Clostridium Pasteurianum Research Articles

Mesoporous Electrodes Enhance the Electrocatalytic Performance of [FeFe]-Hydrogenase.

The metalloenzyme [FeFe]-hydrogenase is of interest to future biotechnologies targeting the production of "green" hydrogen (H2). We recently developed a simple two-step functionalized procedure to immobilize the [FeFe]-hydrogenase from Clostridium pasteurianum ("CpI") on mesoporous indium tin oxide (ITO) electrodes to achieve elevated H2 production with high operational stability, with current densities of 8 mA cm-2. Here, we use a combination of atomic force microscopy (AFM), scanning electron microscopy (SEM) and electrochemical quartz crystal microbalance (EQCM) to understand how mesoporous ITO stabilizes and activates CpI for electroenzymatic H2 production. Examination of the topography and morphology of the mesoporous ITO surface revealed a hierarchical morphology containing cavities and well-defined nanoparticle agglomerates. Any potential effect of mesoporosity was investigated by comparing the stability and electroenzymatic activity of CpI on mesoporous 'nanoITO' and planar ITO, where we determined that CpI has a higher turnover frequency and absorbs with greater stability (with respect to electroenzymatic activity over time) to nanoITO surfaces.

Mesoporous Electrodes Enhance the Electrocatalytic Performance of [FeFe]‐Hydrogenase

The metalloenzyme [FeFe]‐hydrogenase is of interest to future biotechnologies targeting the production of “green” hydrogen (H2). We recently developed a simple two‐step functionalized procedure to immobilize the [FeFe]‐hydrogenase from Clostridium pasteurianum (“CpI”) on mesoporous indium tin oxide (ITO) electrodes to achieve elevated H2 production with high operational stability, with current densities of 8 mA cm–2. Here, we use a combination of atomic force microscopy (AFM), scanning electron microscopy (SEM) and electrochemical quartz crystal microbalance (EQCM) to understand how mesoporous ITO stabilizes and activates CpI for electroenzymatic H2 production. Examination of the topography and morphology of the mesoporous ITO surface revealed a hierarchical morphology containing cavities and well‐defined nanoparticle agglomerates. Any potential effect of mesoporosity was investigated by comparing the stability and electroenzymatic activity of CpI on mesoporous ‘nanoITO’ and planar ITO, where we determined that CpI has a higher turnover frequency and absorbs with greater stability (with respect to electroenzymatic activity over time) to nanoITO surfaces.

Beyond traditional pathways: Uncovering ultrasonication’s novel routes to enhance hydrogen yields in Clostridium pasteurianum

This study presents a comparative analysis of metabolic end-products and proteomic profiles in Clostridium (C.) pasteurianum during dark fermentation, in the presence and absence of ultrasonication. The study revealed significant changes in the abundance of six proteins associated with dark fermentation pathways, particularly those involved in hydrogen generation pyruvate:ferredoxin oxidoreductase (PFOR) and pyruvate formate lyase (PFL)), glycolysis, butyrate pathway, and adenosine triphosphate (ATP) synthesis. Ultrasonication was efficient at degassing with dissolved hydrogen concentrations reduced from 2.63 ± 0.09 mM to 0.65 ± 0.04 mM. This led to an 11 % increase in produced hydrogen concentration, and a 37 % increase in hydrogen yield. Contrary to expectations, ultrasonication resulted in a deceleration of both glycolytic and hydrogen generation pathways, as evidenced by a decrease in the proteomic expression of key enzymes like pyruvate:ferredoxin oxidoreductase and pyruvate formate lyase. The study also discovered an increased ATP demand and an altered carbon flux through metabolic pathways in C. pasteurianum. This flux initially favoured the acetate pathway due to the sudden ATP demand. The long-term maintenance of this ATP demand required balancing the carbon flux between lactate, acetate, and butyrate pathways, to achieve both a stable redox balance and ATP demand. For the first time, this study revealed opportunities to enhance hydrogen yields using strategies other than enhancing the hydrogen generation pathways, specifically by reducing dissolved hydrogen concentrations.

