Orientation-Controllable Enzyme Cascade on Electrode for Bioelectrocatalytic Chain Reaction.

The accomplishment of concurrent interenzyme chain reaction and direct electric communication in a multienzyme-electrode is challenging since the required condition of multienzymatic binding conformation is quite complex. In this study, an enzyme cascade-induced bioelectrocatalytic system has been constructed using solid binding peptide (SBP) as a molecular binder that coimmobilizes the invertase (INV) and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) cascade system on a single electrode surface. The SBP-fused enzyme cascade was strategically designed to induce diverse relative orientations of coupling enzymes while enabling efficient direct electron transfer (DET) at the FAD cofactor of GDHγα and the electrode interface. The interenzyme relative orientation was found to determine the intermediate delivery route and affect overall chain reaction efficiency. Moreover, interfacial DET between the fusion GDHγα and the electrode was altered by the binding conformation of the coimmobilized enzyme and fusion INVs. Collectively, this work emphasizes the importance of interenzyme orientation when incorporating enzymatic cascade in an electrocatalytic system and demonstrates the efficacy of SBP fusion technology as a generic tool for developing cascade-induced direct bioelectrocatalytic systems. The proposed approach is applicable to enzyme cascade-based bioelectronics such as biofuel cells, biosensors, and bioeletrosynthetic systems utilizing or producing complex biomolecules.

Task-specific polymeric membranes to achieve high gas-liquid mass transfer.

In the current study, Polyimide (P84)-based polymeric membranes were fabricated and used as spargers in the bubble column reactor (BCR) to get a high gas-liquid mass transfer (GL-MT) rate of oxygen in water. Different polymeric membranes were fabricated by incorporating polyvinyl pyrrolidone (PVP) as a porogen and a Zeolitic Imidazolate Framework (ZIF-8) to induce high porosity and hydrophobicity in the membranes. The GL-MT efficiency of membranes was evaluated by measuring the overall volumetric mass transfer coefficient (kLa) of oxygen in air. The kLa of O2 (in air) was measured by supplying the gas through a fixed membrane surface area of 11.94 cm2 at a fixed gas flow rate of 3L/min under atmospheric pressure. The results revealed that adding porogen and ZIF-8 increased the porosity of the membranes compared to the pure polymeric membranes. In comparison, the ZIF-8 (3 wt%) based membrane showed the highest porosity (80%), hydrophobicity (95° contact angle) and kLa of oxygen in air (241.2 h-1) with 78% saturation in only 60 s. ZIF-8 based membranes showed the potential to increase the amount of dissolved oxygen in BCR by reducing the bubble size, increasing the number of bubbles, and improving the hydrophobicity. The study showed that ZIF-8 based membrane diffusers are expected to produce high GL-MT in microbial syngas fermentation. To the best of our knowledge, this is the first study on the fabrication and application of polymeric membranes for GL-MT applications. Further research should be conducted under real fermentation conditions to assess the practicality of the system to support substrate utilization, microbial growth, and product formation.

Protocol for construction and characterization of direct electron transfer-based enzyme-electrode using gold binding peptide as molecular binder.

Here, we present a protocol for constructing direct electron transfer (DET)-based enzyme-electrodes using gold-binding peptide (GBP). We describe fusion of four GBPs to flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα), as model oxidoreductase, to generate four GDHγα variants. We then detail the measurements of catalytic and bioelectrochemical properties of these GDHγα variants on electrode together with surface morphology of GDHγα variants immobilized on gold surface. This protocol is useful for construction and validation of enzyme-based electrocatalytic system. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

Metabolism perturbation Causedby the overexpression of carbon monoxide dehydrogenase/Acetyl-CoA synthase gene complex accelerated gas to acetate conversion rate ofEubacterium limosumKIST612.

