Formate Hydrogenlyase Research Articles

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

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

FhlA is a Formate Binding Protein.

Escherichia coli uses glycolysis and mixed acid fermentation and produces formate as by product. One system E. coli uses for formate oxidation is formate hydrogen lyase complex (FHL). The expression of the FHL complex is dependent on formate and regulated by the transcriptional regulator FhlA. The structure of FhlA is composed of three domains. The N-terminal domain is putatively responsible for formate binding and FhlA oligomerization as a tetramer, the central portion of FhlA contains a AAA+ domain that hydrolyzes ATP, and the C-terminal domain binds DNA. Formate enhances FhlA-mediated expression of FHL; however, FhlA direct interaction with formate has never been demonstrated. Formate-protein interactions are challenging to assess, due to the small and ubiquitous nature of the molecule. Here, we have developed three techniques to assess formate-protein interaction. We use these techniques to confirm that FhlA binds formate in the N-terminal domain in vitro, and that this interaction is partially dependent on residues E183 and E363, consistent with previous reports. This study is a proof of concept that these techniques can be used to assess other formate-protein interactions.

Core Transcriptome of Hydrogen Producing Marine Vibrios Reveals Contribution of Glycolysis in Their Efficient Hydrogen Production.

Pyruvate (Pyr) is the end product of the glycolysis pathway. Pyr is also renewable and is further metabolized to produce formate, which is the precursor of H2, via pyruvate formate lyase (PFL) under anaerobic conditions. The formate is excluded and re-imported via the formate channel and is then converted to H2 via the formate hydrogenlyase (FHL) complex. In H2 producing marine vibrios, such as Vibrio tritonius and Vibrio porteresiae in the Porteresiae clade of the family Vibrionaceae, apparent but inefficient H2 production from Pyr has been observed. To elucidate the molecular mechanism of why this inefficient H2 production is observed in Pry-metabolized marine vibrio cells and how glycolysis affects those H2 productions of marine vibrios, the "Core Transcriptome" approach to find common gene expressions of those two major H2 producing Vibrio species in Pyr metabolism was first applied. In the Pyr-metabolized vibrio cells, genes for the "Phosphoenolpyruvate (PEP)-Pyruvate-Oxalate (PPO)" node, due to energy saving, and PhoB-, RhaR-, and DeoR-regulons were regulated. Interestingly, a gene responsible for oxalate/formate family antiporter was up-regulated in Pyr-metabolized cells compared to those of Glc-metabolized cells, which provides new insights into the uses of alternative formate exclusion mechanics due to energy deficiencies in Pyr-metabolized marine vibrios cells. We further discuss the contribution of the Embden-Meyerhof-Parnas (EMP) pathway to efficient H2 production in marine vibrios.

Proton conductance and regulation of proton/potassium fluxes in Escherichia coli FhlA-lacking cells during fermentation of mixed carbon sources

Escherichia coli uptake potassium ions with the coupling of proton efflux and energy utilization via proton FOF1-ATPase. In this study contribution of formate hydrogen lyase (FHL) complexes in the proton/potassium fluxes and the formation of proton conductance (CMH+) were investigated using fhlA mutant strain. The proton flux rate (JH+) decreased in fhlA by ∼ 25 % and ∼70 % during the utilization of glucose and glycerol, respectively, at 20 h suggesting H+ transport via or through FHL complexes. The decrease in JK+ in fhlA by ∼40 % proposed the interaction between FHL and Trk secondary transport system during mixed carbon fermentation. Moreover, the usage of N,N′-dicyclohexylcarbodiimide (DCCD) demonstrated the mediation of FOF1-ATPase in this interaction. CMH+ was 13.4 nmol min−1 mV−1 in WT at 20 h, which decreased by 20 % in fhlA. Taken together, FHL complexes have a significant contribution to the modulation of H+/K+ fluxes and the CMH + for efficient energy transduction and regulation of the proton motive force during mixed carbon sources fermentation.

