Hydrogenase Activity Research Articles

New insights into microalgal photobiological hydrogen production: Potential role of aggregation forms in modulating the hydrogenase activity and metabolic properties of microalgae during hydrogen production

Photobiological hydrogen production of microalgae is highly influenced by environmental conditions, and the appropriate microalgal aggregation form is conducive to microalgae overcome environmental limitations and increase their hydrogen production capacity. In this study, the effects of three aggregation methods including self-flocculation, biofilm attachment, and gel immobilization on the photobiological hydrogen production of both prokaryotic Synechocystis sp. and eukaryotic Chlamydomonas reinhardtii were investigated. The results indicated that microalgal aggregates formed via gel immobilization exhibited superior physicochemical properties such as porosity, integrity, and oxygen diffusion coefficients, creating favorable conditions for enhanced hydrogenase activity. Upon gel-immobilization, hydrogen production of Synechocystis sp. and C. reinhardtii exhibited an increase of 1.89 and 2.02-folds, respectively, compared with suspended microalgae. Concurrently, hydrogenase activities increased to 2.07 and 32.9 H2/(mg chlorophyll·h), respectively. The gel-immobilized aggregate form prompted the microalgae to maintain a relatively high photosynthetic activity, with the electron transfer rate reaching more than 1.58 folds of the control. Notably, three aggregated forms also accelerated oxygen consumption, and intracellular organic matter degradation, thereby optimizing electron utilization by microalgal hydrogenase. Furthermore, aggregation up-regulated hydrogenase gene expression in both species, particularly in gel-immobilized groups (2.34- and 4.14-folds increase compared with the control for Synechocystis sp. and C. reinhardtii, respectively). Subsequently, significantly upregulated gene expression related to oxidative phosphorylation and antioxidant systems was observed in C. reinhardtii aggregates, correlating with its relatively higher increase in hydrogen production compared with Synechocystis sp. aggregates. This study elucidated the mechanism by which aggregation strengthens microalgal hydrogen production and offers insights crucial for its industrialization.

Limosilactobacillus Regulating Microbial Communities to Overcome the Hydrolysis Bottleneck with Efficient One-Step Co-Production of H2 and CH4.

The efficient co-production of H2 and CH4 via anaerobic digestion (AD) requires separate stages, as it cannot yet be achieved in one step. Lactic acid bacteria (LAB) (Limosilactobacillus) release H2 and acetate by enhancing hydrolysis, potentially increasing CH4 production with simultaneous H2 accumulation. This study investigated the enhanced effect of one-step co-production of H2 and CH4 in AD by LAB and elucidated its enhancement mechanisms. The results showed that 236.3 times increase in H2 production and 7.1 times increase in CH4 production are achieved, resulting in profits of 469.39 USD. Model substrates lignocellulosic straw, sodium acetate, and H2 confirmes LAB work on the hydrolysis stage and subsequent sustainable volatile fatty acid production during the first 6 days of AD. In this stage, the enrichment of Limosilactobacillus carrying bglB and xynB, the glycolysis pathway, and the high activity of protease, acetate kinase, and [FeFe] hydrogenase, jointly achieved rapid acetate and H2 accumulation, driving hydrogenotrophic methanogenesis dominated. From day 7 to 24, with enriched Methanosarcina, and increased methenyltetrahydromethanopterin hydrogenase activity, continuously produced acetate led to the mainly acetoclastic methanogenesis shift from hydrogenotrophic methanogenesis. The power generation capacity of LAB-enhanced AD is 333.33 times that of China's 24,000m3 biogas plant.

The intracellular accumulation of iron coincides with enhanced biohydrogen production by Thermoanaerobacterium thermosaccharolyticum

The aim of this study was to investigate the effects of different doses (10–200 mg/L) of magnetite (FeO·Fe2O3), Fe0, Ni0, and FeCl3 on the efficiency of dark fermentative H2 production from cheese whey using Thermoanaerobacterium thermosaccharolyticum SP-H2. Energy conversion efficiency and hydrogen yield increased significantly by 22.7 % and 34.8 %, respectively, when 10 mg/L of magnetite was added, and hydrogenase activity increased by 23.6 % when 100 mg/L of Fe0 was added compared to the control (p < 0.001). Using scanning electron microscopy and energy dispersive X-ray spectroscopy, it was found that during the early exponential phase of hydrogen evolution, T. thermosaccharolyticum cells accumulated up to 11.2 times more Fe (up to 2.8 wt%) in the presence of optimal concentrations of Fe additives compared to the control. Intracellular iron accumulation and hydrogen yield were positively correlated with acetate concentration (p < 0.001), suggesting an efficient acetate pathway for dark fermentation. During the stationary growth phase, with almost no H2 production, the intracellular iron content decreased by up to 67.9 %. The phenomenon of changes in the amount of metal in cells, depending on the phase of hydrogen production, was demonstrated for the first time. The findings suggest that intracellular metals can be used in cell metabolism to produce H2, and the mechanisms behind this process need further investigation. Overall, this study provides valuable insights into the optimal forms and doses of metals for enhancing biohydrogen production, as well as possible research directions for the potential use of metals by microorganisms during additive-assisted dark fermentation of carbohydrate-rich wastes.