Microbial conversion of carbon monoxide (CO) to acetate is a promising upcycling strategy for carbon sequestration. Herein, we demonstrate that CO conversion and acetate production rates of Eubacterium limosum KIST612 strain can be improved by in silico prediction and in vivo assessment. The mimicked CO metabolic model of KIST612 predicted that overexpressing the CO dehydrogenase (CODH) increases CO conversion and acetate production rates. To validate the prediction, we constructed mutant strains overexpressing CODH gene cluster and measured their CO conversion and acetate production rates. A mutant strain (ELM031) co-overexpressing CODH, coenzyme CooC2 and ACS showed a 3.1 × increased specific CO oxidation rate as well as 1.4 × increased specific acetate production rate, compared to the wild type strain. The transcriptional and translational data with redox balance analysis showed that ELM031 has enhanced reducing potential from up-regulation of ferredoxin and related metabolism directly linked to energy conservation.

Methanol supply speeds up synthesis gas fermentation by methylotrophic-acetogenic bacterium, Eubacterium limosum KIST612.

This study analyzed the effect of methanol on the metabolism of syngas components (i.e., H2 and CO) by the syngas fermenting acetogenic strain E. limosum KIST612. The culture characteristics and relevant proteomic expressions (as fold changes) were carefully analyzed under CO/CO2 and H2/CO2 conditions with and without methanol addition, as well as, under methanol/CO2 conditions. The culture characteristics (specific growth rate and H2 consumption rate) under H2/CO2 conditions were greatly enhanced in the presence of methanol, by 4.0 and 2.7 times, respectively. However, the promoting effect of methanol was not significant under CO/CO2 conditions. Proteomic fold changes in most enzyme expression levels in the Wood-Ljungdahl pathway and chemiosmotic energy conservation also exhibited high correspondence between methanol and H2/CO2 but not between methanol and CO/CO2. These findings suggest the advantages of methanol addition to H2/CO2 for biomass enhancement and faster consumption of gaseous substrates during syngas fermentation.

Biosensing and electrochemical properties of flavin adenine dinucleotide (FAD)-Dependent glucose dehydrogenase (GDH) fused to a gold binding peptide.

In the present work, direct electron transfer (DET) based biosensing system for the determination of glucose has been fabricated by utilizing gold binding peptide (GBP) fused flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Burkholderia cepacia. The GBP fused FAD-GDH was immobilized on the working electrode surface of screen-printed electrode (SPE) which consists of gold working electrode, a silver pseudo-reference electrode and a platinum counter electrode, to develop the biosensing system with compact design and favorable sensing ability. The bioelectrochemical and mechanical properties of GBP fused FAD-GDH (GDH-GBP) immobilized SPE (GDH-GBP/Au) were investigated. Here, the binding affinity of GDH-GBP on Au surface, was highly increased after fusion of gold binding peptide and its uniform monolayer was formed on Au surface. In the cyclic voltammetry (CV), GDH-GBP/Au displayed significantly high oxidative peak currents corresponding to glucose oxidation which is almost c.a. 10-fold enhanced value compared with that from native GDH immobilized SPE (GDH/Au). As well, GDH-GBP/Au has shown 92.37% of current retention after successive potential scans. In the chronoamperometry, its steady-state catalytic current was monitored in various conditions. The dynamic range of GDH-GBP/Au was shown to be 3-30 mM at 30 °C and exhibits high selectivity toward glucose in whole human blood. Additionally, temperature dependency of GDH-GBP/Au on DET capability was also investigated at 30-70 °C. Considering this efficient and stable glucose sensing with simple and easy sensor fabrication, GDH-GBP based sensing platform can provide new insight for future biosensor in research fields that rely on DET.

Behavior of CO-water mass transfer coefficient in membrane sparger-integrated bubble column for synthesis gas fermentation.

The gas-liquid mass transfer coefficient (kLa) of O2 was investigated in a bubble column reactor (BCR) using a sintered gas filter (SF), ceramic membrane module (CMM), and hollow fiber membrane module (HFM), which have different ranges of gas supply areas. kLa was enhanced by increasing flow rate in all of the spargers. Different responses when changing the gas supply area were obtained depending on the sparger type. Average values of kLa that were 52 and 258% higher were obtained using a CMM-integrated BCR compared to SFs and HFMs. CO-water kLa was investigated using CMMs for application to gas fermentation. The CO-water kLa ranged from 28.3 to 113.7/h under the experimental conditions. Based on the experimental data from CO and O2, a model to predict kLa was constructed for CMM-integrated BCRs. A dimensionless number indicating a gas supply area of the sparger was newly defined and included in the developed model.