Cyanobacteriochromes from Gloeobacterales Provide New Insight into the Diversification of Cyanobacterial Photoreceptors

The phytochrome superfamily comprises three groups of photoreceptors sharing a conserved GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) domain that uses a covalently attached linear tetrapyrrole (bilin) chromophore to sense light. Knotted red/far-red phytochromes are widespread in both bacteria and eukaryotes, but cyanobacteria also contain knotless red/far-red phytochromes and cyanobacteriochromes (CBCRs). Unlike typical phytochromes, CBCRs require only the GAF domain for bilin binding, chromophore ligation, and full, reversible photoconversion. CBCRs can sense a wide range of wavelengths (ca. 330–750 nm) and can regulate phototaxis, second messenger metabolism, and optimization of the cyanobacterial light-harvesting apparatus. However, the origins of CBCRs are not well understood: we do not know when or why CBCRs evolved, or what selective advantages led to retention of early CBCRs in cyanobacterial genomes. In the current work, we use the increasing availability of genomes and metagenome-assembled-genomes from early-branching cyanobacteria to explore the origins of CBCRs. We reaffirm the earliest branches in CBCR evolution. We also show that early-branching cyanobacteria contain late-branching CBCRs, implicating early appearance of CBCRs during cyanobacterial evolution. Moreover, we show that early-branching CBCRs behave as integrators of light and pH, providing a potential unique function for early CBCRs that led to their retention and subsequent diversification. Our results thus provide new insight into the origins of these diverse cyanobacterial photoreceptors.

Isolation and characterization of Enterococcus faecalis isolate VT-H1: A highly efficient hydrogen-producing bacterium from palm oil mill effluent (POME)

Facultative hydrogen-producing bacteria hold immense potential for fermentative hydrogen and valuable metabolite production. In this study, a mesophilic hydrogen-producing bacterium was isolated from palm oil mill effluent (POME) and identified as Enterococcus faecalis isolate VT-H1 using hybrid sequencing technologies. Our investigation encompassed the growth of the isolate and its capacity for hydrogen and volatile fatty acid (VFA) production. The results revealed that optimal growth occurred when glucose was utilized at 37 °C and initial pH of 7.0, resulting in a final OD600 of 2.58. The isolate demonstrated exceptional hydrogen production capabilities, achieving a maximum cumulative hydrogen yield of 2.22 mol-H2/mol-glucose at 30 °C and pH 7.0, outperforming previously reported E. faecalis strains by 5.84-fold. Kinetic analysis revealed favorable parameters for hydrogen production at 30 °C, with maximum hydrogen production (Hm = 2.63 mol-H2/mol-glucose), maximum hydrogen production rate (Rm = 0.03 mol-H2/mol-glucose·h), and lag-phase time (LPT = 18.34 h). Additionally, E. faecalis isolate VT-H1 exhibited significant butyric acid production (63.0 mM) alongside acetic acid (7.0 mM). Genome analysis unveiled the presence of key enzymes associated with hydrogen production, including formate hydrogen lyase (FHL) complex with formate dehydrogenase subunit alpha (FDH-α), bidirectional [NiFe] hydrogenase, NADH-dependent oxidoreductase subunit E, and NADH-quinone oxidoreductase subunit F. Moreover, genes associated with butyric acid production were also identified. These findings highlight the potential of E. faecalis isolate VT-H1 as a promising candidate for sustainable hydrogen and VFA production. Its unique characteristics, distinct genetic profile, and exceptional hydrogen and VFA production capabilities bring exciting opportunities for future biotechnological applications and further research to optimize and scale-up its potential for industrial use.

Renewable hydrogen production and storage via enzymatic interconversion of CO2 and formate with electrochemical cofactor regeneration.