Bioaugmentation with Clostridium pasteurianum for high-yield continuous bio-hydrogen production in a dynamic membrane bioreactor

This study demonstrated the feasibility of achieving high-yield continuous bio-H2 production through bioaugmentation with Clostridium pasteurianum. C. pasteurianum was introduced during the initial granulation stage in a dynamic membrane bioreactor (DMBR) inoculated with heat-treated digester sludge. Following bioaugmentation, the average hydrogen yield (HY) reached 3.07 ± 0.13 mol H2/mol hexoseconsumed, with a hydraulic retention time (HRT) of 2 h. This high-yield and high-rate hydrogen production performance was achieved alongside a high concentration of suspended and granular biomass as 21.02 ± 1.01 g VSS/L. The DM played a crucial role in prolonging the solid retention time of the suspended biomass to 12.15 h. C. pasteurianum emerged as the predominant species, constituting 92.3 % of the total bacterial population. This dominance significantly contributed to achieving the high HY, reaching 77 % of the theoretical maximum HY (4 mol H2/mol hexoseconsumed) by shifting the metabolic pathway from non-H2 production pathways to hydrogen-producing acetate. However, during further continuous operation, the hydrogen production pathway was gradually shifted towards the butyrate pathway, resulting in a decreased HY of 2.26 mol H2/mol hexoseconsumed along with the decreased population of C. pasteurianum. Additional bioaugmentation of C. pasteurianum during the maturational granulation stage did not impact the metabolic pathways or microbiome significantly, as the added strain was supposed to be washed out. This study provides insights into achieving high-yield continuous bio-H2 production through bioaugmentation with C. pasteurianum during the initial granulation stage. Further studies are necessary to sustain C. pasteurianum-dominated high HY with the acetate pathway during prolonged operation.

Ultrasonication-induced metabolic pathway shifts and reduced electron carrier washout with biomass enhanced hydrogen yield in a continuous stirred tank reactor

Biohydrogen is pivotal for hydrogen economy, providing a renewable, carbon–neutral source of hydrogen from organic waste and biomass. It addresses environmental concerns and supports circular economy principles. However, challenges like managing metabolic pathways and dissolved hydrogen hinder attainment of higher hydrogen yields. This study explored the impact of organic loading rates (OLR) and ultrasonication on glucose fermentation by Clostridium pasteurianum for biohydrogen production.Results showed ultrasonication, particularly at 10 g/L.d OLR, increased hydrogen yield from 1.04 to 1.28 mol-H₂/mol-glucose. Higher OLR boosted biogas production, and at 10 g/L.d OLR, ultrasonication improved hydrogen purity from 46% to 53%, enhancing yield by 13%. C. pasteurianum shifted metabolic pathways under higher OLR, increasing lactate production to address redox imbalance. However, ultrasonication negatively affected lactate and acetate reutilisation, redirecting carbon flux from lactate to butyrate, increasing hydrogen yield.Combining ultrasonication and increased OLR enhanced glycolysis carbon flux by 14.5% and reduced biomass carbon flux by 10.5%. Hydrogen yield, influenced by glycolysis carbon flux, reached 82% under ultrasonication and increased OLR. Ultrasonication mitigated inhibitory effects on electron carrier molecule re-oxidation, emphasising the need to balance biomass, dissolved hydrogen concentration, and OLR for optimal hydrogen recovery.Despite decreased acetate/butyrate ratio and unchanged theoretical hydrogen production with ultrasonication, hydrogen yield increased. This likely resulted from reduced electron carrier molecule wastage with biomass, influenced by higher biomass concentration and ultrasonication. The study underscored preventing electron carrier molecule washout for higher hydrogen production and recommends using batch reactors over continuous stirred tank reactors in dark fermentation operations.

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.

Influence of fumarate on interspecies electron transfer and the metabolic shift induced in Clostridium pasteurianum by Geobacter sulfurreducens.

