Molar-scale formate production via enzymatic hydration of industrial off-gases

Decarbonizing the steel industry, a major CO 2 emitter, is crucial for achieving carbon neutrality 1,2 . Escaping the grip of CO combustion methods, a key contributor to CO 2 discharge is a seemingly simple yet formidable challenge on the path to industrial-wide net-zero carbon emissions 1,3–5 . Here we suggest the enzymatic CO hydration (enCOH), inspired by the biological Wood ‒ Ljungdahl pathway, enabling e�cient CO 2 �xation. By employing the highly e�cient, inhibitor-robust CO dehydrogenase (ChCODH2) and formate dehydrogenase (MeFDH1), we achieved spontaneous enCOH, to convert industrial off-gases into formate with 100% selectivity. This process operates seamlessly under mild conditions (room temperature, neutral pH), regardless of varying CO/CO 2 ratios. Notably, the direct utilization of �ue gas without pretreatment yielded various formate salts, including ammonium formate, at concentrations nearing two molars. Operating the 10 L-scale immobilized enzyme reactor at the steel mill resulted in the production of high-purity formate powder after facile puri�cation, thus demonstrating the potential for decarbonizing the steel industry.


Main
The steel/iron industry is one of the primary contributors to greenhouse gas emissions in the manufacturing eld (24% of total CO 2 emissions) along with electricity generation and transportation 1,2 .
With the increasing demand for steel and iron predicted to continue, substantial reductions in CO 2 emissions are imperative (annually 3 Gt CO 2 emission by 2050) 1,3,4 .A major portion of these emissions originates from the combustion of CO, a byproduct of iron production, releasing 2 tons of CO 2 per ton of manufactured steel 1,3,5 .Due to CO's toxicity, its current method of disposal through combustion results in considerable CO 2 discharge, exacerbating emissions.Hence, the diversion or transformation of CO into value-added compounds without resorting to combustion emerges as a prospective solution towards industry-wide decarbonization, and by implication, a substantial reduction in CO 2 emissions.
The conversion of CO gas into commodity chemicals is largely facilitated via metal-catalyzed chemical processes 6,7 .Among the two prevalent reactions, Fischer-Tropsch Synthesis (FTS) emerges as the preferred conduit for converting CO from the steel and iron industry, as opposed to the CO 2 -generating Water-Gas-Shift (WGS).Nevertheless, these processes necessitate harsh operational conditions and substantial energy inputs, in addition to requiring high-purity CO gas.In contrast, bioconversion of CO is performed under milder conditions, thus demanding less energy input.Several pioneering advancements have demonstrated the e cacy of biocatalytic CO utilization, including LanzaTech's ethanol fermentation derived from the acetogenic Wood-Ljungdahl (WL) pathway, and the Muller group's formate production via H 2 -dependent CO 2 reductase (HDCR) 8,9 .The WL pathway, with its linear progression from CO 2 to formic acid (Eastern branch) and CO (Western branch) 10,11 , presents a simpler implementation route compared to intricate circular CO 2 xation cycles, such as the Calvin Benson Bassham (CBB) cycle (involving multiple enzymes and reactions, Supplementary Fig. 1) 12 .Its application appears particularly promising for sulfur-containing off-gases, as the WL pathway's enzymes have evolved in sulfur-rich environments 13,14 .Despite these promising strides, obstacles still remain: oxygen and toxic substances such as cyanide can impair stability, and low carbon xation yield poses further challenges.While HDCR represents a promising system for CO 2 conversion, it demands a continuous hydrogen supply to transform CO 2 effectively.Bioconversion of industrial off-gas necessitates addressing issues such as inhibition by gas impurities (CN -, SO x , NO x , O 2 ), lower carbon yields, and slower conversion rates at lower CO concentrations 12,[15][16][17][18][19][20][21][22] .
Addressing these challenges, we suggest the engineered in-vitro enzymatic reaction, inspired by the reverse Western branch of the WL pathway, to e ciently exploit industrial CO off-gas containing varied gas components and toxic impurities (Fig. 1a): Reverse Western branch: CO + H 2 O → CO 2 + 2 e -+ 2 H + (catalyzed by CO dehydrogenase) Eastern branch: CO 2 + 2 e -+ 2 H + → HCOOH (catalyzed by formate dehydrogenase) The designed reaction demonstrates favorable thermodynamics, presenting a promising route for the direct utilization of real industrial off-gas.Importantly, our enzyme-catalyzed CO hydration (enCOH) reaction, formulated as CO + H 2 O → HCOOH (formic acid), occurs without any additional CO 2 emission (Fig. 1b).The standard Gibbs free energy of the enCOH reaction, ΔG 0 ′= -17.4 kJ/mol at 25℃ and pH 7.0, further substantiates its thermodynamic feasibility (Supplementary Table 1).Toxic impurities typically found in ue gas, such as SO x , NO x , H 2 S, and CN -, that usually deactivate metal catalysts and necessitate complex, expensive pre-separation processes [23][24][25][26] , surprisingly exhibit negligible inhibition effects on both CO dehydrogenase (CODH) and formate dehydrogenase (FDH) involved in the enCOH reaction (Supplementary Fig. 2), making it facilely applicable to direct industrial off-gas.
Here, we report on an enCOH reaction operational at atmospheric temperature and pressure, facilitating molar-scale formate production from industrial off-gas of steel manufacturing and waste plastic gasi cation.This process exhibits enduring stability in both laboratory settings and the semi-pilot scale installation within the steel mill's operational eld.

