Rate Of Product Formation Research Articles

Electrochemical CO2 conversion to formic acid using engineered enzymatic catalysts in a batch reactor

Formic acid is one of the most valuable fuel products for the capture and conversion of CO2 due to its unique usage in fuel cells and hydrogen storage. Electrochemically mediated conversion of CO2 to FA has its advantages over the traditional Kemira process, in that high temperatures and pressures are not required, cutting on manufacturing and operating costs. However, selectivity of the metal catalysts used in CO2 conversion to certain products remains limited. Here, an engineered enzymatic catalyst is employed to convert CO2 into formic acid (in the form of formate) in a batch reactor. This work seeks to maximize both formate production and Coulombic efficiency, which is achieved primarily through: (1) adjustment of the operating voltage, (2) implementation of an O2 scavenger to mitigate competition with dissolved O2, and (3) control of system pH to maintain a stable operating range for the catalyst. Peak formate production and peak efficiency achieved in long-term experiments (> 40 h) were 225 mM and 91 %, respectively, both of which show promise of strong metrics for CO2 conversion to formate. The optimal operating cathode voltage was shown to be below the baseline voltage of − 0.75 V vs. Ag/AgCl. In short-term experiments (≈ 1 h), operating below this range, at approximately − 0.85 V, could achieve efficiencies of 100 %. By implementing sodium thiosulfate as an O2 scavenger, the rate of formate production improved by over 4 ×. Long-term retention of the efficiency, however, remains an issue to be addressed due to the enzyme sensitivity to pH and increased current at higher values of acid used on the anode.

Effects of Mineral Admixtures on the Evolution of Static Yield Stress of Different Composite Pastes

The effects of different mineral admixtures on the evolution of static yield stress of common composite cementitious material paste and ultra-high-performance concrete (UHPC)-based paste were investigated. The results show that there are obvious differences in the role of mineral admixtures in the common paste and the UHPC-based paste. Adding mineral admixtures can change the initial static yield stress of the paste by affecting the particle size, particle shape and the charged particles. The addition of mineral admixtures with small particle size such as silica fume and ultrafine slag can increase the initial static yield stress of common paste but reduce that of UHPC-based paste. Adding mineral admixtures changes the evolution of static yield stress of the paste by affecting the particle spacing and the formation and growth rate of hydration products. In turn, the addition of ultrafine slag or silica fume increases the growth of the static yield stress of common paste. Adding slag, fly ash or fly ash microbeads successively reduces the static yield stress of common paste at the later stage. Affected by the content of PCE, the static yield stress of UHPC-based paste containing fly ash microbeads, slag, ultrafine slag and fly ash increases sequentially compared with the blank group at the later stage. The effect of silica fume with different dosages on the evolution of static yield stress of UHPC-based paste is significantly different.

Investigation of Cu/TiO2 synthesis methods and conditions for CO2 photocatalytic reduction via conversion of bicarbonate/carbonate to formate

This work is aimed at improving the photocatalytic activity of TiO2 in the reduction of CO2. TiO2 and Cu/TiO2 at 5 wt% copper bulk loading were synthesised using the sol-gel method at a pH of 0.4 and 1.5. The synthesised pure TiO2 samples, alongside the commercial TiO2 (Degussa® P25), were also doped with copper via the incipient wetness method. The low pH and the presence of copper inhibited the anatase-rutile transformation. Copper existed in Cu(I) and Cu(II) states in all the doped samples, with the Cu(I) to Cu(II) ratio intimately linked with the anatase to rutile ratio. The UPS data have shown the type II band offset between TiO2 and CuO. The performance of the samples prepared was tested through the reduction of sodium bicarbonate-carbonate buffered at pH > 8. Commercial TiO2 impregnated with Cu demonstrated the highest formate production rate of 173 µmol·g−1·h−1 at pH 11. This study demonstrated the synergetic effect of the anatase-rutile mixture of phases, the reaction pH, and the copper dopant in improving the photocatalytic activity of TiO2.