The H-cluster of [FeFe] Hydrogenases: Its Enzymatic Synthesis and Parallel Inorganic Semisynthesis.

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

Enhancing dark fermentative hydrogen production from wheat straw through synergistic effects of active electric fields and enzymatic hydrolysis pretreatment

Hydrogen, a clean and sustainable energy source, faces challenges from energy-intensive pre-processing technologies. This study explores the synergistic enhancement of active electric fields on enzymolysis of wheat straw and hydrogen production through dark fermentation. The active electric field enzymolysis system improved the adsorption capacity of wheat straw to cellulase, increasing cellulase activity by 18.0 %, causing a 39.1 % increase in reducing sugar content. In the active fermentation system, Clostridium_sensu_stricto_1 activity was enhanced in the first stage, increasing hydrogenase activity by 23.0 %, prolonging the first hydrogen production peak. Elevated reducing sugars were observed in the second stage, with Prevotella_9 and Bacteroides becoming the dominant hydrogen-producing bacteria in the third stage, leading to a second hydrogen production peak. Overall, cumulative hydrogen production was enhanced by 50.9 % compared to the control. The synergistic pretreatment with an active electric field and cellulase provides a novel approach for efficiently utilizing wheat straw.

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.

Application of recyclable iron-based supplementation for promoting biohydrogen and ethanol co-production

Supplementation additives is a promising approach in enhancing hydrogen production during fermentation processes, owing to the advantages of simple operation and low energy consumption. Exploring efficient and recyclable additives is a pivotal issue for its industrial applications. In this study, a magnetic recyclable iron-based biochar (FeBCs) was synthesized by one-step co-pyrolysis of straw biomass and K2FeO4 to enhance hydrogen production of Ethanoligenens harbinense. Results showed that the biochar derived by pyrolysis at 1000 °C (FeBC1000) established the highest H2 yield (2.09 mol·H2/mol·glucose) and ethanol yield (82.35 ± 3.27 mM) being 41.21 % and 22.27 % higher than that of the control group, respectively. The addition of FeBCs facilitated the substrate consumption and the excretion of protein and carbohydrate in extracellular polymeric substance (EPS). Magnetic FeBC1000 displayed the remarkable reusability, and the H2 yield was 33.59 % higher than that of the control group after being recycled for three times. The mechanism of FeBC1000 enhancing hydrogen production was that it promoted hydrogenase activity by reducing the redox potential, enhancing electron transfer, buffering the pH, releasing ferrous ions, and promoting cell immobilization. Overall, the study provided a recyclable and efficient magnetic additive for biohydrogen production, and operate a new direction for comprehensive utilization of straw with low energy input.

Mechanism of ZnFe2O4 NPs on biohydrogen production by Clostridium pasteurianum

The biohydrogen production in Dark fermentation (DF) could be enhanced by ZnFe2O4 nanomaterials (NPs). The optimum conditions were obtained by a response surface method. Eventually, a biohydrogen production of 2645 mL/L was obtained after 48 h of batch culture, which is 116% higher than control without ZnFe2O4 NPs. The mechanism was investigated by a multi-scale method. In bioreactor scale, ZnFe2O4 NPs could enhance the glucose metabolic ability of C. pasteurianum to promote cell growth and biohydrogen production. The metabolic pathways of biohydrogen production were shifted from the butyric acid-type to the ethanol-type. In cellular scale, ZnFe2O4 NPs could reduce bacterial flagella assembly. In macromolecule scale, the activities of hydrogenase and ethanol dehydrogenase were increased. In genetic scale, the differentially expressed genes mainly involved in biohydrogen production, like those encoded Fe-only hydrogenase, ethanol dehydrogenase, and nitrogenase were up-regulated. The results could provide theoretical and technical supports for the development of commercial biohydrogen process.

