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.

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.

High performance enzyme fuel cells using a genetically expressed FAD-dependent glucose dehydrogenase α-subunit of Burkholderia cepacia immobilized in a carbon nanotube electrode for low glucose conditions.

FAD-dependent glucose dehydrogenase (FAD-GDH) of Burkholderia cepacia was successfully expressed in Escherichia coli and subsequently purified in order to use it as an anode catalyst for enzyme fuel cells. The purified enzyme has a low Km value (high affinity) towards glucose, which is 463.8 µM, up to 2-fold exponential range lower compared to glucose oxidase. The heterogeneous electron transfer coefficient (Ks) of FAD-GDH-menadione on a glassy carbon electrode was 10.73 s(-1), which is 3-fold higher than that of GOX-menadione, 3.68 s(-1). FAD-GDH was able to maintain its native glucose affinity during immobilization in the carbon nanotube and operation of enzyme fuel cells. FAD-GDH-menadione showed 3-fold higher power density, 799.4 ± 51.44 µW cm(-2), than the GOX-menadione system, 308.03 ± 17.93 µW cm(-2), under low glucose concentration, 5 mM, which is the concentration in normal physiological fluid.

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.

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.

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.

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.

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.

Treatment of alcohol distillery wastewater using a Bacteroidetes-dominant thermophilic microbial fuel cell.

Simultaneous electricity generation and distillery wastewater (DWW) treatment were accomplished using a thermophilic microbial fuel cell (MFC). The results suggest that thermophilic MFCs, which require less energy for cooling the DWW, can achieve high efficiency for electricity generation and also reduce sulfate along with oxidizing complex organic substrates. The generated current density (2.3 A/m(2)) and power density (up to 1.0 W/m(2)) were higher than previous wastewater-treating MFCs. The significance of the high Coulombic efficiency (CE; up to 89%) indicated that electrical current was the most significant electron sink in thermophilic MFCs. Bacterial diversity based on pyrosequencing of the 16S rRNA gene revealed that known Deferribacteres and Firmicutes members were not dominant in the thermophilic MFC fed with DWW; instead, uncharacterized Bacteroidetes thermophiles were up to 52% of the total reads in the anode biofilm. Despite the complexity of the DWW, one single bacterial sequence (OTU D1) close to an uncultured Bacteriodetes bacterium became predominant, up to almost 40% of total reads. The proliferation of the D1 species was concurrent with high electricity generation and high Coulombic efficiency.

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.

Complete genome sequencing of Lactobacillus acidophilus 30SC, isolated from swine intestine.

Lactobacillus acidophilus 30SC has been isolated from swine intestines and considered a probiotic strain for dairy products because of its ability to assimilate cholesterol and produce bacteriocins. Here, we report the complete genome sequence of Lactobacillus acidophilus 30SC (2,078,001 bp) exhibiting strong acid resistance and enhanced bile tolerance.

Complete genome sequence of a carbon monoxide-utilizing acetogen, Eubacterium limosum KIST612.

Eubacterium limosum KIST612 is an anaerobic acetogenic bacterium that uses CO as the sole carbon/energy source and produces acetate, butyrate, and ethanol. To evaluate its potential as a syngas microbial catalyst, we have sequenced the complete 4.3-Mb genome of E. limosum KIST612.

Bifunctional silver nanoparticle cathode in microbial fuel cells for microbial growth inhibition with comparable oxygen reduction reaction activity.

Organic contamination of water bodies in which benthic microbial fuel cells (benthic MFCs) are installed, and organic crossover from the anode to the cathode of membraneless MFCs, is a factor causing oxygen depletion and substrate loss in the cathode due to the growth of heterotrophic aerobic bacteria. This study examines the possible use of silver nanoparticles (AgNPs) as a cathodic catalyst for MFCs suffering from organic contamination and oxygen depletion. Four treated cathodes (AgNPs-coated, Pt/C-coated, Pt/C+AgNPs-coated, and plain graphite cathodes) were prepared and tested under high levels of organics loading. During operation (fed with 50 mM acetate), the AgNPs-coated system showed the highest DO concentration (0.8 mg/L) in the cathode area as well as the highest current (ranging from 0.04 to 0.12 mA). Based on these results, we concluded that (1) the growth of oxygen-consuming heterotrophic microbes could be inhibited by AgNPs, (2) the function of AgNPs as a bacterial growth inhibitor resulted in a greater increase of DO concentration in the cathode than the other tested cathode systems, (3) AgNPs could be applied as a cathode catalyst for oxygen reduction, and as a result (4) the MFC with the AgNPs-coated cathode led to the highest current generation among the tested MFCs.

Microbial fuel cells for energy self-sufficient domestic wastewater treatment-a review and discussion from energetic consideration.

As the microbial fuel cell (MFC) technology is getting nearer to practical applications such as wastewater treatment, it is crucial to consider the different aspects that will make this technology viable in the future. In this paper, we provide information about the specifications of an energy self-sufficient MFC system as a basis to extrapolate on the potential benefits and limits of a future MFC-based wastewater treatment plant. We particularly emphasize on the importance of two crucial parameters that characterize an MFC: its electromotive force (E (emf)) and its internal resistance (R (int)). A numerical projection using state-of-art values (E (emf) = 0.8 V and R (int) = 5 Ω) emphasized on the difficulty at this moment to reach self-sufficiency using a reasonable number of MFCs at the laboratory scale. We found that a realistic number of MFCs to provide enough voltage (=5 V) at a sufficient current (=0.8 A) to power a pump requiring 4 W would be of 13 MFCs in series and 10 stacks of MFCs in parallel, resulting in a total number of 130 MFCs. That would result in a treatment capacity of 144 L of domestic wastewater (0.5 g-COD L(-1)) per day. The total MFC system would be characterized by an internal resistance of 6.5 Ω.

Microbial community differences between propionate-fed microbial fuel cell systems under open and closed circuit conditions.

We report the electrochemical characterization and microbial community analysis of closed circuit microbial fuel cells (CC-MFCs) and open circuit (OC) cells continuously fed with propionate as substrate. Differences in power output between MFCs correlated with their polarization behavior, which is related to the maturation of the anodophilic communities. The microbial communities residing in the biofilm growing on the electrode, biofouled cation-exchange membrane and anodic chamber liquor of OC-and CC-MFCs were characterized by restriction fragment length polymorphism screening of 16S rRNA gene clone libraries. The results show that the CC-MFC anode was enriched in several microorganisms related to known electrochemically active and dissimilatory Fe(III) reducing bacteria, mostly from the Geobacter spp., to the detriment of Bacteroidetes abundant in the OC-MFC anode. The results also evidenced the lack of a specific pelagic community in the liquor sample. The biofilm growing on the cation-exchange membrane of the CC-MFC was found to be composed of a low-diversity community dominated by two microaerophilic species of the Achromobacter and Azovibrio genus.

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.

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.

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.