Enzymatic hydration of CO to formate
The enCOH reaction initiates with CO as the starting substrate, leading to CO 2 production through CO oxidation catalyzed by CODH.The subsequent spontaneous reaction catalyzed by FDH results in the generation of formic acid, with a theoretical yield of 1 mole of formic acid per 1 mole of CO (Fig. 1b).To ensure e cient enCOH, it is preferable for a complete cycle reaction to take place through the regeneration of the electron mediator in the preceding one-pot reaction.Examination of arti cial mediators, such as viologens, was conducted as prior option (Supplementary Table 2) since electron carrier proteins (such as ferredoxin) are too speci c to be employed for both CODHs and FDHs originated from various strains 27 .As the alternative, the various viologens (MV, methyl viologen; BV, benzyl viologen; EV, ethyl viologen) and natural mediator (NAD, nicotinamide adenine dinucleotide) were investigated based on their reaction energies, which were calculated based on redox potential and standard Gibbs free energy at pH 7 (Supplementary Table 1 and Supplementary Fig. 3).This suggests that MV and EV are more e cient mediators than BV or NAD since those can supply enough energy to convert CO 2 to formate.To con rm the functionality of our proposed enCOH system involving CODH and FDH and to select a suitable enzyme combination set through experiment, we compared formate production with three commercially available mediators in their oxidized forms (BV ox , EV ox , NAD + ) due to easiness compared to MV in handling.Candidate enzymes that exhibit fast reaction rates, oxygen tolerance, and are advantageous for large-scale protein production were considered 20,26,[28][29][30][31][32][33][34][35] : for CODH, e cient CO-oxidizing ChCODH2 (Ch, Carboxydothermus hydrogenoformans) and ToCODH (To, Thermococcus onnurineus NA1), and O 2 -tolerant ChCODH4; for FDH, e cient CO 2 -reducing TsFDH (Ts, Thiobacillus sp.KNK65MA), O 2 -tolerant MeFDH1 (Me, Methylobacterium extorquens), and RcFDH (Rc, Rhodobacter capsulatus).Upon conducting reactions with 99.998% CO (v/v), formate production for 48 h was observed only when employing mediator EV and MeFDH1 (Fig. 1c and Supplementary Fig. 4).Interestingly, the highest formate production was displayed in the following order for CODH: ChCODH2 (40.2 mM), ToCODH (7.7 mM), and ChCODH4 (2.6 mM) when combined with MeFDH1.RcFDH, devoid of the stabilizer KNO 3 , and TsFDH, lacking metal in the active site, showed no formate production in any combination with CODHs and mediators.
Further, to elucidate these differences in productivity, we measured the catalytic properties of each enzyme as a function of the mediator in reaction mimicry environment (pH 6.5, 30°C), as shown in Table 1 and Supplementary Fig. 5.Among the three tested mediators, the CODHs displayed no activities for NAD + , and FDHs showed very low activities for reduced BV, implying that the overall reaction rate of enCOH using these mediators would be considerably slow.In contrast, the use of EV enables COoxidizing activity in all CODHs tested and high CO 2 -reducing activities in two metalloenzyme FDHs (MeFDH1 and RcFDH), suggesting that EV is the most effective mediator.When using EV as an electron mediator, the highest CO oxidizing and CO 2 -reducing activities were observed in ChCODH2 (290 U/mg using EV ox as the electron acceptor) and MeFDH1 (66 U/mg using EV red as the electron donor), respectively, among the six candidate enzymes.Therefore, we concluded that high CO oxidation activity of ChCODH2 and superior CO 2 reduction activity of MeFDH1 led the highest formate concentration (40.2 mM) after 48 h from the reaction initiated in a closed system (Fig. 1c).These ndings suggest that ChCODH2 and MeFDH1 are the best-performing set among tested enzymes for enCOH.
A more in-depth examination of the yield and selectivity of the reaction involving ChCODH2 and MeFDH1 was carried out by conducting enzymatic conversion of the gas substrate CO to formate in closed serum bottles (Fig. 1d).The amounts of CO, CO 2 , and formate were measured for 36 h in a closed system to estimate the yield of CO to formate.As a result, we observed that the formate gradually increased as CO was consumed, and after 30 h, all the CO had been converted to formate.During the reaction starting points within 12 h, the initial formate production exhibited the slightly lower yield until CO 2 accumulated to approximately 1 mM.This is likely attributed to MeFDH1's modest CO 2 a nity at a level of 3.9 mM (Supplementary Table 3).However, approaching the end point of the reaction, the consumed CO amount was completely matched with the amount of produced formate, which suggested the stoichiometric conversion between CO and formate (Fig. 1d and Supplementary Fig. 6).Furthermore, to verify whether the enzymatic reaction products of enCOH were genuinely a result of carbon monoxide combining with water to generate formic acid, we examined the substrate and product masses using isotopes ( 13 CO).
Isotopic labeling experiments con rmed that HCOO -was derived from 13 CO and D 2 O (Fig. 1e).The molar mass of H 13 COO -was detected at 45.98 g/mol, comparable to that of H 12 COO -(44.98 g/mol) obtained from the 12 CO/H 2 O condition.Similarly, the H 2 O/D 2 O experiment with the same ratio showed a molar mass of D 12 COO -at 45.98 g/mol.These results demonstrate that the carbon and hydrogen atoms of formate originated from CO and water, respectively.