In previous studies, it was demonstrated that co-culturing Clostridium pasteurianum and Geobacter sulfurreducens triggers a metabolic shift in the former during glycerol fermentation. This shift, attributed to interspecies electron transfer and the exchange of other molecules, enhances the production of 1,3-propanediol at the expense of the butanol pathway. The aim of this investigation is to examine the impact of fumarate, a soluble compound usually used as an electron acceptor for G. sulfurreducens, in the metabolic shift previously described in C. pasteurianum. Experiments were conducted by adding along with glycerol, acetate and different quantities of fumarate in co-cultures of G. sulfurreducens and C. pasteurianum. Ametabolic shift was exhibited in all the co-culture conditions. This shift was more pronounced at higher fumarate concentrations. Additionally, we observed G. sulfurreducens growing even in the absence of fumarate and utilizing small amounts of this compound as an electron donor rather than an electron acceptor in the co-cultures with high fumarate addition. This study provided evidence that interspecies electron transfer continues to occur in the presence of a soluble electron acceptor, and the metabolic shift can be enhanced by promoting the growth of G. sulfurreducens.

Properties of the iron-sulfur cluster electron transfer relay in an [FeFe]-hydrogenase that is tuned for H2 oxidation catalysis

[FeFe]-hydrogenases catalyze the reversible oxidation of H2 from electrons and protons at an organometallic active site cofactor named the H-cluster. In addition to the H-cluster, most [FeFe]-hydrogenases possess accessory FeS cluster (F-cluster) relays that function in mediating electron transfer with catalysis. There is significant variation in the structural properties of F-cluster relays amongst the [FeFe]-hydrogenases, however it is unknown how this variation relates to the electronic and thermodynamic properties, and thus the electron transfer properties, of F-cluster relays. Clostridium pasteurianum [FeFe]-hydrogenase II (CpII), exhibits a large catalytic bias for H2 oxidation (compared to H2 production), making it a notable system for examining if F-cluster properties contribute to the overall function and efficiency of the enzyme. By applying a combination of multifrequency and potentiometric EPR, we resolved two EPR signals with distinct power- and temperature-dependent properties at g = 2.058 1.931 1.891 (F2.058) and g = 2.061 1.920 1.887 (F2.061), with assigned midpoint potentials of -140 ± 18 mV and -406 ± 12 mV vs. NHE, respectively. Spectral analysis revealed features consistent with spin-spin coupling between the two [4Fe-4S] F-clusters, and possible functional models are discussed that account for the contribution of coupling to the electron transfer landscape. The results signify the interplay of electronic coupling and free energy properties and parameters of the FeS clusters to the electron transfer mechanism through the relay and provide new insight as to how relays functionally complement the catalytic directionality of active sites to achieve highly efficient catalysis.

Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

Design of a microbial photoheterotrophic consortia for biohydrogen production under nongrowing conditions: Insight into microbial associations

Physiological and metabolic behavior of photoheterotrophic mixed cultures (PHMCs), monocultures of Rhodopseudomonas palustris, Clostridium pasteurianum and Syntrophomonas wolfei and a designed microbial consortium (DMC), consisting of these microorganisms that emulates natural consortia were studied. Growing and photoheterotrophic nongrowing conditions to simulate the use of dark fermentative effluents were used. Under growing conditions, C. pasteurianum showed higher bioH2 production (8.5 mmol) and molar bioH2 yield (21 mmoLH2/gCOD), while R. palustris did not produce this gas. Under nongrowing conditions, DMC reached the highest bioH2 production (10 mmol) and bioH2 yield (78.6 mmoLH2/gCOD) together with Poly-hydroxy-alkanoates (PHA), followed for PHMC-C2 (7.5; 59.6) and the monocultures. Higher bioH2 and PHA production of the PHMC and the DMC suggest that these conditions promote the use of nonconventional energy pathways, and interactions among microbial populations that allow for the survival and sustain bioH2 production. This study forms the basis for more in-depth studies of the metabolic behavior of photosynthetic natural and designed cultures.