The urgent need to reduce CO2 emissions has motivated the development of CO2 capture and utilization technologies. An emerging application is CO2 transformation into storage chemicals for clean energy carriers. Formic acid (FA), a valuable product of CO2 reduction, is an excellent hydrogen carrier. CO2 conversion to FA, followed by H2 release from FA, are conventionally chemically catalyzed. Biocatalysts offer a highly specific and less energy intensive alternative. CO2 conversion to formate is catalyzed by formate dehydrogenase (FDH), which usually requires a cofactor to function. Several FDHs have been incorporated in bioelectrochemical systems where formate is produced by the biocathode and the cofactor is electrochemically regenerated. H2 production from formate is also catalyzed by several microorganisms possessing either formate hydrogenlyase or hydrogen-dependent CO2 reductase complexes. Combination of these two processes can lead to a CO2-recycling cycle for H2 production, storage, and release with potentially lower environmental impact than conventional methods.

Green synthesis of nickel ferrite nanoparticles for efficient enhancement of lignocellulosic hydrolysate-based biohydrogen production

To improve the lignocellulosic hydrolysate-based production of biohydrogen, bimetallic nickel ferrite nanoparticles (NPs) with small particle size, cubic shape, high stability, and biocompatibility are synthesized using an Eichhornia crassipes extract at a low annealing temperature of 400 °C and added to the lignocellulosic hydrolysate fermentation by Klebsiella sp. WL1316. The optimal addition of 30 mg/L gsNiFe2O4 NPs accounts for the highest cumulative hydrogen production of 5544.86 ± 37.03 mL/L and improvement of 112.32% ± 1.86% at 24 h, while also resulting in the highest improvement of hydrogenase and formate-hydrogen lyase activities up to 102.11% ± 13.73% and 62.99% ± 4.66% compared to the Control treatment, respectively. Moreover, the conversion efficiencies of glucose, xylose, and substrate are enhanced upon addition of gsNiFe2O4 NPs, reaching values higher than 96% in the presence of 30 mg/L gsNiFe2O4 NPs. At the same time, the hydrogen yield converted from the substrate (Y(H2/S)) and biomass converted from the substrate (Y(B/S)) are also improved. In addition, the alteration of soluble metabolic products, especially significant changes in formic acid and ethanol concentrations compared to the control, increases the flux in the formate-hydrogen lytic pathway for hydrogen evolution, thereby promoting the substrate conversion level to hydrogen gas.

Formate and hydrogen in hydrothermal vents and their use by extremely thermophilic methanogens and heterotrophs.

Extremely thermophilic methanogens in the Methanococci and heterotrophs in the Thermococci are common in deep-sea hydrothermal vents. All Methanococci use H2 as an electron donor, and a few species can also use formate. Most Methanococci have a coenzyme F420-reducing formate dehydrogenase. All Thermococci reduce S0 but have hydrogenases and produce H2 in the absence of S0. Some Thermococci have formate hydrogenlyase (Fhl) that reversibly converts H2 and CO2 to formate or an NAD(P)+-reducing formate dehydrogenase (Nfd). Questions remain if Methanococci or Thermococci use or produce formate in nature, why only certain species can grow on or produce formate, and what the physiological role of formate is? Formate forms abiotically in hydrothermal fluids through chemical equilibrium with primarily H2, CO2, and CO and is strongly dependent upon H2 concentration, pH, and temperature. Formate concentrations are highest in hydrothermal fluids where H2 concentrations are also high, such as in ultramafic systems where serpentinization reactions occur. In nature, Methanococci are likely to use formate as an electron donor when H2 is limiting. Thermococci with Fhl likely convert H2 and CO2 to formate when H2 concentrations become inhibitory for growth. They are unlikely to grow on formate in nature unless formate is more abundant than H2 in the environment. Nearly all Methanococci and Thermococci have a gene for at least one formate dehydrogenase catalytic subunit, which may be used to provide free formate for de novo purine biosynthesis. However, only species with a membrane-bound formate transporter can grow on or secrete formate. Interspecies H2 transfer occurs between Thermococci and Methanococci. This and putative interspecies formate transfer may support Methanococci in low H2 environments, which in turn may prevent growth inhibition of Thermococci by its own H2. Future research directions include understanding when, where, and how formate is used and produced by these organisms in nature, and how transcription of Thermococci genes encoding formate-related enzymes are regulated.