Screening of enhanced biohydrogen production from anaerobic fermentation amended with nano-sized barium ferrite supported by aluminum oxide

Anaerobic fermentation has attracted wide attention to effectively reduce carbon footprint and alleviate potential energy shortages, which is being regarded as a sustainable and promising technology to obtain high biohydrogen production from widespread organic wastes. However, biohydrogen commercial uptake is now dominantly inhibited by the low hydrogen yield and process instability. In this work, Al2O3/BaFe2O4 nanoparticles (NPs) are successfully applied to anaerobic fermentation for investigating their effects on hydrogen yield, activity of bacteria and microbial community structure. The benefits of Al2O3/BaFe2O4 NPs with respect to microbial metabolism, reactivity of enzymes and process optimization are systematically researched through batch experiments. In addition, various characterizations are used to verify the physical and chemical properties of Al2O3/BaFe2O4 NPs. The results show that the activity of hydrogenases and microbe metabolism can be significantly inhibited by excess addition of Al2O3/BaFe2O4 NPs (150 mg/L and 200 mg/L) while the optimum addition 100 mg/L of Al2O3/BaFe2O4 NPs can boost the biohydrogen yield by 49% via facilitating microorganism growth and enhancing key enzymatic activities, which indicate that the Al2O3/BaFe2O4 NPs display a beneficial influence on electron transfer efficiency and process stability. The existence of Fe3+, Al3+and Ba2+ derived from the corrosion of Al2O3/BaFe2O4 NPs by organic acids synergistically improve the microbial transmembrane transportation and substrate utilization, which contribute to higher conversion rate of glucose into acetate, butyrate and H2. Meanwhile, the complementary functions of Fe3+ and Al3+ can powerfully shorten the lag phase and promote the glycolysis, which finally accelerate the dark fermentation process. Compared with the control group, Clostridium_sensu_stricto_1 is selectively enriched by the addition of Al2O3/BaFe2O4 NPs, which keep heavily involved in the synthesis of H2. On balance, this study provides an effective strategy to enhance hydrogen productivity and maintain process stability, which offer the valuable information for the sustainable utilization of sewage sludge.

Enhanced H2 photoproduction in sodium sulfite-treated Chlamydomonas reinhardtii by optimizing wavelength of pulsed light

Photosynthetic hydrogen induced by Chlamydomonas reinhardtii presents a promising pathway for producing clean and renewable biofuel. Whereas, this process is transient owing to the repression of [FeFe]-hydrogenase by the oxygen co-evolved in photosystem II. Pulsed light is employed to prevent O2 accumulation and the activation of Calvin-Benson-Bassham cycle for sustainable H2. While light quality significantly influences photosynthesis, the optimal wavelength for pulsed light-induced H2 production remains not fully elucidated. Continuous irradiation at 660–670 nm performs poorly in H2 production due to the activation of PSII, which is responsible for water oxidation. However, in this study, O2 was removed using a combination of pulsed light and sulfite treatment. Among six light regimes with different wavelengths, pulsed light peaking at 660 nm resulted in the highest photo-H2 evolution, reaching approximately 180 mL L−1 within three days. This value was 1.4 times higher than that achieved under conventionally white pulsed light. Exposure to 660 nm red pulsed light not only sustained high activity of PSII and hydrogenase but also reduced the gene expression levels of ribulose-1,5-bisphosphate carboxylase/oxygenase and ferredoxin-NADP+ reductase, which was supposed to attenuate the competing effects of CBB cycle. This finding optimizes an efficient light source for pulsed light-induced H2 production and demonstrates a great potential for refining pulsed light parameters to improve H2 production in algae in the future.

Strategies to enhance biohydrogen production from microalgae: A comprehensive review

Microalgae represent a promising renewable feedstock for the sustainable production of biohydrogen. Their high growth rates and ability to fix carbon utilizing just sunlight, water, and nutrients make them well-suited for this application. Recent advancements have focused on improving microalgal hydrogen yields and cultivation methods. This review aims to summarize recent developments in microalgal cultivation techniques and genetic engineering strategies for enhanced biohydrogen production. Specific areas of focus include novel microalgal species selection, immobilization methods, integrated hybrid systems, and metabolic engineering. Studies related to microalgal strain selection, cultivation methods, metabolic engineering, and genetic manipulations were compiled and analyzed. Promising microalgal species with high hydrogen production capabilities such as Synechocystis sp., Anabaena variabilis, and Chlamydomonas reinhardtii have been identified. Immobilization techniques like encapsulation in alginate and integration with dark fermentation have led to improved hydrogen yields. Metabolic engineering through modulation of hydrogenase activity and photosynthetic pathways shows potential for enhanced biohydrogen productivity. Considerable progress has been made in developing microalgal systems for biohydrogen. However, challenges around process optimization and scale-up remain. Future work involving metabolic modeling, photobioreactor design, and genetic engineering of electron transfer pathways could help realize the full potential of this renewable technology.