A simultaneous gas feeding and cell-recycled reaction (SGCR) system to achieve biomass boosting and high acetate titer in microbial carbon monoxide fermentation.

This study employed a simultaneous gas feeding and cell-recycled reaction (SGCR) system to ferment CO using Eubacterium limosum KIST612. A bubble column reactor was equipped with an ex-situ hollow fiber membrane module to enable cell recycling. The internal gas circulation rate was adjusted by controlling the pump speed to provide sufficient gas supplement to the microorganism. Gas feedings were conducted by either the use of a gas-tight bag (Batch), a pressurized gas cylinder (Continuous), or a sequential combination of the two (Mixed feeding). Mixed feeding mode achieved higher biomass (9.7 g/L) and acetate (9.8 g/L) concentrations than Batch mode (3.2 g/L biomass and 7.0 g/L acetate) or Continuous mode (5.0 g/L biomass and 8.1 g/L acetate). The high acetate titer in Mixed feeding mode was achieved due to the high concentration of cells secured in a short time at the initial operation stage and maintaining a high specific growth rate.

Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer.

Direct electron transfer (DET) between enzymes and electrodes is a key issue for practical use of bioelectrocatalytic devices as a bioenergy process, such as enzymatic electrosynthesis, biosensors, and enzyme biofuel cells. To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current was transferred to final electron acceptor due to energy loss at enzyme-electrode interface. This study describes the design and construction of a synthetic glucose dehydrogenase (GDH; α and γ subunits) combined with a gold-binding peptide at its amino or carboxy terminus for direct contact between enzyme and electrode. The fused gold-binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto a Au electrode. Depending on the fused site of binding peptide to the enzyme complex, nine combinations of recombinant GDH proteins on the electrode show significantly different direct electron-transfer efficiency across the enzyme-electrode interface. The fusion of site-specific binding peptide to the catalytic subunit (α subunit, carboxy terminus) of the enzyme complex enabled apparent direct electron transfer (DET) across the enzyme-electrode interface even in the absence of the electron-transfer subunit (i.e., ß subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of ca. (-)0.46 V vs Ag/AgCl was associated with the appearance of an flavin adenine dinucleotide (FAD)/FADH2 redox wave and a stabilized bioelectrocatalytic current of more than 100 µA, determined from chronoamperometric analysis. Electron recovery was 7.64%, and the catalytic current generation was 249 µA per GDH enzyme loading unit (U), several orders of magnitude higher than the values reported previously. These observations corroborated that the last electron donor facing to electrode was controlled to be in close proximity without electron-transfer intermediates and the native affinity for glucose was preserved. The design and construction of the site-specific "sticky-ended" proteins without loss of catalytic activity could be applied to other redox enzymes having a buried active site.

Determination of volumetric gas-liquid mass transfer coefficient of carbon monoxide in a batch cultivation system using kinetic simulations.

A mathematical model of microbial kinetics was introduced to predict the overall volumetric gas-liquid mass transfer coefficient (kLa) of carbon monoxide (CO) in a batch cultivation system. The cell concentration (X), acetate concentration (Cace), headspace gas (Nco and [Formula: see text] ), dissolved CO concentration in the fermentation medium (Cco), and mass transfer rate (R) were simulated using a variety of kLa values. The simulated results showed excellent agreement with the experimental data for a kLa of 13/hr. The Cco values decreased with increase in cultivation times, whereas the maximum mass transfer rate was achieved at the mid-log phase due to vigorous microbial CO consumption rate higher than R. The model suggested in this study may be applied to a variety of microbial systems involving gaseous substrates.

Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing, Butyrate-Forming Acetogen, Eubacterium limosum KIST612.

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na(+) dependent. Consistent with the finding of a Na(+)-dependent Rnf complex is the presence of a conserved Na(+)-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H2 + CO2. The energetic benefit may be one reason that butyrate is formed only from CO but not from H2 + CO2.