The influence of Pd4Cr6@(NH2)M-MOF(Cr) catalyst on components of formic acid dehydrogenation gaseous products

ABSTRACT In the dehydrogenation of formic acid, catalyst has a great influence on the composition and formation rate of gaseous products. However, the hydrogen used in fuel cells has strict requirements on the content of carbon monoxide impurities. Therefore, it is very important to study the relationship between catalyst components and their synergistic effects and the composition of gaseous products. The role of Pd-Cr bimetallic nanoparticles supported on amino modified Cr-based metal-organic framework catalyst (Pd4Cr6@(NH2)M-MOF(Cr)) in the formic acid dehydrogenation was explored through comparative experiments and catalyst characterization. Results show that both Cr-based metal-organic framework (MOF(Cr)) and amino-modified Cr-based metal-organic framework ((NH2)M-MOF(Cr)) have no effect on promoting the generation of gaseous products. The presence of Cr and NH2 groups in Pd4Cr6@(NH2)M-MOF(Cr) greatly improves the formation rate of H2 and CO2 products, the gas production rate of Pd4Cr6@(NH2)M-MOF(Cr) is 2.89 times that of Pd4@(NH2)M-MOF(Cr), and the gas production rate of Pd4Cr6@(NH2)M-MOF(Cr) is 6.26 times of Pd4Cr6@MOF(Cr). However, the formation rate of H2 increases more in the presence of Cr. Synergy of Pd-Cr and Pd-NH2 in the Pd4Cr6@(NH2)M-MOF(Cr) catalyst promotes CO formation.

Boosting electrocatalytic CO2 reduction to formate via carbon nanofiber encapsulated bismuth nanoparticles with ultrahigh mass activity

Electrochemical CO2 conversion is one of the most promising technologies to achieve carbon neutrality. However, it still suffers from some nonnegligible challenges on low production rate and unsatisfied current densities for potential large-scale applications. Herein, we prepare ultrasmall Bi nanoparticles uniformly encapsulated in the carbon nanofibers through electrospinning techniques, which is denoted as Bi/CNFs-900. Gratifyingly, this Bi/CNFs-900 catalyst demonstrates excellent performance and stability on CO2 electro-reduction in a broad potential window. Specifically, it can produce formate with a Faradaic efficiency over 90% and a high partial current density of –235.3 mA cm−2 at −1.23 V vs. RHE in a flow-cell. Furthermore, the confinement effect of carbon nanofibers largely restricts the severe aggregation of bismuth nanoparticles during synthesis as well as electrolysis procedure, which greatly increases the accessible active sites and decreases the actual mass fraction of bismuth composition. Consequently, Bi/CNFs-900 not only achieves ultrahigh mass activity of –1.6 A mgBi−1, but also possesses an unprecedented formate production rate of 4403.3 μmol h−1 cm−2. DFT calculations and in situ Raman spectroscopy further uncover the possible reaction mechanism for CO2 reduction toward formate. These results could provide an economical and industrial-viable strategy for the preparation of electrocatalysts in CO2 reduction.

Solvent-free synthesis of nanostructured TiO2 in a continuous flow spinning disc reactor for application to photocatalytic reduction of CO2

Nanostructured TiO2 catalysts were synthesised via the sol-gel method using solvent-free titanium (IV) n-butoxide and acidified water at pH 1 in a continuous flow spinning disc reactor (SDR). The influence of disc rotational speed, total flow rate of the reagents, and molar hydrolysis ratio (molar ratio of the acidified water to the titanium (IV) n-butoxide) on the particle size, phase distribution, band gap energy and photocatalytic activity for CO2 reduction was studied. Increasing the disc rotational speed from 400 ​rpm to 1400 ​rpm results in highly sheared, uniformly mixed thin films where small particles (up to ca. 40 ​nm mean diameter) with narrow particle size distribution (polydispersity index of up to 0.5) are formed even at the lower molar hydrolysis ratio of 113. Increasing the molar hydrolysis ratio from 113 to 301 favours anatase phase transformation to rutile phase, thus improving photocatalytic activity. Larger TiO2 particles from the SDR are associated with an increase in their band gap energy whilst doping with copper narrows the band gap energy from 3.00 ​eV down to 2.53 ​eV.The photocatalytic performance of the TiO2 nanoparticles was evaluated for CO2 reduction in the form of bicarbonate ions using a meso-structured photocatalytic reactor at a TiO2 loading of 0.5 ​g L-1 and flow rate of 4 ​mL min-1. A formate production rate of 500 ​μmol ​g-1 ​h-1 is achieved after 2 ​h of irradiation (λ ​= ​254 ​nm) on a bare TiO2 catalyst, with no apparent trend observed with SDR operating conditions used in the production of the nanoparticles. However, for copper-doped TiO2, there is a clear correlation between the anatase to rutile ratio and formate production rate.