Flocculation-enhanced photobiological hydrogen production by microalgae: Flocculant composition, hydrogenase activity and response mechanism

The practical application of microalgal photobiological hydrogen was limited by its low yield and short production time. In this study, the promotion of inorganic flocculants (polymeric aluminum chloride, PAC) and organic flocculants (cationic polyacrylamide, CPAM) to the aggregate formation and photobiological hydrogen production of prokaryotic microalgae (Synechocystis sp.) and eukaryotic microalgae (Chlamydomonas reinhardtii) were investigated. The effectiveness of PAC-CPAM composite flocculants in promoting aggregation was compared to single PAC or CPAM, with flocculation efficiencies exceeding 92.2 % for both microalgal strains. After aggregate formation, hydrogen production in Synechocystis and C. reinhardtii increased by 65.9 % and 80.2 %, respectively, compared with the control (without flocculation). Their hydrogenase activity rose to 1.14 and 28.2 μmol H2/(mg chlorophyll·h), respectively. Aggregation sustained the photosynthetic activity of both Synechocystis and C. reinhardtii, with the photosynthetic system electron transfer rate (ETR) 1.54–1.72 times higher than that of the control. The formation of aggregates enhanced microalgal respiration, accelerating the reduction of oxygen content, and increasing hydrogenase activity. Notably, the increased respiration in Synechocystis and C. reinhardtii aggregates supplied more electrons, enhancing hydrogen production by consuming intracellular organic matter. Aggregate formation upregulated the expression of Synechocystis and C. reinhardtii hydrogenase genes by average 2.1-fold and 2.5-fold, respectively. Simultaneously, the expression of genes related to photosynthetic system Ⅱ, ferredoxin, pyruvate ferredoxin-oxidoreductase, and oxidative phosphorylation were upregulated, with a significant effect observed in C. reinhardtii aggregates. The results of this study advanced the utilization and industrialization of microalgae-based green hydrogen production, contributing to carbon neutrality.

Electron syntrophy between mixed hydrogenogens and Geobacter metallireducens boosted dark hydrogen fermentation: Clarifying roles of electroactive extracellular polymeric substances

To modulate the electron transfer behavior of hydrogen-producing bacteria (HPB) for enhanced hydrogen production, Geobacter metallireducens culture (GM) was introduced as an electron syntrophy partner and redox balance regulator in dark fermentation systems with hydrogen-producing sludge (HPS) as inoculum. The highest hydrogen yield was 306.5 mL/g-COD at the GM/HPS volatile solids ratio of 0.08, which was 65.2 % higher than the HPS group. The multi-layered extracellular polymeric substances (EPS) of GM played a significant role in promoting hydrogen production, with c-type cytochromes probably serving as electroactive functional components. The addition of GM significantly improved the NADH/NAD+ ratio, electron transport system activity, hydrogenase activity, and electrochemical properties of HPS. Furthermore, the microbial community structure and metabolic functions were optimized due to the potential syntrophic interaction between Clostridium sensu stricto (dominant HPB) and Geobacter, thus promoting hydrogen production. This study provided novel insights into the interactions among exoelectrogens, electroactive EPS, and mixed HPB.

Gold nanoparticles activate hydrogenase synthesis and improve heterotrophic growth of Ralstonia eutropha H16.

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H2 and CO2. Hydrogenases (Hyds) are synthesized under lithoautotrophic, or energy-limited heterotrophic conditions and are used in enzyme fuel cells (EFC) as anodic catalysts. The effects of chemically synthesized gold nanoparticles (Au-NPs) on R. eutropha H16 growth, oxidation-reduction potential (ORP) kinetics, and H2-oxidizing Hyd activity were investigated in this study. Atomic force microscopy showed that thin, plate-shaped Au-NPs were in the nanoscale range with an average size of 5.68 nm. Compared with growth in medium without Au-NPs (control), the presence of Au-NPs stimulated growth, and resulted in a decrease in ORP to negative values. H2-oxidizing activity was not detected in the absence of Au-NPs, but activity was significantly induced (12 U/g CDW) after 24 h of growth with 18 ng/ml, increasing a further 4-fold after 72 h of growth. The results demonstrate that Au-NPs primarily influence the membrane-bound Hyd. In contrast to R. eutropha, Au-NPs had a negligible or negative effect on the growth, Hyd activity, and H2 production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs.

