of interest.

Oxidative stress in adipose tissue may alter the secretion pattern of adipocytokines and potentially promote atherosclerosis. However, the therapeutic role of hydrogen in adipose tissue under oxidative stress remains unclear. In this study, subcutaneous adipose tissue (SCAT) was collected from the mid-thoracic wounds of 12 patients who underwent open-heart surgery with a mid-thoracic incision. The adipose tissue was then immersed in a culture medium dissolved with hydrogen, which was generated using a hydrogen-generating device. The weight of the adipose tissue was measured before and after hydrogenation, and the tissue was immunostained for nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and superoxide dismutase (SOD), which are markers of oxidative stress. The immunostaining results showed that HO-1 and Nrf2 expression levels were significantly decreased in the hydrogenated group, whereas SOD expression levels increased, but did not attain statistical significance. Image analysis of adipose tissue revealed that a reduction in adipocyte size. Furthermore, hydrogenated adipose tissue showed a trend toward increased gene expression levels of adiponectin and decreased gene expression levels of chemerin, an adipocytokine involved in adipogenesis. These results demonstrated the therapeutic potential of hydrogen gas for oxidative stress in adipose tissue and for reducing adipocyte size., (© 2024. The Author(s).)

Biofuel can be generated by different organisms using various substrates. The green alga Chlorococcum humicola OQ934050 exhibited the capability to photosynthesize carbonate carbon, maybe via the activity of carbonic anhydrase enzymes. The optimum treatment is C:N ratio of 1:1 (0.2 mmoles sodium carbonate and 0.2 mmoles sodium nitrate) as it induced the highest dry mass (more than 0.5 mg.mL -1 ). At this combination, biomass were about 0.2 mg/mL -1 carbohydrates, 0.085 mg/mL -1 proteins, and 0.16 mg/mL -1 oil of this dry weight. The C/N ratios of 1:1 or 10:1 induced up to 30% of the Chlorococcum humicola dry mass as oils. Growth and dry matter content were hindered at 50:1 C/N and oil content was reduced as a result. The fatty acid profile was strongly altered by the applied C.N ratios. The defatted leftovers of the grown alga, after oil extraction, were fermented by a newly isolated heterotrophic bacterium, identified as Bacillus coagulans OQ053202, to evolve hydrogen content as gas. The highest cumulative hydrogen production and reducing sugar (70 ml H 2 /g biomass and 0.128 mg/ml; respectively) were found at the C/N ratio of 10:1 with the highest hydrogen evolution efficiency (HEE) of 22.8 ml H 2 / mg reducing sugar. The optimum treatment applied to the Chlorococcum humicola is C:N ratio of 1:1 for the highest dry mass, up to 30% dry mass as oils. Some fatty acids were induced while others disappeared, depending on the C/N ratios. The highest cumulative hydrogen production and reducing sugar were found at the C/N ratio of 10:1., (© 2024. The Author(s).)

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 CO 2 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 k cat as well as chimeric versions with promising features to attain gain-of-function variants and applications in various biotechnological processes are also presented.

Background: Clean energy hydrogen (H 2 ) produced from abundant lignocellulose is an alternative to fossil energy. As an essential influencing factor, there is a lack of comparison between constant temperatures (35, 55 and 65 °C) and gradient heating temperature (35 to 65 °C) on the H 2 production regulation potential from lignocellulose-rich straw via high-solid anaerobic digestion (HS-AD). More importantly, the microbial mechanism of temperature regulating H 2 accumulation needs to be investigated., Results: Constant 65 °C led to the lowest lignin residue (1.93%) and the maximum release of cellulose and hemicellulose, and the highest H 2 production (26.01 mL/g VS). H 2 production at 35 and 55 °C was only 14.56 and 24.13 mL/g VS, respectively. In order to further explore the potential of ultra-high temperature (65 °C), HS-AD was performed by gradient heating conditions (35 to 65 °C). However, compared to constant 65 °C, gradient heating conditions led to higher lignin residue (2.49%) and lower H 2 production (13.53 mL/g VS) than gradient heating conditions (47.98%). In addition, metagenomic analysis showed the cellulose/hemicellulose hydrolyzing bacteria and genes (mainly Thermoclostridium, and xynA, xynB, abfA, bglB and xynD), H 2- producing bacteria and related genes (mainly Thermoclostridium, and nifD, nifH and nifK), and microbial movement and metabolic functions were enriched at 65 °C. However, the enrichment of two-component systems under gradient heating conditions resulted in a lack of highly-enriched ultra-high-temperature cellulose/hemicellulose hydrolyzing genera and related genes but rather enriched H 2 consumption genera and genes (mainly Acetivibrio, and hyaB and hyaA) resulting in a weaker H 2 production., Conclusions: The lignin degradation process does not directly determine H 2 accumulation, which was actually regulated by bacteria/genes contributing to H 2 production/consumption. In addition, it is temperature that enhances the hydrolysis process of lignin rather than lignin-degrading enzymes, bacteria and genes by promoting microbial material transfer and metabolism. In terms of temperature, one of the key parameters of HS-AD for H 2 production, we developed an important regulatory strategy, enriched the theoretical basis of temperature regulation for H 2 production to further expanded the research horizon in this field. Video Abstract., (© 2024. The Author(s).)