Steady-state kinetic analysis of reversible enzyme inhibitors: A case study on calf intestine alkaline phosphatase.

Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pKa values based on absorbance at 405nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: Km=40±3µM; Vmax=72.8±1.2µmolmin-1mg protein-1; kcat=9.70±0.16s-1; kcat/Km=2.44±0.16 × 105 M-1s-1 (mean±SEM, N=4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC50 values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N=4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A Ki value for orthovanadate of 51±8nM (mean±SEM, N=3) was determined. Finally, data analysis and statistical design of experiments are discussed.

Biotransformation of 3-cyanopyridine to nicotinic acid using whole-cell nitrilase of Gordonia terrae mutant MN12.

In the present study, the Gordonia terrae was subjected to chemical mutagenesis using ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 5-bromouracil (5-BU) and hydroxylamine with the aim of improving the catalytic efficiency of its nitrilase for conversion of 3-cyanopyridine to nicotinic acid. A mutant MN12 generated with MNNG exhibited increase in nitrilase activity from 0.5U/mgdcw (dry cell weight) (in the wild G. terrae) to 1.33U/mgdcw. Further optimizations of culture conditions using response surface methodology enhanced the enzyme production to 1.2-fold. Whole-cell catalysis was adopted for bench-scale synthesis of nicotinic acid, and 100% conversion of 100mM 3-cyanopyridine was achieved in potassium phosphate buffer (0.1M, pH 8.0) at 40°C in 15min. The whole-cell nitrilase of the mutant MN12 exhibited higher rate of product formation and volumetric productivity, i.e., 24.56g/h/gdcw and 221g/L as compared to 8.95g/h/g dcw and 196.8g/L of the wild G. terrae. The recovered product was confirmed by HPLC, FTIR and NMR analysis with high purity (> 99.9%). These results indicated that the mutant MN12 of G. terrae as whole-cell nitrilase is a very promising biocatalyst for the large-scale synthesis of nicotinic acid.

Solvent Deuterium Isotope Effects of Substrate Reduction by Nitrogenase from Azotobacter vinelandii.

The mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, has been of great interest for understanding the catalytic strategy utilized to reduce dinitrogen to ammonia under ambient temperatures and pressures. The reduction mechanism of nitrogenase is generally envisioned as involving multiple cycles of electron and proton transfers, with the known substrates requiring at least two cycles. Solvent kinetic isotope effect experiments, in which changes of reaction rates or product distribution are measured upon enrichment of solvent with heavy atom isotopes, have been valuable for deciphering the mechanism of complex enzymatic reactions involving proton or hydrogen transfer. We report the distribution of ethylene, dihydrogen, and methane isotopologue products measured from nitrogenase-catalyzed reductions of acetylene, protons, and cyanide, respectively, performed in varying levels of deuterium enrichment of the solvent. As has been noted previously, the total rate of product formation by nitrogenase is largely insensitive to the presence of D2O in the solvent. Nevertheless, the incorporation of H/D into products can be measured for these substrates that reflect solvent isotope effects on hydrogen atom transfers that are faster than the overall rate-determining step for nitrogenase. From these data, a minimal isotope effect is observed for acetylene reduction (1.4 ± 0.05), while the isotope effects for hydrogen and methane evolution are significantly higher at 4.2 ± 0.1 and 4.4 ± 0.1, respectively. These results indicate that there are pronounced differences in the sensitivity to isotopic substitution of the hydrogen atom transfer steps associated with the reduction of these substrates by nitrogenase.

Engineering of Microbial Substrate Promiscuous CYP105A5 for Improving the Flavonoid Hydroxylation

Bacterial cytochrome P450 (CYP) enzymes are versatile biocatalysts that are responsible for the biotransformation of diverse endogenous substances. CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A, regioselective C3′-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. By rational design of substrate-binding pocket based on experimental data, homology modeling, and molecular docking analysis, we enhanced the product formation rate of flavonoids. The double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. Particularly, the L100A/I302A mutant’s kcat/Km values and in vivo conversion rate increased by 1.68-fold and 2.57-fold, respectively, for naringenin. Overall, our result might facilitate the potential use of CYP105A5 for future modification and application in whole-cell biocatalysts for the production of valuable polyphenols.