Comparative Physiology and Genomics of Hydrogen-Producing Vibrios.

The Hyf-type formate hydrogen lyase (FHL) complex was first proposed based on sequence comparisons in Escherichia coli in 1997 (Andrews et al. in Microbiology 143:3633-3647, 1997). The hydrogenase in the Hyf-type FHL was estimated to be a proton-translocating energy-conserving [NiFe]-hydrogenase. Although the structure of FHL is similar to that of complex I, silent gene expression in E. coli has caused delays in unveiling the genetic and biochemical features of the FHL. The entire set of genes required for Hyf-type FHL synthesis has also been found in the genome sequences of Vibrio tritonius in 2015 (Matsumura et al. in Int J Hydrog Energy 40:9137-9146, 2015), which produces more hydrogen (H2) than E. coli. Here we investigate the physiological characteristics, genome comparisons, and gene expressions to elucidate the genetic backgrounds of Hyf-type FHL, and how Hyf-type FHL correlates with the higher H2 production of V. tritonius. Physiological comparisons among the seven H2-producing vibrios reveal that V. porteresiae and V. tritonius, grouped in the Porteresiae clade, show greater capacity for H2 production than the other species. The structures of FHL-Hyp gene clusters were closely related in both Porteresiae species, but differed from those of the other species with the presence of hupE, a possible nickel permease gene. Interestingly, deeper genome comparisons revealed the co-presence of nickel ABC transporter genes (nik) with the Hyf-type FHL gene only on the genome of the Porteresiae clade species. Therefore, active primary Ni transport might be one of the key factors characterizing higher H2 production in V. tritonius. Furthermore, the expression of FHL gene cluster was significantly up-regulated in V. tritonius cells stimulated with formate, indicating that formate is likely to be a control factor for the gene expression of V. tritonius FHL in a similar way to the formate regulon encoding the E. coli FHL.

FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism.

During enterobacterial mixed-acid fermentation, formate is generated from pyruvate by the glycyl-radical enzyme pyruvate formate-lyase (PflB). In Escherichia coli, especially at low pH, formate is then disproportionated to CO2 and H2 by the cytoplasmically oriented, membrane-associated formate hydrogenlyase (FHL) complex. If electron acceptors are available, however, formate is oxidized by periplasmically oriented, respiratory formate dehydrogenases. Formate translocation across the cytoplasmic membrane is controlled by the formate channel, FocA, a member of the formate-nitrite transporter (FNT) family of homopentameric anion channels. This review highlights recent advances in our understanding of how FocA helps to maintain intracellular formate and pH homeostasis during fermentation. Efflux and influx of formate/formic acid are distinct processes performed by FocA and both are controlled through protein interaction between FocA's N-terminal domain with PflB. Formic acid efflux by FocA helps to maintain cytoplasmic pH balance during exponential-phase growth. Uptake of formate against the electrochemical gradient (inside negative) is energetically and mechanistically challenging for a fermenting bacterium unless coupled with proton/cation symport. Translocation of formate/formic acid into the cytoplasm necessitates an active FHL complex, whose synthesis also depends on formate. Thus, FocA, FHL and PflB function together to govern formate homeostasis. We explain how FocA achieves efflux of formic acid and propose mechanisms for pH-dependent uptake of formate both with and without proton symport. We propose that FocA displays both channel- and transporter-like behaviour. Whether this translocation behaviour is shared by other members of the FNT family is also discussed.

Formate hydrogenlyase, formic acid translocation and hydrogen production: dynamic membrane biology during fermentation

Formate hydrogenlyase-1 (FHL-1) is a complex-I-like enzyme that is commonly found in gram-negative bacteria. The enzyme comprises a peripheral arm and a membrane arm but is not involved in quinone reduction. Instead, FHL-1 couples formate oxidation to the reduction of protons to molecular hydrogen (H2). Escherichia coli produces FHL-1 under fermentative conditions where it serves to detoxify formic acid in the environment. The membrane biology and bioenergetics surrounding E. coli FHL-1 have long held fascination. Here, we review recent work on understanding the molecular basis of formic acid efflux and influx. We also consider the structure and function of E. coli FHL-1, its relationship with formate transport, and pay particular attention to the molecular interface between the peripheral arm and the membrane arm. Finally, we highlight the interesting phenotype of genetic mutation of the ND1 Loop, which is located at that interface.

Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli

The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H2 during fermentation, its reversibility, allowing H2-dependent CO2 reduction, and its evolutionary link to respiratory complex I. FHL has been studied for over a century, but its atomic structure remains unknown. Here we report cryo-EM structures of FHL in its aerobically and anaerobically isolated forms at resolutions reaching 2.6 Å. This includes well-resolved density for conserved loops linking the soluble and membrane arms believed to be essential in coupling enzymatic turnover to ion translocation across the membrane in the complex I superfamily. We evaluate possible structural determinants of the bias toward hydrogen production over its oxidation and describe an unpredicted metal-binding site near the interface of FdhF and HycF subunits that may play a role in redox-dependent regulation of FdhF interaction with the complex.

The influence of hydrogen production on the formation of metabolic pathways and regulation of ΔpH in Escherichia coli

Hydrogen (H2) metabolism in Escherichia coli occurs via reversible membrane-associated hydrogenase enzymes (Hyd). Hyd-3 and Hyd-4 with formate dehydrogenase H (FDH-H) form formate hydrogen lyase complexes. The changes of metabolic pathways and ΔpH (pHin-pHex) regulation during fermentation of glucose, glycerol and formate in non H2-producing hypF (lack of all Hyds) and fdhF (lack of FDH-H) mutants at pH 7.5 were investigated. It was shown that specific growth rate was higher by ∼23% in hypF and fdhF, compared to wild type (wt), suggesting the negative effect of H2 on bacterial growth. Moreover, it was shown that H2 generation did not have a vital role in glucose and glycerol utilization rate at 0–72 h. The utilization of external formate was detected in wt (∼2.6 mM) and hypF (∼0.68 mM), but not in fdhF, due to the absence of enzyme responsible for formate metabolism. Nevertheless, the changes in ΔpH were not evident at 3 h. The ratio of generated end-products and regulation of ΔpH at late log (6 h) and exponential phase (24, 72 h) were various in hypF and fdhF due to formate disproportionation in hypF and proton generation, therewith absence of H2 generation. Taken together it can be concluded that bacteria regulate generation of fermentation end-products via balancing the concentration of acids and ethanol to maintain ΔpH and redox potential values. The results obtained are important for development and regulation of H2 production technology when applying mixed carbon sources.

Physiology of formate hydrogenlyase: a complex-I-like enzyme from Proteobacteria

Physiology of formate hydrogenlyase: a complex-I-like enzyme from Proteobacteria

Hydrogen production in the presence of oxygen by Escherichia coli K-12.

Escherichia coli is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O2, E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling and molecular genetics to make advances in the understanding of bacterial formate and H2 metabolism. It is shown that, under anaerobic (anoxic) conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H2 and CO2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H2 and CO2. Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a micro-aerobic (micro-oxic) environment it was possible to analyse aspects of formate and O2 respiration occurring alongside anaerobic metabolism. While cells growing under micro-aerobic conditions generated endogenous formic acid, no H2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H2 production in the presence of O2.

Articles of Significant Interest in This Issue

In a recent study by Roger et al. (e00299-21), genetic engineering of Escherichia coli for constitutive production of its formate hydrogenlyase enzyme resulted in the continuous capture of CO2 and H2 as formate. This platform shows promise for H2 storage as formate, a liquid fuel, using a greenhouse gas.