Photoautotrophic and sustained H2 production by the pgr5 mutant of Chlamydomonas reinhardtii in simulated daily light conditions

Green algae, such as Chlamydomonas reinhardtii, can produce H2 efficiently using their hydrogenases, but these enzymes are O2-sensitive and compete with CO2-fixation for electrons. To overcome these limitations, we previously developed an anaerobic, carbon-limited protocol that keeps the Calvin-Benson cycle inactive, and the evolved O2 is scavenged by an absorbent. The PROTON-GRADIENT REGULATION5 (PGR5)-deficient mutant performs better than the wild type in this system, as it produces at least 2.5-fold more H2 and maintains its photosynthetic apparatus and hydrogenase activity even at the intensity of sunlight. However, as the pgr5 mutant is known to be sensitive to fluctuating light conditions, it is of paramount interest to determine how it reacts to changes in light intensity during H2 production. Therefore, we developed an automated system to monitor H2 production in thin layer cultures under simulated daily light conditions. We found that the pgr5 mutant outperformed the wild type strain by 100 % when light intensity was varied stepwise between 0 and 1000 μmol photons m−2s−1 during the day. Photosynthetic subunits, including PsbA, PSBO, CP47, PetB and PsaA, were fully preserved in the pgr5 mutant, and approximately 29 % of its original hydrogenase activity was sustained after 85 h of H2 production in simulated daily light conditions. Hence, the pgr5 mutant is a promising candidate for H2 production under the adverse light conditions that algae may encounter in bioindustry settings.

Enhanced thermophilic dark fermentation of hydrogen production from food waste by Fe-modified biochar

The industrialization of hydrogen production through dark fermentation of food waste faces challenges, such as low yields and unpredictable fermentation processes. Biochar has emerged as a promising green additive to enhance hydrogen production in dark fermentation. Our study demonstrated that the introduction of Fe-modified biochar (Fe-L600) significantly boosted hydrogen production during thermophilic dark fermentation of food waste. The addition of Fe-L600 led to a remarkable 31.19% increase in hydrogen yield and shortened the time needed for achieving stabilization of hydrogen production from 18 h to 12 h. The metabolite analysis revealed an enhancement in the butyric acid pathway as the molar ratio of acetic acid to butyric acid decreased from 3.09 to 2.69 but hydrogen yield increased from 57.12 ± 1.48 to 76.78 ± 2.77 mL/g, indicating Fe-L600 improved hydrogen yield by regulating crucial metabolic pathways of hydrogen production. The addition of Fe-L600 also promoted the release of Fe2+ and Fe3+ and increased the concentrations of Fe2+ and Fe3+ in the fermentation system, which might promote the activity of hydrogenase and ferredoxin. Microbial community analysis indicated a substantial increase in the relative abundance of Thermoanaerobacterium after thermophilic dark fermentation. The relative abundances of microorganisms responsible for hydrolysis and acidogenesis were also observed to be improved in the system with Fe-L600 addition. This research provides a feasible strategy for improving hydrogen production of food waste and deepens the understanding of the mechanisms of biochar.

Mitochondria: one of the vital hubs for molecular hydrogen's biological functions.

As a novel antioxidant, a growing body of studies has documented the diverse biological effects of molecular hydrogen (H2) in a wide range of organisms, spanning animals, plants, and microorganisms. Although several possible mechanisms have been proposed, they cannot fully explain the extensive biological effects of H2. Mitochondria, known for ATP production, also play crucial roles in diverse cellular functions, including Ca2+ signaling, regulation of reactive oxygen species (ROS) generation, apoptosis, proliferation, and lipid transport, while their dysfunction is implicated in a broad spectrum of diseases, including cardiovascular disorders, neurodegenerative conditions, metabolic disorders, and cancer. This review aims to 1) summarize the experimental evidence on the impact of H2 on mitochondrial function; 2) provide an overview of the mitochondrial pathways underlying the biological effects of H2, and 3) discuss H2 metabolism in eukaryotic organisms and its relationship with mitochondria. Moreover, based on previous findings, this review proposes that H2 may regulate mitochondrial quality control through diverse pathways in response to varying degrees of mitochondrial damage. By combining the existing research evidence with an evolutionary perspective, this review emphasizes the potential hydrogenase activity in mitochondria of higher plants and animals. Finally, this review also addresses potential issues in the current mechanistic study and offers insights into future research directions, aiming to provide a reference for future studies on the mechanisms underlying the action of H2.