Insights into the Molecular Mechanism of Formaldehyde Inhibition of [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are efficient H2 converting biocatalysts that are inhibited by formaldehyde (HCHO). The molecular mechanism of this inhibition has so far not been experimentally solved. Here, we obtained high-resolution crystal structures of the HCHO-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum, showing HCHO reacts with the secondary amine base of the catalytic cofactor and the cysteine C299 of the proton transfer pathway which both are very important for catalytic turnover. Kinetic assays via protein film electrochemistry show the CpI variant C299D is significantly less inhibited by HCHO, corroborating the structural results. By combining our data from protein crystallography, site-directed mutagenesis and protein film electrochemistry, a reaction mechanism involving the cofactor's amine base, the thiol group of C299 and HCHO can be deduced. In addition to the specific case of [FeFe]-hydrogenases, our study provides additional insights into the reactions between HCHO and protein molecules.

Fermentative iron reduction buffers acidification and promotes microbial metabolism in marine sediments

Microbial iron reduction is a crucial process in natural ecosystems, contributing to the cycling of elements and supporting the biological activities of organisms. However, the significance of fermentative iron reduction in marine environments and microbial metabolism remains understudied compared with iron reduction coupled with respiration. The main objective of our study was to investigate the influence of fermentative iron reduction on microbial populations and marine sediment. Our findings revealed a robust iron-reducing activity in the enriched marine sediment, demonstrating a maximum ferrihydrite-reducing rate of 0.063 mmol/h. Remarkably, ferrihydrite reduction exhibited an intriguing pH-buffering effect through the release of OH+ and Fe2+ ions, distinct from fermentation alone. This effect resulted in substantial improvements in glucose consumption (71.4%), bacterial growth (48.1%), and metabolite production (80.8%). To further validate the acidification-buffering and metabolism-promoting effects of ferrihydrite reduction, we conducted iron-reducing experiments using a pure strain, Clostridium pasteurianum DMS525. The observed pH-buffering effect resulted from microbial iron reduction in marine sediment and has potential environmental implications by reducing CO2 emissions, mitigating acidification, and preserving the delicate balance of marine ecosystems.

Influence of the permeate flux on continuous biohydrogen production, permeability, and granulation in a dynamic membrane bioreactor

This study examined the effect of the high permeate flux on continuous biohydrogen production using dynamic membrane bioreactor (DMBR). A lab-scale DMBR was equipped with polyester mesh having two pore sizes, 220 μm and 444 μm, to avoid washout and membrane fouling, respectively. The DMBR was continuously fed with 20 g glucose/L at a hydraulic retention time of 3 h, while the permeate flux increased from 116 to 291 L/m2.h over 58 days, under non-sterile conditions. The highest average hydrogen production rate, hydrogen yield, and daily hydrogen production were observed as 21.7 ± 1.7 L H2/L/d, 1.41 ± 0.05 mol H2/mol hexoseconsumed, and 184.0 ± 9.3 L H2/d, respectively, at the permeate flux of 233 ± 12 L/m2.h, with an average permeability for 444-μm mesh was 25.42 L/m2.h/kPa at an average transmembrane pressure (TMP) of 9 ± 1.2 kPa. Higher permeate flux (≥291 ± 14 L/m2.h) caused severe biomass washout, while lower permeate flux (≤175 ± 9 L/m2.h) resulted in excessive cake layer along with the increased H2 consuming pathways. At the optimum permeate condition, H2-producing granules were formed, which were with an average size of 1875 μm and a size distribution of 1150–2580 μm for 60% of the volume fraction. Although the reactor was inoculated with Clostridium butyricum DSM 10702, the dominant microbial species became Clostridium pasteurianum and Ethanoligenens harbinense as it was operated under non-sterile conditions.

Hydrogen online monitoring based on thermal conductivity for anaerobic microorganisms.