Microbial synthesis gas utilization and ways to resolve kinetic and mass-transfer limitations.

Microbial conversion of syngas to energy-dense biofuels and valuable chemicals is a potential technology for the efficient utilization of fossils (e.g., coal) and renewable resources (e.g., lignocellulosic biomass) in an environmentally friendly manner. However, gas-liquid mass transfer and kinetic limitations are still major constraints that limit the widespread adoption and successful commercialization of the technology. This review paper provides rationales for syngas bioconversion and summarizes the reaction limited conditions along with the possible strategies to overcome these challenges. Mass transfer and economic performances of various reactor configurations are compared, and an ideal case for optimum bioreactor operation is presented. Overall, the challenges with the bioprocessing steps are highlighted, and potential solutions are suggested. Future research directions are provided and a conceptual design for a membrane-based syngas biorefinery is proposed.

Effect of internal pressure and gas/liquid interface area on the CO mass transfer coefficient using hollow fibre membranes as a high mass transfer gas diffusing system for microbial syngas fermentation.

This study proposed a submerged hollow fibre membrane bioreactor (HFMBR) system capable of achieving high carbon monoxide (CO) mass transfer for applications in microbial synthesis gas conversion systems. Hydrophobic polyvinylidene fluoride (PVDF) membrane fibres were used to fabricate a membrane module, which was used for pressurising CO in water phase. Pressure through the hollow fibre lumen (P) and membrane surface area per unit working volume of the liquid (A(S)/V(L)) were used as controllable parameters to determine gas-liquid volumetric mass transfer coefficient (k(L)a) values. We found a k(L)a of 135.72 h(-1) when P was 93.76 kPa and AS/VL was fixed at 27.5m(-1). A higher k(L)a of 155.16 h(-1) was achieved by increasing AS/VL to 62.5m(-1) at a lower P of 37.23 kPa. Practicality of HFMBR to support microbial growth and organic product formation was assessed by CO/CO2 fermentation using Eubacterium limosum KIST612.

Rapid enrichment of (homo)acetogenic consortia from animal feces using a high mass-transfer gas-lift reactor fed with syngas.

A gas-lift reactor having a high mass transfer coefficient (k(L)a = 80.28 h(-1)) for a relatively insoluble gas (carbon monoxide; CO) was used to enrich (homo)acetogens from animal feces. Samples of fecal matter from cow, rabbit, chicken, and goat were used as sources of inoculum for the enrichment of CO and H(2) utilizing microbial consortia. To confirm the successful enrichment, the Hungate roll tube technique was employed to count and then isolate putative CO utilizers. The results of this work showed that CO and H(2) utilizing consortia were established for each inoculum source after 8 days. The number of colony-forming units in cow, rabbit, chicken, and goat fecal samples were 3.83 × 10(9), 1.03 × 10(9), 8.3 × 10(8), and 3.25 × 10(8) cells/ml, respectively. Forty-two colonies from the animal fecal samples were screened for the ability to utilize CO/H(2). Ten of these 42 colonies were capable of utilizing CO/H(2). Five isolates from cow feces (samples 5, 6, 8, 16, and 22) were highly similar to previously unknown (homo)acetogen, while cow-7 has shown 99 % similarity with Acetobacterium sp. as acetogens. On the other hand, four isolates from chicken feces (samples 3, 8, 10, and 11) have also shown high CO/H(2) utilizing activity. Hence, it is expected that this research could be used as the basis for the rapid enrichment of (homo)acetogenic consortia from various environmental sources.

Electricity generation from synthesis gas by microbial processes: CO fermentation and microbial fuel cell technology.

A microbiological process was established to harvest electricity from the carbon monoxide (CO). A CO fermenter was enriched with CO as the sole carbon source. The DGGE/DNA sequencing results showed that Acetobacterium spp. were enriched from the anaerobic digester fluid. After the fermenter was operated under continuous mode, the products were then continuously fed to the microbial fuel cell (MFC) to generate electricity. Even though the conversion yield was quite low, this study proved that synthesis gas (syn-gas) can be converted to electricity with the aid of microbes that do not possess the drawbacks of metal catalysts of conventional methods.