Metabolic division of labor between Acetivibrio thermocellus DSM 1313 and Thermoanaerobacterium thermosaccharolyticum MJ1 enhanced hydrogen production from lignocellulose

In this consortium, DSM 1313 was responsible for degrading lignocellulose by cellulosome, while the highly efficient hydrogen-producing bacterium MJ1 consumed the sugar produced by DSM 1313 to grow and produce more hydrogen. The results showed that the maximum hydrogen production of 259.57 mL/g substrate was obtained at the inoculation ratio (OD600) of 2:1 (DSM 1313:MJ1) and substrate concentration of 10 g/L, 70.84 % higher than pure culture. Furthermore, MJ1 dominated the co-culture system by using various sugars resulting from the biodegradation of substrate, thereby relieving the inhibition of sugar on DSM 1313 and leading to more hydrogen production. In the co-culture system, the value of extracellular oxidation–reduction potential and the ratio of NAD+/NADH was lower than that of pure culture. Additionally, at the gene level, [NiFe]-hydrogenase and [FeFe]-hydrogenase related enzymes were significantly up-regulated, leading to a two-fold increase in hydrogenase activity of co-culture compared with pure culture.

Comparative genomic analysis reveals electron transfer pathways of Thermoanaerobacterium thermosaccharolyticum: Insights into thermophilic electroactive bacteria

Microbial extracellular respiration is an important energy metabolism on earth, which is significant for the elemental biogeochemical cycle. Herein, extracellular Fe(III) and electrode respiration were confirmed in Thermoanaerobacterium thermosaccharolyticum MJ2. The intra/extracellular electron transfer (IET/EET) mechanism of MJ2 was investigated by comparative genomic analysis for the first time. Morphological characterization and electrochemical properties of anode illustrated that MJ2 generated bio-electricity by forming a biofilm. The respiration chain inhibition and enzyme activity tests showed that hydrogenase with cytochrome c (Cyt-c) was involved in IET of MJ2. Noteworthily, the exogenous Cyt-c increased hydrogenase activity to promote bio-electricity generation by 92.84 %. The Cyt-c gene synteny between MJ2 and another well-known exoelectrogen (Thermincola potens JR) indicated that Cyt-c bound to the outer membrane mediated the formation of biofilm involved in EET of MJ2. This study broadened the understanding of microbial extracellular respiration diversity and provided new insights to explore the electron transfer pathways of exoelectrogens.

Unraveling the role of polymeric ferric sulfate in promoting propionic acid in anaerobic sludge hydrogen production fermentation

Polymerized ferric sulfate (PFS) is widely employed in the pretreatment of industrial wastewater and sludge dewatering. Its widespread use results in its accumulation in waste activated sludge (WAS) and may impact anaerobic fermentation (AF). This work fills the gap in the effects and mechanisms of PFS on hydrogen (H2) production from AF under mesophilic and thermophilic conditions. PFS (40 g/kg total solids, TS) reduced cumulative H2 production by 59.2% (mesophilic conditions) and 52.6% (thermophilic conditions) (p < 0.001), whereas propionic acid production increased by 299.2% and 192.0%, respectively. Model-based analysis showed that PFS inhibited solubilization, hydrolysis, and acidification below 10 g/kg TS but promoted them above 10 g/kg TS. Changes in the ratio of the two forms of nicotinamide adenine dinucleotide (NADH/NAD+) and hydrogenase activity led to unfavorable pathways for H2 production. Thermophilic fermentation exhibited strong tolerance to PFS due to the increased release of extracellular polymeric substances (EPSs). By analyzing 16S-rRNA gene operational taxonomic units (OTUs), the effects of environmental factors on microbial communities and microbial interactions under the combined effects of PFS and temperature were identified, revealing the important factors affecting H2 production. Sludge containing PFS was not conducive to anaerobic H2 production; however, mesophilic fermentation with PFS may be a potential way to recover propionic acid with low energy consumption. This study provided insights into the effects of potential contaminants in WAS on AF for H2 production and information for the effective disposal and resource utilization of WAS.