enCOH conversion of industrial off-gases to formate
An e cient enCOH system requires simultaneous catalysis of CO and CO 2 substrates by both CODH and FDH enzymes at their best performance.To achieve this, factors such as substrate and product inhibition, physicochemical conditions like pH, temperature, and buffer, as well as the enzyme ratio, should be considered 36 .Interestingly, no inhibitory cross-talks were observed between the formate product on ChCODH2 and the CO substrate on MeFDH1 (Supplementary Fig. 7).This is likely due to the tungsten (W)-containing active site of FDH being tolerant to CO 37 and the size of formate hindering its passage through the substrate tunnel to access the nickel (Ni)-containing active site of CODH (Supplementary Fig. 8).In addition to the inhibition effect, the pH pro le revealed that both enzymes were active between pH 6.5-8, as displayed in Supplementary Fig. 9a.A pH of 6.5 was chosen for enCOH because the overall reaction rate was governed by the enzymatic reaction of MeFDH1, which exhibited lower activity compared to ChCODH2 (Table 1).In terms of temperature, enzyme activity progressively increased within the 30-70°C range.However, based on kinetic stability, activity was better maintained at temperatures below 30°C (Supplementary Fig. 9b-d).Long-term, it is deemed more costeffective to sustain consistent activity at lower temperatures rather than having high activity at elevated temperatures.The impact of various buffers (sodium phosphate, bis-Tris propane, imidazole, MOPS (3-(N-morpholino) propanesulfonic acid)) at 50 mM concentrations appeared minimal, and a higher enzyme ratio of ChCODH2 to FDH proved to be bene cial since a higher EV red /EV ox ratio is preferred for CO 2 reduction catalyzed by FDH (Supplementary Fig. 9e-f).Based on these ndings, we concluded that both enzymes, ChCODH2 and MeFDH1, can function effectively together under ambient/neutral conditions.
In steel mill ue gas applications, it is essential for the two enCOH enzymes to be tolerant to potential inhibitors, such as O 2 , HCN (0.3-1.5 g/Nm 3 ) and SO x (0.1-0.8 g/Nm 3 ), which are present in the ue gas of steel mill 38 .For O 2 , this challenge can be addressed by employing the recently developed O 2 -tolerant ChCODH2 A559W 26,39 and the well-established O 2 -tolerant MeFDH1 20 .Apart from this, when examining the inhibitory effect, both ChCODH2 and MeFDH1 retained their activities at aqueous cyanide and bisul te concentrations corresponding to the range found in real ue gas (~ 0.05 µM of cyanide, ~ 4 µM of bisul te).However, those enzyme activities declined when exposed to excessively high levels of cyanide and bisul te (IC 50 of ChCODH2 to cyanide is 2 µM and MeFDH1 to cyanide is 600 µM), despite the fact that ue gas from steel mills contains levels far below these IC 50 values (Supplementary Fig. 2).
ChCODH2 undergoes competitive inhibition by cyanide due to the binding of cyanide to the Ni atom of the catalytic center (Ni-Fe-S cluster), whereas MeFDH1 exhibits greater tolerance to cyanide 16 .No de nitive inhibition mechanism caused by bisul te on the two enzymes has been identi ed, and resistance to bisul te is considerably higher than that of cyanide.
Leveraging this resilience, the production of formate using an O 2 -tolerant enzyme-based enCOH (ChCODH2 A559W and MeFDH1) 20,26 was evaluated in a closed reactor using real ue gas.Three representative waste gases, including COG (Coke Oven Gas), BFG (Blast Furnace Gas), and LDG (Linz-Donawitz Gas) from a Hyundai steel mill in Korea (Supplementary Table 4), and waste gas from mixed plastics (SRF, solid recovered fuel) from the gasi er of Korea Institute of Energy Research (KIER) were applied for the enCOH reaction (Fig. 2a).A mass balance analysis for CO consumption and formate production was conducted by measuring CO levels in the gas phase and formate concentrations in the aqueous phase.Despite variations in CO content and complex gas compositions of the supplied gases, all CO molecules were converted to formate, corroborating the theoretical yield of the enCOH reaction.As expected, LDG with higher carbon contents yielded a higher amount of formate (~ 460 µmol) on the while COG with the lowest carbon contents resulted in lower formate production (~ 93 µmol).This observation supports that an inexpensive LDG is a suitable waste gas for conversion into fuels and chemicals, due to the high content of recyclable CO/CO 2 .Based on the gas composition of CO/CO 2 , we have classi ed waste real gas into three categories: COG/BFG, equivalent ratios; LDG, high CO/low CO 2 ; SRF-derived gas, low CO/high CO 2 .As shown in Fig. 2b, the enCOH transformed the low CO content in waste plastic-derived gas into formate.These ndings demonstrate that enCOH can effectively utilize CO across diverse gas compositions, owing to its high substrate a nity for CO 26 .COG, BFG, LDG and SRF-derived gas have various compositions of gaseous components other than CO/CO 2 , but they had no signi cant adverse effects on the enzymatic conversion of enCOH.For example, COG has a high H 2 content 26, 40,41 , but it had no impact (Fig. 2a-b and Supplementary Fig. 10).Based on this, it is expected that enCOH can utilize real waste gas with various compositions.
To achieve cost-effective enzymatic formate production, the activity of enCOH enzymes must be stably maintained during the conversion of CO to formate when LDG gas with higher CO/CO 2 was applied for long term operation.A repeated reuse experiment was conducted, immobilizing CODH and FDH on Ni-NTA (Fig. 2c-d).Consequently, formate productivity was sustained without sacri cing enzyme activity for over ten reuse cycles.This suggests that stable chemical production is attainable through enCOH using real ue gas, due to the enzymes' resilience toward toxic inhibitors such as cyanide and bisul te.

