Prevalence of Escherichia coli in electrogenic biofilm on activated carbon in microbial fuel cell.

For a better understanding of the distribution of depth-dependent electrochemically active bacteria at in the anode zone, a customized system in a microbial fuel cell (MFC) packed with granular activated carbon (GAC) was developed and subsequently optimized via electrochemical tests. The constructed MFC system was sequentially operated using two types of matrice solutions: artificially controlled compositions (i.e., artificial wastewater, AW) and solutions obtained directly from actual sewage-treating municipal plants (i.e., municipal wastewater, MW). Notably, significant difference(s) of system efficiencies between AW or MW matrices were observed via performance tests, in that the electricity production capacity under MW matrices is < 25% that of the AW matrices. Interestingly, species of Escherichia coli (E. coli) sampled from the GAC bed (P1: deeper region in GAC bed, P2: shallow region of GAC near electrolytes) exhibited an average relative abundance of 75 to 90% in AW and a relative abundance of approximately 10% in MW, while a lower relative abundance of E. coli was found in both the AW and MW anolyte samples (L). Moreover, similar bacterial communities were identified in samples P1 and P2 for both the AW and MW solutions, indicating a comparable distribution of bacterial communities over the anode area. These results provide new insights into E. coli contribution in power production for the GAC-packed MFC systems (i.e., despite the low contents of Geobacter (> 8%) and Shewanella (> 1%)) for future applications in sustainable energy research. KEY POINTS: • A microbial community analysis for depth-dependence in biofilm was developed. • The system was operated with two matrices; electrochemical performance was assessed. • E. coli spp. was distinctly found in anode zone layers composed of activated carbon.

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.

Regulatory transcription factor (CooA)-driven carbon monoxide partial pressure sensing whole-cell biosensor.

We designed and constructed a whole-cell biosensor capable of detecting the presence and quantity of carbon monoxide (CO) using the CO regulatory transcription factor. This biosensor utilizes CooA, a CO-sensing transcription regulator that activates the expression of carbon monoxide dehydrogenase (CODH), to detect the presence of CO and respond by triggering the expression of a GUS reporter protein (ß-glucuronidase). The GUS reporter protein is expressed from a CO-induced CooA-binding promoter (PcooF) by CooA and enables the effective colorimetric detection of CO. An Escherichia coli strain used to validate the biosensor showed growth and GUS activity under anaerobic conditions; this study used the inert gas (Ar) to create anaerobic conditions. The pBRCO biosensor could successfully detect the presence of CO in the headspace. Moreover, the GUS-specific activity of pBRCO according to the CO strength as partial pressure followed Michaelis-Menten kinetics (R2 = 0.98). It was confirmed that the GUS-specific activity of pBRCO increased linearly up to 30.39 kPa (R2 = 0.98), and thus, a quantitative analysis of CO concentration (i.e., partial pressure) was possible.

Acetogen and acetogenesis for biological syngas valorization.

The bioconversion of syngas using (homo)acetogens as biocatalysts shows promise as a viable option due to its higher selectivity and milder reaction conditions compared to thermochemical conversion. The current bioconversion process operates primarily to produce C2 chemicals (e.g., acetate and ethanol) with sufficient technology readiness levels (TRLs) in process engineering (as midstream) and product purification (as downstream). However, the economic feasibility of this process could be improved with greater biocatalytic options in the upstream phase. This review focuses on the Wood-Ljungdahl pathway (WLP) which is a biological syngas-utilization pathway, redox balance and ATP generation, suggesting that the use of a specific biocatalysts including Eubacterium limosum could be advantageous in syngas valorization. A pertinent strategy to mainly produce chemicals with a high degree of reduction is also provided with examples of flux control, mixed cultivation and mixotrophy. Finally, this article presents future direction of industrial utilization of syngas fermentation.

Genome-Based Reclassification of Strain KIST612, Previously Classified as Eubacterium limosum, into a New Strain of Eubacterium callanderi.