The hydrogen molecule can effectively regulate plant growth and development, improving plant resistance to abiotic stresses. However, studies regarding the optimal concentration of hydrogen and the associated mechanisms of action in organisms are lacking. This study showed that the maximum germination rate of radish seeds decreased from 90 % to 50 % under the stress of cadmium ions (Cd 2+ ), and hydrogen nanobubble (NB) water significantly alleviated the stress effect of Cd 2+ on radish seed germination. A hydrogen concentration of 0.8 ppm had the best effect, reducing Cd 2+ accumulation in radish seeds by 63.23 % and increasing the maximum germination rate from 50 % to 65 %. At concentrations exceeding 1.2 ppm, the beneficial effect of hydrogen was weakened or even reversed. Consequently, we integrated the concept of the oxidative window into a REDOX balance model and demonstrated that an appropriate hydrogen concentration can effectively maintain the REDOX state within organisms. Transcriptome sequencing analysis revealed that hydrogen NB water modulated Cd 2+ absorption and accumulation in seeds by regulating cell wall components, alleviating oxidative stress through oxidoreductase activity, and enhancing nutrient synthesis and metabolism. This collectively alleviated the inhibitory effect of Cd 2+ on seed germination. This study is helpful for further understanding the effect of hydrogen concentration on the REDOX balance of seed germination, providing a theoretical basis for selecting hydrogen concentration to improve its effectiveness in agricultural fields., Competing Interests: Declaration of Competing Interest TThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although the past decade has witnessed a rapid development of oxidoreductase-mimicking nanozymes, the mimicry of cofactors that play key roles in mediating electron and proton transfer remains limited. This study explores how surface Au-H species conjugated to Au nanoparticles (NPs) that imitate formate dehydrogenase (FDH) can serve as cofactors, analogous to NADH in natural enzymes, offering diverse possibilities for FDH-mimicking Au nanozymes to mimic various enzymes. Once O 2 is present, Au-H species assist Au NPs to complete the on-demand H 2 O 2 generation for cascade reactions. Alternatively, when oxidizing organic molecules are introduced as substrates, Au-H species confer nitro reductase- and aldehyde reductase-like activities on Au NPs under anaerobic conditions. Furthermore, similar to the dehydrogenase-NADH complex, Au NPs possessing Au-H species are gifted with esterase-like activity for ester hydrolysis. By revealing that Au-H species are prosthetic groups for FDH-mimicking Au nanozymes, this work may inspire explorations into future self-generated cofactor mimics for nanozymes, thereby circumventing the need for exogenous cofactors.

Microbial electrolysis cell (MEC) is gaining importance not only for effectively treating wastewater but also for producing hydrogen. The up-flow microbial electrolysis cell (UPMEC) is an innovative approach to enhance the efficiency, and substrate degradation. In this study, a baffled UPMEC with an anode divided into three regions by inserting the baffle (sieve) plates at varying distances from the cathode was designed. The effect of process parameters, such as flow rate (10, 15, and 20 mL/min), electrode area (50, 100, and 150 cm 2 ), and catholyte buffer concentration (50, 100, and 150 mM) were investigated using distillery wastewater as substrate. The experimental results showed a maximum of 0.6837 ± 0.02 mmol/L biohydrogen at 150 mM buffer, with 49 ± 1.0% COD reduction using an electrode of area 150 cm 2 . The maximum current density was 1335.94 mA/m 2 for the flow rate of 15 mL/min and surface area of 150 cm 2 . The results showed that at optimized flow rate and buffer concentration, maximum hydrogen production and effective treatment of wastewater were achieved in the baffled UPMEC. PRACTITIONER POINTS: Biohydrogen production from distillery wastewater was investigated in a baffled UPMEC. Flowrate, concentration and electrode areas significantly influenced the hydrogen production. Maximum hydrogen (0.6837±0.02mmol/L.day) production and COD reduction (49±1.0%) was achieved at 15 mL/min. Highest CHR of 95.37±1.9 % and OHR of 4.6±0.09 % was observed at 150 mM buffer concentration., (© 2024 Water Environment Federation.)