Scaling up of enCOH conversion for steel mill eld implementation
To explore the applicability of the enCOH system for continuous ow reactions, a customized 100 mL enzyme reactor was designed and tested (Fig. 3a and Supplementary Fig. 11).Since formic acid production caused a continuous pH drop due to low pKa value (pKa = 3.745), a neutralizing agent was required to keep constant pH.We operated a 100 mL-scale reactor for enCOH under pH 6.5 control by continuously purging LDG and evaluated the productivity and e ciency of the enCOH reaction using three representative neutralizers: NaOH, KOH, and NH 4 OH (Fig. 3b).In all cases with different neutralizers, formate production remained stable for 64 h, yielding approximately 1.1 M (for NaOH), 1.2 M (for KOH), or 1.8 M (for NH 4 OH).The resulting formate salts-sodium (HCOONa, 88.8% purity), potassium (HCOOK, 94.6% purity), and ammonium (HCOONH 4 , 92.1% purity)-were easily obtained (Fig. 3c and Supplementary Fig. 12).Through this process, the use of NH 4 OH not only yields high productivity but also generates ammonium formate, a notable compound that is challenging to produce via conventional industrial processes 42,43 and is synthesized easily through enzymatic reactions.Applying a catalyst to formate salts is anticipated to facilitate e cient hydrogen production, positioning them as promising hydrogen carriers 44 .
To evaluate the biocatalytic e ciency of the enCOH system, we compared it with recent bioconversion technologies employed for generating value-added products from CO x gaseous sources (Fig. 3d and Supplementary Table 5).The enCOH system enables the production of high formate concentrations and notable productivity even using real ue gas, however few reported works have been tested with real offgas.This highlights the potential utility of the enCOH system for industrial applications.
Finally, the scaled-up 10 L reactor was designed for eld operation of the enCOH reaction (Fig. 3e and Supplementary Fig. 13).The reactor was installed on-site to utilize LDG from Hyundai Steel (Dangjin, Korea).We observed that approximately 298.5 g of formate was produced during the 69.5-hour enCOH reaction under ambient conditions, as shown in Fig. 3f.Additionally, we demonstrated the repeated reuse of enCOH biocatalysts (Fig. 3g).Unexpectedly, a temporary gas-supply interruption (at 25 h) and low temperature in the actual eld environment negatively affected formate productivity since electric temperature control system could not be installed due to strict safety regulation of steel mill.Nonetheless, the enCOH system's stress test results indicate that the actual waste by-product gas can be effectively applied to produce the valuable chemical formate.Given the system's minimal susceptibility to variations in CO content, catalytic inhibitors, and gas composition, the enCOH reaction represents a promising way for transforming real ue gas into valuable products in industrial applications.