Tri-Reforming of Methane: Thermodynamics, Operating Conditions, Reactor Technology and Efficiency Evaluation—A Review

The production of syngas with optimal energy usage, a minimal environmental impact, and an adjustable H2/CO molar ratio is possible using tri-reforming of methane (TRM). Despite the number of studies dedicated to the TRM process, this process is still in its infancy, with many technical obstacles to overcome. Except for its kinetics and catalysts, which have been reviewed elsewhere, the TRM process is evaluated thoroughly in this work. First, feasibility studies of TRM and the TRM process are presented. Second, the impacts of various operating conditions on the rate of gas conversions, syngas production, and coke formation are discussed. Third, different reactor configurations are compared. This review then goes through the energy and energetic efficiency, economic, environmental, and safety aspects of the TRM process. Finally, a research path for the future is suggested.

Transition Metal-Free Hydrogenolysis of Anilines to Arenes Mediated by Lithium Hydride.

Hydrodenitrogenation (HDN) of nitrogen-containing organic compounds such as aniline and its derivatives is of scientific interest and practical importance. Major efforts have been devoted to the development and understanding of transition metal-mediated chemical processes. Herein, we report a fundamentally different strategy using a transition metal-free material, that is, lithium hydride (LiH) enabling the hydrogenolysis of aniline to benzene and ammonia via a chemical looping approach. Aniline reacts with LiH to form lithium anilide, and subsequently, the hydrogenolysis of lithium anilide yields benzene and ammonia and regenerates LiH to complete the loop. This LiH-mediated chemical looping HDN process stands in sharp contrast to the transition metal-catalyzed or -mediated processes, which commonly lead to the complete hydrogenation of aromatic rings. A highly denitrogenated product formation rate of 2623 μmol·g-1·h-1 is achieved for the hydrogenolysis of lithium anilide at 300 °C and 10 bar H2, which exceeds the catalytic rate of transition metal catalysts. Computational studies reveal that the scission of C-N bonds is facilitated by a Li-mediated nucleophilic attack of hydride to the α-sp2C atom of aniline. This work not only provides a distinctive chemical looping route for HDN, but also opens up materials space for the denitrogenation of anilines.

Bifunctional hydroformylation on heterogeneous Rh-WOx pair site catalysts.

Metal-catalysed reactions are often hypothesized to proceed on bifunctional active sites, whereby colocalized reactive species facilitate distinct elementary steps in a catalytic cycle1-8. Bifunctional active sites have been established on homogeneous binuclear organometallic catalysts9-11. Empirical evidence exists for bifunctional active sites on supported metal catalysts, for example, at metal-oxide support interfaces2,6,7,12. However, elucidating bifunctional reaction mechanisms on supported metal catalysts is challenging due to the distribution of potential active-site structures, their dynamic reconstruction and required non-mean-field kinetic descriptions7,12,13. We overcome these limitations by synthesizing supported, atomically dispersed rhodium-tungsten oxide (Rh-WOx) pair site catalysts. The relative simplicity of the pair site structure and sufficient description by mean-field modelling enable correlation of the experimental kinetics with first principles-based microkinetic simulations. The Rh-WOx pair sites catalyse ethylene hydroformylation through a bifunctional mechanism involving Rh-assisted WOx reduction, transfer of ethylene from WOx to Rh and H2 dissociation at the Rh-WOx interface. The pair sites exhibited >95% selectivity at a product formation rate of 0.1 gpropanal cm-3 h-1 in gas-phase ethylene hydroformylation. Our results demonstrate that oxide-supported pair sites can enable bifunctional reaction mechanisms with high activity and selectivity for reactions that are performed in industry using homogeneous catalysts.

Boosting selective photocatalytic CO2 reduction to CO over Dual-core@shell structured Bi2O3/Bi2WO6@g-C3N4 catalysts with strong interaction interface

The strong interaction between hetero materials interface is crucial to boost the transfer efficiency of photogenerated electrons. Herein, the dual-core@shell structured Bi2O3/Bi2WO6@g-C3N4 (BB@CN) photocatalysts were fabricated by the co-grind and calcination method. The separation/transfer efficiencies of photogenerated electron-hole pairs could be improved via the EXAFS (Extended X-ray Absorption Fine Structure) proved strong interaction of core/shell interface. The BB@CN catalysts with a strong interaction interface exhibit high activity for CO2-to-CO, and the BB@10CN (10 g melamine is used) catalyst has the largest formation rate (263.7 µmol g−1) and selectivity (97.0%) of CO product. Based on the results of in-situ DRIFTS (Diffuse Reflectance Infrared Fourier Transform Spectroscopy) and Density Functional Theory calculation, the photoconversion mechanism of CO2-to-CO has been proposed: the COOH* formation is the rate-determined step of CO2 conversion, and the selectivity of CO product is dependent on its suitable desorption energy. It inspires the fabrication strategy of efficient hetero photocatalysts with a strong interaction interface for selective photocatalytic CO2 conversion.