Co-production of hydrogen and ethyl acetate in Escherichia coli

BackgroundEthyl acetate (C4H8O2) and hydrogen (H2) are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products renders the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved, but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO2.ResultsWe engineered Escherichia coli towards improved conversion of formate into H2 and CO2 by inactivating the formate hydrogen lyase repressor (hycA), both uptake hydrogenases (hyaAB, hybBC) and/or overexpressing the hydrogen formate lyase activator (fhlA), in an acetate kinase (ackA) and lactate dehydrogenase (ldhA)-deficient background strain. Initially 10 strains, with increasing number of modifications were evaluated in anaerobic serum bottles with respect to growth. Four reference strains ΔldhAΔackA, ΔldhAΔackA p3-fhlA, ΔldhAΔackAΔhycAΔhyaABΔhybBC and ΔldhAΔackAΔhycAΔhyaABΔhybBC p3-fhlA were further equipped with a plasmid carrying the heterologous ethanol acyltransferase (Eat1) from Wickerhamomyces anomalus and analyzed with respect to their ethyl acetate and hydrogen co-production capacity. Anaerobic co-production of hydrogen and ethyl acetate via Eat1 was achieved in 1.5-L pH-controlled bioreactors. The cultivation was performed at 30 °C in modified M9 medium with glucose as the sole carbon source. Anaerobic conditions and gas stripping were established by supplying N2 gas.ConclusionsWe showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70% of the pathway maximum for ethyl acetate and hydrogen, and propose in situ product removal via gas stripping as efficient technique to isolate the products of interest.

Green-synthesized nickel oxide nanoparticles enhances biohydrogen production of Klebsiella sp. WL1316 using lignocellulosic hydrolysate and its regulatory mechanism

Green synthesis of nickel oxide nanoparticle (NiO-NP) from Eichhornia crassipes (Ec) extract was novelly conducted, and regulatory effect of this NP on fermentative hydrogen production was evaluated. Characterization of the Ec-NiO-NP revealed their cubic and spherical shape, small diameter (9.1 ± 2.6 nm) and high purity. The maximum cumulative hydrogen production and hydrogen yield Y(H2/S) reached 4842.19 ± 23.43 mL/L and 101.45 ± 3.32 mL/gsubstrate, respectively, in the presence of 20 mg/L Ec-NiO-NP, which were 47.29% and 37.78% higher than the control without NPs addition. Evaluation of glucose and xylose utilization efficiency as well as key node metabolites further established the potential of Ec-NiO-NP to improve the reducing sugar conversion and metabolic flux distribution in hydrogen synthesis pathway. Furthermore, addition of 20 mg/L Ec-NiO-NP resulted in enhanced hydrogenase activity with a maximum increase of 623% comparing to the control, and led to changes in the gene expression of both hydrogenase and formate-hydrogen lyase, which play important roles in promoting hydrogen production at different stages of fermentation. This study demonstrates that supplementation with green-synthesized Ec-NiO-NP effectively improves fermentative hydrogen production and regulates key node metabolites alteration and functional gene expression.

H2-dependent formate production by hyperthermophilic Thermococcales: an alternative to sulfur reduction for reducing-equivalents disposal.

Removal of reducing equivalents is an essential catabolic process for all microorganisms to maintain their internal redox balance. The electron disposal by chemoorganotrophic Thermococcales generates H2 by proton reduction or H2S in presence of S0. Although in the absence of S0 growth of these (hyper)thermopiles was previously described to be H2-limited, it remains unclear how Thermococcales could be present in H2-rich S0-depleted habitats. Here, we report that 12 of the 47 strains tested, distributed among all three orders of Thermococcales, could grow without S0 at 0.8 mM dissolved H2 and that tolerance to H2 was always associated with formate production. Two conserved gene clusters coding for a formate hydrogenlyase (FHL) and a putative formate dehydrogenase-NAD(P)H-oxidoreductase were only present in H2-dependent formate producers, and were both systematically associated with a formate dehydrogenase and a formate transporter. As the reaction involved in this alternative pathway for disposal of reducing equivalents was close to thermodynamic equilibrium, it was strongly controlled by the substrates-products concentration ratio even in the presence of S0. Moreover, experimental data and thermodynamic modelling also demonstrated that H2-dependent CO2 reduction to formate could occur within a large temperature range in contrasted hydrothermal systems, suggesting it could also provide an adaptive advantage.