Discussion
The tailored enCOH's accomplishment in the waste C1 conversion signi es a crucial leap forward in industrial biotechnology and renewable energy elds 45 .This feasible technology facilitates the transformation of ue CO into formate at a near 2-molar scale, demonstrating enzyme engineering's potent role in crafting novel industrial biocatalysts.Formate's role as a hydrogen carrier underscores its signi cance in CO 2 reduction and sustainable energy generation 46,47 .The production of formate from industrial off-gases and plastic-derived gases bolsters sustainable energy and chemical production while diminishing greenhouse gas emissions 9,48 .This oxygen-tolerant enzymatic technology showcases potential for e cient mixed waste gas conversion into valuable formate, advancing industrial CO 2 reduction and sustainable hydrogen carrier production.
The implementation of 10 L-scale enzyme reactor at the eld of steel company signals an important step in its advancement, indicating its potential for scalability and integration into industrial processes.Major steel companies in South Korea (POSCO and Hyundai Steel) are now exploring opportunities for incorporating this technology into their processes.This not only emphasizes the versatility and applicability of enzyme engineering but also its potential in promoting sustainability within industrial processes 17,21 .
In conclusion, the enzymatic molar-scale production of formate from complex industrial off-gases using a rationally designed in-vitro reaction exempli es a notable stride in sustainable energy production systems and the realm of industrial biotechnology.The potential rami cations of this technology are considerable, providing invaluable insights for scienti c, environmental, and industrial sectors seeking to reduce greenhouse gas emissions and support sustainable industrial processes.
For kinetic analysis, cells were lysed either by sonication in an anaerobic chamber (COY Laboratory Products Inc., Michigan, USA) or by a homogenizer (GEA Mechanical Equipment, Parma, Italy) for larger enCOH reaction (100mL reactor) preparations.Lysis buffers varied depending on the enzyme: CODHexpressing cells utilized a 50 mM sodium phosphate buffer (pH 8.0) of 300 mM NaCl, 2 mM DTE (dithioerythritol), and 2 µM resazurin; FDH-expressing cells employed a 50 mM MOPS (3-(Nmorpholino)propanesulfonic acid) buffer (pH 7.0) containing 300 mM NaCl, 2 mM DTE, and 2 µM resazurin.After lysis, the supernatant from centrifugation (11,000 rpm, 4°C, 20 min) was mixed with Ni-NTA agarose beads and incubated for 15 min in an anaerobic chamber.The enzyme-bound beads were washed and directly used for enCOH reactions or eluted for further assays.The elution buffers were similar to the lysis buffers but contained 20-250 mM imidazole for CODH and 20-300 mM imidazole for FDH.An additional 10 mM KNO 3 was used for RcFDH puri cation.
For FDH activity assay, the reaction was initiated by adding 1 µg of enzyme to a 200 mM sodium phosphate buffer (pH 6.5) containing 100 mM sodium bicarbonate, 0.1 mM reduced mediators (BV red , EV red , NADH), and 2 mM DTE at 30°C.Activity was estimated by tracking the initial rate of decline in absorbance for each reduced mediator spectrophotometrically.Reduced viologens, BV red and EV red , were prepared anaerobically by mixing 200 mM oxidized viologens with 1 g of zinc and ltering through a 0.45 µm syringe lter 52 .All experiments were performed in triplicate.