Biomethanation represents a promising approach for biomethane production, with biofilm-based processes like trickle bed reactors (TBRs) being among the most efficient solutions. However, maintaining stable performance can be challenging, and both pure and mixed culture approaches have been applied to address this. In this study, inocula enriched with hydrogenotrophic methanogens were introduced to to TBRs as bioaugmentation strategy to assess their impacts on the process performance and microbial community dynamics. Metagenomic analysis revealed a metagenome-assembled genome belonging to the hydrogenotrophic genus Methanobacterium, which became dominant during enrichment and successfully colonized the TBR biofilm after bioaugmentation. The TBRs achieved a biogas production with > 96 % methane. The bioaugmented reactor consumed additional H 2. This may be due to microbial species utilizing CO 2 and H 2 via various CO 2 reduction pathways. Overall, implementing bioaugmentation in TBRs showed potential for establishing targeted species, although challenges remain in managing H 2 consumption and optimizing microbial interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The global climate change mainly caused by fossil fuels combustion promotes that zero-carbon hydrogen production through eco-friendly methods has attracted attention in recent years. This investigation explored the biohydrogen production by co-fermentation of corn straw (CS) and excess sludge (ES), as well as comprehensively analyzed the internal mechanism. The results showed that the optimal ratio of CS to ES was 9:1 (TS) with the biohydrogen yield of 101.8 mL/g VS, which was higher than that from the mono-fermentation of CS by 1.0-fold. The pattern of volatile fatty acids (VFAs) indicated that the acetate was the most preponderant by-product in all fermentation systems during the biohydrogen production process, and its yield was improved by adding appropriate dosage of ES. In addition, the content of soluble COD (SCOD) was reduced as increasing ES, while concentration of NH 4 + -N showed an opposite tendency. Microbial community analysis revealed that the microbial composition in different samples showed a significant divergence. Trichococcus was the most dominant bacterial genus in the optimal ratio of 9:1 (CS/ES) fermentation system and its abundance was as high as 41.8%. The functional genes prediction found that the dominant metabolic genes and hydrogen-producing related genes had not been significantly increased in co-fermentation system (CS/ES = 9:1) compared to that in the mono-fermentation of CS, implying that enhancement of biohydrogen production by adding ES mainly relied on balancing nutrients and adjusting microbial community in this study. Further redundancy analysis (RDA) confirmed that biohydrogen yield was closely correlated with the enrichment of Trichococcus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δ 2 H), carbon (δ 13 C), and nitrogen (δ 15 N) stable isotope ratios are usually adopted. However, previous studies have not fully accounted for exchangeable hydrogens, which could affect these evaluations. Here, we performed standard δ 13 C, δ 15 N, and δ 2 H analyses on bulk samples. Additionally, we conducted δ 2 H analysis on α-cellulose and cellulose nitrate samples to investigate whether the heterogeneity of exchangeable hydrogens among plant species influences the assessment of nutritional modes. The δ 2 H of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δ 13 C, its δ 2 H was higher than those of the autotrophs. Notably, among all response variables, δ 15 N and δ 2 H exhibited high abilities for discriminating the nutritional modes of rhizoctonia-associated orchids. These results indicate that a time-efficient bulk sample analysis is an effective method for evaluating plant nutritional modes, as the heterogeneity of exchangeable hydrogens does not significantly impact the estimation. Using δ 15 N and δ 2 H benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Despite being classified as microaerophilic microorganisms, most Campylobacter species can grow anaerobically, using formate or molecular hydrogen (H 2 ) as electron donors, and various nitrogenous and sulfurous compounds as electron acceptors. Herein, we showed that both l-asparagine (l-Asn) and l-aspartic acid (l-Asp) bolster H 2 -driven anaerobic growth in several Campylobacter species, whereas the d-enantiomer form of both asparagine (d-Asn) and aspartic acid (d-Asp) only increased anaerobic growth in Campylobacter concisus strain 13826 and Campylobacter ureolyticus strain NCTC10941. A gene annotated as racD encoding for a putative d/l-Asp racemase was identified in the genome of both strains. Disruption of racD in Cc13826 resulted in the inability of the mutant strain to use either d-enantiomer during anaerobic growth. Hence, our results suggest that the racD gene is required for campylobacters to use either d-Asp or d-Asn. The use of d-Asp by various human opportunistic bacterial pathogens, including C. concisus, C. ureolyticus, and also possibly select strains of Campylobacter gracilis, Campylobacter rectus and Campylobacter showae, is significant, because d-Asp is an important signal molecule for both human nervous and neuroendocrine systems. To our knowledge, this is the first report of pathogens scavenging a d-amino acid essential for human health., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Hydrogen-ethanol co-production can significantly improve the energy conversion efficiency of corn stalk (CS). In this study, with CS as the raw material, the co-production characteristics of one-step and two-step photo-fermentation hydrogen production (PFHP) and ethanol production were investigated. In addition, the gas and liquid characteristics of the experiment were analyzed. The kinetics of hydrogen-ethanol co-production was calculated, and the economics of hydrogen and ethanol were analyzed. Results of the experiments indicated that the two-step hydrogen-ethanol co-production had the best hydrogen production performance when the concentration of CS was 25 g/L. The total hydrogen production was 350.08 mL, and the hydrogen yield was 70.02 mL/g, which was 2.45 times higher than that of the one-step method. The efficiency of hydrogen-ethanol co-production was 17.79 %, which was 2.76 times more efficient than hydrogen compared to fermentation with hydrogen. The result provides technical reference for the high-quality utilization of CS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)