The strain KIST612, initially identified as E. limosum, was a suspected member of E. callanderi due to differences in phenotype, genotype, and average nucleotide identity (ANI). Here, we found that E. limosum ATCC 8486T and KIST612 are genetically different in their central metabolic pathways, such as that of carbon metabolism. Although 16S rDNA sequencing of KIST612 revealed high identity with E. limosum ATCC 8486T (99.2%) and E. callanderi DSM 3662T (99.8%), phylogenetic analysis of housekeeping genes and genome metrics clearly indicated that KIST612 belongs to E. callanderi. The phylogenies showed that KIST612 is closer to E. callanderi DSM 3662T than to E. limosum ATCC 8486T. The ANI between KIST612 and E. callanderi DSM 3662T was 99.8%, which was above the species cut-off of 96%, Meanwhile, the ANI value with E. limosum ATCC 8486T was not significant, showing only 94.6%. The digital DNA-DNA hybridization (dDDH) results also supported the ANI values. The dDDH between KIST612 and E. callanderi DSM 3662T was 98.4%, whereas between KIST612 and E. limosum ATCC 8486T, it was 57.8%, which is lower than the species cut-off of 70%. Based on these findings, we propose the reclassification of E. limosum KIST612 as E. callanderi KIST612.

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).

Control of carbon monoxide dehydrogenase orientation by site-specific immobilization enables direct electrical contact between enzyme cofactor and solid surface.

Controlling the orientation of redox enzymes on electrode surfaces is essential in the development of direct electron transfer (DET)-based bioelectrocatalytic systems. The electron transfer (ET) distance varies according to the enzyme orientation when immobilized on an electrode surface, which influences the interfacial ET rate. We report control of the orientation of carbon monoxide dehydrogenase (CODH) as a model enzyme through the fusion of gold-binding peptide (gbp) at either the N- or the C-terminus, and at both termini to strengthen the binding interactions between the fusion enzyme and the gold surface. Key factors influenced by the gbp fusion site are described. Collectively, our data show that control of the CODH orientation on an electrode surface is achieved through the presence of dual tethering sites, which maintains the enzyme cofactor within a DET-available distance (<14 Å), thereby promoting DET at the enzyme-electrode interface.

Peptide sequence-driven direct electron transfer properties and binding behaviors of gold-binding peptide-fused glucose dehydrogenase on electrode.

Oriented enzyme immobilization on electrodes is crucial for interfacial electrical coupling of direct electron transfer (DET)-based enzyme-electrode systems. As inorganic-binding peptides are introduced as molecular binders and enzyme-orienting agents, inorganic-binding peptide-fused enzymes should be designed and constructed to achieve efficient DET. In this study, it is aimed to compare the effects of various gold-binding peptides (GBPs) fused to enzymes on electrocatalytic activity, bioactivity, and material-binding behaviors. Here, GBPs with identical gold-binding properties but different amino acid sequences were fused to the FAD-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) to generate four GDHγα variants. The structural, biochemical, mechanical, and bioelectrochemical properties of these GDHγα variants immobilized on electrode were determined by their fused GBPs. Our results confirmed that the GBP type is vital in the design, construction, and optimization of GBP-fused enzyme-modified electrodes for facile interfacial DET and practical DET-based enzyme-electrode systems.

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.

Functional Expression of a Mo-Cu-Dependent Carbon Monoxide Dehydrogenase (CODH) and Its Use as a Dissolved CO Bio-microsensor.

Herein, we report the heterologous expression in Escherichia coli of a Mo-Cu-containing carbon monoxide dehydrogenase (Mo-Cu CODH) from Hydrogenophaga pseudoflava, which resulted in an active protein catalyzing CO oxidation to CO2. By supplying the E. coli growth medium with Na2MoO4 (Mo) and CuSO4 (Cu), the Mo-Cu CODH metal cofactors precursors, the expressed L-subunit was found to have CO-oxidation activity even without the M- and S- subunits. This successful expression of CO-oxidizing-capable single L-subunit provides direct evidence of its role as the catalytic center of Mo-Cu CODH that has not been discovered and studied before. Subsequently, we used the expressed protein to construct a CO bio-microsensor based on a newly developed fast and sensitive Clark-type CO2 transducer using an aprotic solvent/ionic liquid electrolyte. The CO bio-microsensor exhibited a linear response to CO concentration in the 0-9 µM range, with a limit of detection (LOD) of 15 nM CO. The sensor uses a mixture of Mo-Cu CODH's L-subunit/Mo, Cu cofactors/methylene blue, confined in the enzyme chamber that is placed in front of a CO2 transducer. The optimized sensor's sensitivity and performance were retained to levels of at least 80% for 1 week of continuous polarization and operation in an aqueous medium. We have also demonstrated the use of an alkaline front-trap solution to make a completely O2/CO2 interference-free microsensor. The CO bio-microsensor developed in this study is potentially useful as an analytical tool for the detection of trace CO in dissolved form for monitoring dissolved CO concentration dynamics in natural or synthetic systems.