Enzyme catalytic properties
Kinetic analysis of enzyme activity was performed using varying concentrations of electron mediators (0.5-8 mM for CODH-mediated CO oxidation and 0.006-0.1 mM for FDH-mediated CO 2 reduction) in conditions identical to the activity assays.Kinetic parameters for BV, EV, and NAD(H) were determined using the Hanes-Woolf plot.Enzyme activity pro les across different pH (6-8) and temperature (30-70°C) conditions were assessed using pH-adjusted buffers and 200 mM sodium phosphate buffer (pH 6.5), respectively.Inhibition of CODH activity by formate was evaluated using a CO-saturated sodium phosphate buffer (pH 6.5) with 20 mM EV ox , 2 mM DTE, 2 µM resazurin and varying formate concentrations (0-1 M).The inhibitory effect of CO on FDH activity was assayed in a sodium phosphate buffer (pH 6.5) supplemented with 100 mM sodium bicarbonate, 0.1 mM EV red , 2 mM DTE, 2 µM resazurin and different CO concentrations (0-1 mM).By Henry's law, a 99.998% (v/v) CO purged buffer at room temperature and pressure contains approximately 1 mM CO.To assess enzyme inhibition by KCN and NaHSO 3 , the enzymes (4 µg) were anaerobically incubated with varying concentrations of inhibitors (10 nM-20 mM) for 30 min in a sodium phosphate buffer (pH 6.5).The residual enzyme activities were subsequently measured.All experiments were performed in triplicate.