Flow/flame and emissions fields of premixed oxy-methane stratified flames in a dual annular counter-rotating swirl burner

Dual Annular Counter-Rotating Swirl (DACRS) premixed oxy-methane flames for nine different cases were studied numerically in a Dual-Stage Lean Premixed (DLPM) combustor for gas turbine combustion applications. The combustor consists of two streams, a primary central stream and a secondary annular one swirling in opposing directions. For all cases, the primary and secondary oxygen fraction (OF: O2%vol. in O2/CO2 oxidizer) was maintained at 30%, and the equivalence ratios (φ) of the primary and secondary streams were set to 0.9 and 0.55, respectively. Three different velocities were investigated for the primary central stream, and for each primary stream velocity, three different secondary velocities were considered to explore flow/flame interactions. The results show that: for all primary flow velocities, decreasing the secondary flow velocity led to the flame becoming thin and slightly elongated. The zone of peak local temperatures was concentrated in the flame core downstream of the center-body near the primary flow inlet as the secondary flow velocity was reduced. The magnitude of axial velocity has an effect on the flow distribution up to the first one-third section of the combustor. The mixture velocity varies inversely with Da number towards the combustor's exit. The thermal effect of secondary stream velocity starts around 40% away from the burner surface. Species distributions show that complete combustion is achieved and excess oxygen leaves the combustor with products due to the lean mixture of the secondary flow stream. The peak of product formation rate (PFR) is affected by flow streams velocities. Stable flames with reduced emissions were obtained over wide range of inlet velocities indicating the effectiveness of DLPM combustion approach for wider operability of gas turbine combustors.

The use of isothermal titration calorimetry for the assay of enzyme activity: Application in higher education practical classes.

Determination of enzyme activity is crucial for discovery, research, and development in life sciences. The activity of enzymes is routinely determined using spectrophotometric assays that measure rates of substrate consumption or product formation. Though colorimetric-based detection systems are simple, rapid, and economical to perform, the majority of enzymes are unsuitable for this technique as their substrates/products do not absorb in the UV or visible range. This limitation can be addressed by the use of coupled-enzyme assays or artificial chromogenic substrates; however these approaches have their own drawbacks. Here, we describe a method based on the use of an isothermal titration calorimeter (ITC) to measure the heat produced or absorbed during any enzyme-catalyzed reaction. The concept of calorimetric enzyme assays was demonstrated for the determination of enzyme hexokinase activity, which cannot be monitored colorimetrically without first coupling it to another enzymatic reaction. The assay is suitable for incorporation into undergraduate laboratory classes, providing students with an appreciation for; the versatility and ease of use of ITC assays; ITC as a flexible generic method for exploring the functional characteristics of uncharacterized enzymes; an activity detection parameter suitable for enzymes that either have no straightforward colorimetric methods available or require the use of nonartificial chromogenic substrates.

Strong Intermolecular Interactions Induced by High Quadrupole Moments Enable Excellent Photostability of Non‐Fullerene Acceptors for Organic Photovoltaics

AbstractUnderstanding degradation mechanisms of organic photovoltaics (OPVs) is a critical prerequisite for improving device stability. Herein, the effect of molecular structure on the photostability of non‐fullerene acceptors (NFAs) is studied by changing end‐group substitution of ITIC derivatives: ITIC, ITIC‐2F, and ITIC‐DM. Using an assay of in situ spectroscopy techniques and molecular simulations, the photodegradation product of ITIC and the rate of product formation are identified, which correlates excellently to reported device stability, with ITIC‐2F being the most stable and ITIC‐DM the least. The choice of acceptor is found to affect both the donor polymer (PBDB‐T) photostability and the morphological stability of the bulk heterojunction blend. Molecular simulations reveal that NFA end‐group substitution strongly modulates the electron distribution within the molecule and thus its quadrupole moment. Compared to unsubstituted‐ITIC, end‐group fluorination results in a stronger, and demethylation a weaker, molecular quadrupole moment. This influences the intermolecular interactions between NFAs and between the NFA and the polymer, which in turn affects the photostability and morphological stability. This hypothesis is further tested on two other high quadrupole acceptors, Y6 and IEICO‐4F, which both show impressive photostability. The strong correlation observed between NFA quadrupole moment and photostability opens a new synthetic direction for photostable organic photovoltaic materials.