H2 -producing microorganisms are a promising source of sustainable biohydrogen. However, most H2 -producing microorganisms are anaerobes, which are difficult to cultivate and characterize. While several methods for measuring H2 exist, common H2 sensors often require oxygen, making them unsuitable for anaerobic processes. Other sensors can often not be operated at high gas humidity. Thus, we applied thermal conductivity(TC) sensors and developed a parallelized, online H2 monitoring for time-efficient characterization of H2 production by anaerobes. Since TC sensors are nonspecific for H2 , the cross-sensitivity of the sensors was evaluated regarding temperature, gas humidity, and CO2 concentrations. The systems' measurement range was validated with two anaerobes: a high H2 -producer (Clostridium pasteurianum) and a low H2 -producer (Phocaeicola vulgatus). Online monitoring of H2 production in shake flask cultivations was demonstrated, and H2 transfer rates were derived. Combined with online CO2 and pressure measurements, molar gas balances of the cultivations were closed, and an anaerobic respiration quotient was calculated. Thus, insight into the effect of medium components and inhibitory cultivation conditions on H2 production with the model anaerobes was gained. The presented online H2 monitoring method can accelerate the characterization of anaerobes for biohydrogen production and reveal metabolic changes without expensive equipment and offline analysis.

A (S)-3-Hydroxybutyrate Dehydrogenase Belonging to the 3-Hydroxyacyl-CoA Dehydrogenase Family Facilitates Hydroxyacid Degradation in Anaerobic Bacteria.

Ketone bodies, including acetoacetate, 3-hydroxybutyrate, and acetone, are produced in the liver of animals during glucose starvation. Enzymes for the metabolism of (R)-3-hydroxybutyrate have been extensively studied, but little is known about the metabolism of its enantiomer (S)-3-hydroxybutyrate. Here, we report the characterization of a novel pathway for the degradation of (S)-3-hydroxybutyrate in anaerobic bacteria. We identify and characterize a stereospecific (S)-3-hydroxylbutyrate dehydrogenase (3SHBDH) from Desulfotomaculum ruminis, which catalyzes the reversible NAD(P)H-dependent reduction of acetoacetate to form (S)-3-hydroxybutyrate. 3SHBDH also catalyzes oxidation of d-threonine (2R, 3S) and l-allo-threonine (2S, 3S), consistent with its specificity for β-(3S)-hydroxy acids. Isothermal calorimetry experiments support a sequential mechanism involving binding of NADH prior to (S)-3-hydroxybutyrate. Homologs of 3SHBDH are present in anaerobic fermenting and sulfite-reducing bacteria, and experiments with Clostridium pasteurianum showed that 3SHBDH, acetate CoA-transferase (YdiF), and (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) are involved together in the degradation of (S)-3-hydroxybutyrate as a carbon and energy source for growth. (S)-3-hydroxybutyrate is a human metabolic marker and a chiral precursor for chemical synthesis, suggesting potential applications of 3SHBDH in diagnostics or the chemicals industry. IMPORTANCE (R)-3-hydroxybutyrate is well studied as a component of ketone bodies produced by the liver and of bacterial polyesters. However, the biochemistry of its enantiomer (S)-3-hydroxybutyrate is poorly understood. This study describes the identification and characterization of a stereospecific (S)-3-hydroxylbutyrate dehydrogenase and its function in a metabolic pathway for the degradation of (S)-3-hydroxybutyrate as a carbon and energy source in anaerobic bacteria. (S)-3-hydroxybutyrate is a mammalian metabolic marker and a precursor for chemical synthesis and bioplastics, suggesting potential applications of these enzymes in diagnostics and biotechnology.

Heterologous 1,3-Propanediol Production Using Different Recombinant Clostridium beijerinckii DSM 6423 Strains.