Microbial fuel cell driven mineral rich wastewater treatment process for circular economy by creating virtuous cycles.

The aim of this work is to study for concurrent harvesting bioelectricity and struvite mineral from mineral rich wastewater containing with nitrogen (N) and phosphorous (P) contents using MFCs and a chemical precipitation system. Whole reaction was constructed to sequentially run hybrid reactor (consisting of MFCs and struvite precipitation), gravitational sedimentation, nitrogen purging and MFCs. The MFCs generated around 6.439 ± 0.481 mA and 2.084 ± 0.310 mW as Imax and Pmax, respectively under 2g/l of COD. More than 70% of C source, and around 95% of P and N sources have been removed. Struvite mineral was precipitated in the hybrid reactor after the injection of Mg2+ and collected in sedimentation tank. Economic feasibility and beneficial concerns were carefully investigated, and it is proposed for applications in the "decentralised treatment process" of agriculture and livestock wastewater in order to realise circular and strong economy in agriculture by creating virtuous cycles.

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.

Controlling Voltage Reversal in Microbial Fuel Cells.

Microbial fuel cell (MFC) systems have been developed for potential use as power sources, along with several other applications, with bacteria as the prime factor enabling electrocatalytic activity. Limited voltage and current production from unit cells limit their practical applicability, so stacking multiple MFCs has been proposed as a way to increase power production. Special attention is paid to voltage reversal (VR), a common occurrence in stacked MFCs, and to identifying the mechanisms underlying this phenomenon. We also proposed realistic perspectives on stacked MFCs in an effort to control and suppress VR by balancing the kinetics in the system, such as using enriched electroactive microorganisms or altering the circuitry mode.

Dissolved carbon monoxide concentration monitoring platform based on direct electrical connection of CO dehydrogenase with electrically accessible surface structure.

CO dehydrogenase (CODH) employed in a dissolved CO biosensor development study harbors a solvent-exposed cofactor capable of DET to electrode. Here, CODH was immobilized on arrays of AuNPs of various dimensions to determine the effect of the size and shape of the electrode surface on the direct electrical connection between CODH and electrode surface. The results showed the degree of proximity between the CODH cofactor and electrode surface, which varied with AuNP size and caused significant changes to the electrical connection at the interface as well as to the substrate accessibility. Consequently, a high-density nanoscale SRS was fabricated on electrode to further facilitate direct electrical connection as well as to enable distribution of CODH into monolayer or near-monolayer for lowering the barrier of CO diffusion toward enzyme. The findings show the feasibility of controlling the direct electrical connection between CODH and the electrode as well as controlling the substrate accessibility.

Determination of optimum electrical connection mode for multi-electrode-embedded microbial fuel cells coupled with anaerobic digester for enhancement of swine wastewater treatment efficiency and energy recovery.

In this work, three multi-electrode-embedded microbial fuel cells (MFCs) were connected sequentially and operated in series and parallel modes, fed by effluent of an anaerobic digester continuously operated using swine wastewater. The anaerobic digester achieved ~0.75 CH4 L d-1 while removing 71.2% of COD and 0.8% of ammonia, which was comparable to the literatures reported. The MFCs removed additional COD from the anaerobic digester effluent, achieving the lowest concentration in the last unit, leading to a voltage reversal in the serially-connected unit. The MFCs connected and operated in parallel mode showed the highest power density of ~25 W m-3, which is 18% higher compared to the one operated in series mode. These results definitively show that differences in substrate concentrations among MFC units are inevitable with sequential flow. Further, a parallel connection mode of operation is necessary to achieve stable, long-term power generation from MFC units, without any electrical malfunction.

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.