HPLC and GC analysis
In this study, formate (reaction product), was analyzed using high-performance liquid chromatography (HPLC), while gaseous components including CO (substrate), CO 2 (intermediate), and other gases such as H 2 and N 2 , were quanti ed via gas chromatography (GC) to monitor their changes during the enzymatic reactions.Immediately after sampling, formate from the enCOH reaction solution was quenched using a 6 M H 2 SO 4 solution in a 10:1 ratio.HPLC measurements utilized an Aminex HPX-87H column (Bio-Rad, California, USA) and an RID-20A refractive index detector (Shimadzu, Kyoto, Japan) (mobile phase: 5 mM H 2 SO 4 , ow rate: 0.6 mL•min -1 ).
Gas content in the headspace of the serum bottle from enCOH experiments was analyzed using a 7890B GC-TCD system (Agilent Technologies, California, USA).A 0.5 mL gas sample was obtained using a gastight syringe (Trajan Scienti c and Medical, Victoria, Australia).Gas chromatographic analyses employed a Carboxen-1000 column (Supelco, Missouri, USA) with Ar as the carrier gas ( ow rate: 30 mL•min -1 ).
The oven temperature was initially set at 35°C for 5 min, and then ramped to 225°C at a rate of 20°C•min -1 .All experiments were performed in triplicate.

ESI-MS, FT-IR, and NMR analysis
The isotopic identity of enCOH products was con rmed using electrospray ionization mass spectrometry (ESI-MS).Reaction mixtures, prepared with 13 CO (99% atom 13 C, Cambridge Isotope Laboratories, USA) or D 2 O (99.9% atom D, Sigma-Aldrich, USA), yielded formates with distinct masses.Either 12 CO or 13 CO was introduced into a reaction buffer (10 mM EV ox , 2 mM DTE, 2 µM resazurin; for D 2 O experiments, up to 50% (v/v) D 2 O) in a serum bottle.Reactions were initiated by injecting 2,000 U ChCODH2 and 20 U MeFDH1, and quenched after 24 h with 10 M KOH for ESI-MS analysis.The ESI-MS (JMS-T100LP, JEOL, Japan) operated in negative mode, set at -2,000 V needle voltage, 250℃ desolvation chamber, -30 V and 80℃ ori ce 1, -5 V ring lens, -10 V ori ce 2, and 100 V ion guide RF.A survey TOF MS experiment scanned m/z 10 to 100 for isotope-labeled formate.
To characterize formate salts produced from enCOH reactions, Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) were employed.For three formate salts (sodium, potassium, ammonium) analysis, FT-IR was used.Those salts were produced through immobilized

Evaluation of enCOH enzyme candidates
In order to identify the most effective enzymes for the enCOH reaction, combinations of various enzymes and electron mediators were examined.For CODHs, 100 µg each of ChCODH2, ChCODH4, and ToCODH were tested.As for FDHs, 250 µg each of MeFDH1, RcFDH, and TsFDH were evaluated.Serum bottles containing a 200 mM bis-Tris propane buffer (pH 6.5), supplemented with 10 mM electron mediators (BV ox , EV ox , NAD + ), 2 mM DTE, and 2 µM resazurin, were purged with 99.998% (v/v) CO gas for 1 h.
Reactions at room temperature were initiated by enzyme injection, and the formate concentration was measured after 48 h.For the time course analysis, the conditions remained the same, employing ChCODH2, MeFDH1, and EV ox .The reaction was monitored over time using both HPLC and GC.All experiments were performed in triplicate.