1,3-propanediol (1,3-PDO) is a valuable basic chemical, especially in the polymer industry to produce polytrimethylene terephthalate. Unfortunately, the production of 1,3-PDO mainly depends on petroleum products as precursors. Furthermore, the chemical routes have significant disadvantages, such as environmental issues. An alternative is the biobased fermentation of 1,3-PDO from cheap glycerol. Clostridium beijerinckii DSM 6423 was originally reported to produce 1,3-PDO. However, this could not be confirmed, and a genome analysis revealed the loss of an essential gene. Thus, 1,3-PDO production was genetically reinstalled. Genes for 1,3-PDO production from Clostridium pasteurianum DSM 525 and Clostridium beijerinckii DSM 15410 (formerly Clostridium diolis) were introduced into C. beijerinckii DSM 6423 to enable 1,3-PDO production from glycerol. 1,3-PDO production by recombinant C. beijerinckii strains were investigated under different growth conditions. 1,3-PDO production was only observed for C. beijerinckii [pMTL83251_Ppta-ack_1,3-PDO.diolis], which harbors the genes of C. beijerinckii DSM 15410. By buffering the growth medium, production could be increased by 74%. Furthermore, the effect of four different promoters was analyzed. The use of the constitutive thlA promoter from Clostridium acetobutylicum led to a 167% increase in 1,3-PDO production compared to the initial recombinant approach.

Facile Functionalization of Carbon Electrodes for Efficient Electroenzymatic Hydrogen Production.

Enzymatic electrocatalysis holds promise for new biotechnological approaches to produce chemical commodities such as molecular hydrogen (H2). However, typical inhibitory limitations include low stability and/or low electrocatalytic currents (low product yields). Here we report a facile single-step electrode preparation procedure using indium-tin oxide nanoparticles on carbon electrodes. The subsequent immobilization of a model [FeFe]-hydrogenase from Clostridium pasteurianum ("CpI") on the functionalized carbon electrode permits comparatively large quantities of H2 to be produced in a stable manner. Specifically, we observe current densities of >8 mA/cm2 at -0.8 V vs the standard hydrogen electrode (SHE) by direct electron transfer (DET) from cyclic voltammetry, with an onset potential for H2 production close to its standard potential at pH 7 (approximately -0.4 V vs. SHE). Importantly, hydrogenase-modified electrodes show high stability retaining ∼92% of their electrocatalytic current after 120 h of continuous potentiostatic H2 production at -0.6 V vs. SHE; gas chromatography confirmed ∼100% Faradaic efficiency. As the bioelectrode preparation method balances simplicity, performance, and stability, it paves the way for DET on other electroenzymatic reactions as well as semiartificial photosynthesis.

Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway.

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN- ) ligands. Extrinsic CO and CN- , however, inhibit hydrogenases. The mechanism by which CN- binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN- -treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN- and CO ligands and to show that extrinsic CN- binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN- treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally.

Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference

The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.

Respiratory electrogen Geobacter boosts hydrogen production efficiency of fermentative electrotroph Clostridium pasteurianum

Electrogens and electrotrophs are microorganisms that generate and consume electricity in a bioelectrochemical system, respectively. However, the influence of respiratory electrogen Geobacter on the hydrogen production efficiency of fermentative electrotroph Clostridium pasteurianum remains unclear. Herein, cocultures with Geobacter sulfurreducens and C. pasteurianum were successfully established during glucose fermentation. Compared with those in C. pasteurianum monoculture alone, the maximum rate and yield of hydrogen production in the coculture increased by 122.2% and 28.92%, respectively. Meanwhile, substrate conversation efficiency elevated by 50.6%. The acetic and butyric acid fermentation pathways in the cocultures were also promoted relative to those in the monoculture. Carbon and electron balance analysis also unraveled that acetate production in syntrophic coculture accounted for approximately-two times more carbon and electron contributions than that in the monoculture. A direct manner mediated by pili-like structures or cell contacts was built between the two electroactive bacteria during the electric syntropy. Extracellular electron inputting from G. sulfurreducens shifted the fermentative pattern of C. pasteurianum, which resulted in an enhanced acetic acid fermentation pathway accompanied by a higher hydrogen yield. These findings provide new insights into electric syntrophy between respiratory electrogenic G. sulfurreducens and fermentative electrotrophic C. pasteurianum. A new strategy for hydrogen enhancement by coculturing hydrogen-producing electrotrophic bacteria with electrogenic microorganisms is also explored.