Alkane Oxidation with H2O2 Catalyzed by Dicopper Complex with 6-hpa Ligand: Mechanistic Insights as Key Features for Methane Oxidation

Abstract Alkane oxidations with H2O2 catalyzed by copper complexes [Cu2(µ-OH)(6-hpa)]3+ (1) and [Cu(MeCN)(tpa)]2+ (2) were examined. In the oxidation of cyclohexane (CyH), cyclohexyl hydroperoxide (CyO2H) was formed as the first product and converted to cyclohexanol (CyOH) with PPh3. The turnover frequency (TOF/h) and turnover number (TON) of 1 are 150 and 1030, respectively. The kinetic studies showed that the product formation rate, d[CyO2H]/dt, is proportional to [1] and [H2O2], and partly to [Et3N] and [H2O]. Solvent kinetic isotope effect kH2O/kD2O was 2.2, showing that a H2O molecule is involved in the rate-limiting step. tert-BuO2H disturbs the formation of a di(hydroperoxo) intermediate [Cu2(O2H)2(6-hpa)]2+ to reduce the d[CyO2H]/dt. The active species [Cu2(O•)(O2•)(6-hpa)]2+ was detected by CSI MS. The inhibitory effects of a radical trap reagent DMPO and CO gas revealed that 1 suppresses the HO• formation. Methane oxidation with H2O2 catalyzed by 1, 2, and related complexes was conducted using a high-pressure reactor. Key features for the high catalytic activity of 1 in the methane oxidation are the complex-based active species [Cu2(O•)(O2•)(6-hpa)]2+ capable of cleaving the strong C-H bond of methane and the long catalyst life enabled by the suppression of the HO• formation.

Bioconversion of CO to formate by artificially designed carbon monoxide:formate oxidoreductase in hyperthermophilic archaea

Ferredoxin-dependent metabolic engineering of electron transfer circuits has been developed to enhance redox efficiency in the field of synthetic biology, e.g., for hydrogen production and for reduction of flavoproteins or NAD(P)+. Here, we present the bioconversion of carbon monoxide (CO) gas to formate via a synthetic CO:formate oxidoreductase (CFOR), designed as an enzyme complex for direct electron transfer between non-interacting CO dehydrogenase and formate dehydrogenase using an electron-transferring Fe-S fusion protein. The CFOR-introduced Thermococcus onnurineus mutant strains showed CO-dependent formate production in vivo and in vitro. The maximum formate production rate from purified CFOR complex and specific formate productivity from the bioreactor were 2.2 ± 0.2 μmol/mg/min and 73.1 ± 29.0 mmol/g-cells/h, respectively. The CO-dependent CO2 reduction/formate production activity of synthetic CFOR was confirmed, indicating that direct electron transfer between two unrelated dehydrogenases was feasible via mediation of the FeS-FeS fusion protein.

Quantification of Biocatalytic Transformations by Single Microbial Cells Enabled by Tailored Integration of Droplet Microfluidics and Mass Spectrometry.

Improving the performance of chemical transformations catalysed by microbial biocatalysts requires a deep understanding of cellular processes. While the cellular heterogeneity of cellular characteristics, such as the concentration of high abundant cellular content, is well studied, little is known about the reactivity of individual cells and its impact on the chemical identity, quantity, and purity of excreted products. Biocatalytic transformations were monitored chemically specific and quantifiable at the single‐cell level by integrating droplet microfluidics, cell imaging, and mass spectrometry. Product formation rates for individual Saccharomyces cerevisiae cells were obtained by i) incubating nanolitre‐sized droplets for product accumulation in microfluidic devices, ii) an imaging setup to determine the number of cells in the droplets, and iii) electrospray ionisation mass spectrometry for reading the chemical contents of individual droplets. These findings now enable the study of whole‐cell biocatalysis at single‐cell resolution.