Enzymatic reaction in sealed serum bottles
The enCOH reactions were performed at room temperature in 120 mL serum bottles sealed with rubber, containing 200 mM sodium phosphate buffer (pH 6.5), 10 mM EV ox , 2 mM DTE, and 2 µM resazurin.
Bottles were purged for 1 h with test gases (99.998% (v/v) CO or real ue gases, including coke-oven gas (COG), blast-furnace gas (BFG), Linz-Donawitz converter gas (LDG), or solid recovered fuel (SRF)-1.During operation, LDG from the steel mill was continuously supplied via compressor into the 15 L reactor, which held 10 L of 50 mM sodium phosphate solution (pH 6.5), supplemented with 1 mM EV ox , 2 mM DTE, 3.5 kU immobilized ChCODH2 A559W, and 12.5 kU immobilized MeFDH1.The pH was maintained at 6.5 by adding 10 N NaOH as a neutralizing agent.

Salt formate separation
Following 100 mL reactor operation, solution was ltered through a sintered glass lter, followed by an activated carbon bed (10 g, Darco-G60, Sigma-Aldrich, USA).The adsorptive properties of activated carbon enabled selective formate separation, considering the reported a nity of viologens for activated carbon 55,56 .The recovered solution was dried in a convection oven at 90°C overnight to yield powdered solids.The purity and identity of the produced formate were veri ed using HPLC, FT-IR, and NMR analyses, respectively.* Kinetic data were assayed at 30°C, pH 6.5.The kinetic parameters were calculated by tting the initial rates obtained at six different BV/EV concentrations (0.0625-32 mM) to the Hanes-Woolf equation using SigmaPlot 10.0.All enzymatic activities were determined in triplicate (see details in the Methods section).† The values of k cat were calculated from V max for BV and EV.
ND was noted when the speci c activity was under 1 U/mg.isotopes, 13 CO and D 2 O. 13 CO gas and D 2 O were used for testing and the molecular weight of produced formate through enCOH was determined by mass spectrometry.98.9 % (v/v) of carbon is 12 C and 99.9 % (v/v) of hydrogen is 1 H, leading most formate to have 45 g/mol of molar mass 18,19 .
Figure 2 E cient formate production through enCOH using real ue gas in a closed system.a, In uence of using real ue gas (BFG, COG, LDG) on enCOH.The industrial ue gases were used in enCOH.The CO (blue) consumption, CO 2 (orange) and formate (purple) production were measured in time course.b, In uence of using plastic-derived gas (SRF, solid recovered fuel) on enCOH.The gas derived from SRF gasi cation was used in enCOH.The CO (blue), CO 2 (orange), and formate (purple) were observed.c, A schematic for experimental procedure.Enzymes with a His 6 -tag was immobilized to a Ni-NTA resin for 10 cycles of reuse with no severe losses in conversion rate.The reaction was performed at room temperature using a Figure 3

enzymes ( 35 U
of Ni-NTA immobilized ChCODH2 A559W and 125 U of Ni-NTA MeFDH1) in a reaction buffer with 10 mM EV ox , 2 mM DTE, 2 µM resazurin.FT-IR spectra were acquired on a Nicolet iS50 spectrometer (Thermo Fisher Scienti c Inc., USA) in attenuated total re ection mode. 1 H-NMR spectra were acquired using a Bruker 400 MHz AVANCE III HD spectrometer (Massachusetts, USA) at ambient temperature, with D 2 O as the reference (D 2 O, δ = 4.69 ppm; formate, δ = 8.37 ppm).

Figures Figure 1
Figures rubber-sealed serum bottle (115 mL) with LDG-purged reaction buffer (20 mM EV ox , 200 mM sodium phosphate buffer pH 6.5 with purged LDG gas for 1 h).The immobilized enzyme was subsequently recovered through a disposable open column, washed twice with 200 mM sodium phosphate buffer pH 6.5, and reused after each reaction.d, Repeated reuse of enzyme for enCOH with LDG.By using CODH and FDH which are immobilized on Ni-NTA agarose beads, the reusability of enzymes was tested.The CO (blue) consumption, CO 2 (orange) and formate (purple) production were completed in every n h.

Table 1 Table 1 .
Catalytic properties of CODHs and FDHs.Kinetic properties of the associated enzymes were quanti ed under the given enzymatic reaction conditions.Speci c activities were determined at 20 mM mediator in sodium phosphate buffer saturated with CO (30°C, pH 6.5).Values are the means ± standard variation, n = 3. a