External Electron Acceptor Research Articles

Systematic investigation of anode materials for microbial fuel cells with the model organism G. sulfurreducens

Different carbon and metal-based anode materials for microbial fuel cells were systematically investigated with a pure culture of the model organism G. sulfurreducens. The highest limiting current density of 756±15μAcm−2 at −0.253±0.037V vs. SCE was achieved with graphite foil using a step-wise galvanostatic technique. But also the application of completely different anode materials such as activated carbon cloth, stainless steel and graphite felt led to similar high limiting current densities, suggesting that G. sulfurreducens is able to use a large range of substantially different anode materials as external electron acceptor. Additionally, we could show that a step-wise galvanostatic technique to record polarization curves yields similar current densities as potentiostatic control at −0.400V vs. SCE with the investigated carbon-based materials. In case of stainless steel these techniques yield slightly different results, presumably due to an effect related to the material's surface properties.

Heliobacteria Reveal Fermentation As a Key Pathway for Mercury Reduction in Anoxic Environments.

The accumulation of mercury (Hg) in rice, a dietary staple for over half of the world's population, is rapidly becoming a global food safety issue. Rice paddies support the anaerobic production of toxic methylmercury that accumulates in plant tissue, however the microbial controls of Hg cycling in anoxic environments remain poorly understood. In this study, we reveal a novel reductive Hg metabolism in a representative of the family Heliobacteria ( Heliobacterium modesticaldum Ice1) that we confirm in model chemotrophic anaerobes. Heliobacteria served as our initial model because they are a family of spore-forming fermentative photoheterotrophs commonly isolated from terrestrial environments. We observed that H. modesticaldum reduced up to 75% of HgII under phototrophic or fermentative conditions. Fermentative HgII reduction relied on the ability of cells to oxidize pyruvate whereas phototrophic HgII reduction could be supported even in the absence of a carbon source. Inhibiting pyruvate fermentation eliminated HgII reduction in all chemotrophic strains tested, whereas phototrophic cells remained unaffected. Here we propose a non mer-operon dependent mechanism for Hg0 production in anoxic environments devoid of light where external electron acceptors are limited. These mechanistic details provide the foundation for novel bioremediation strategies to limit the negative impacts of Hg pollution.

Elucidation of the intra- and inter-molecular electron transfer pathways of glucoside 3-dehydrogenase

Glucoside 3‑dehydrogenase (G3DH) is a flavin adenine dinucleotide (FAD)-containing oxidoreductase that catalyzes the oxidation of the hydroxy group on the C-3 position of pyranose and shows broad substrate specificity by oxidizing many saccharides. Due to unique site specificity and wide substrate specificity, G3DHs can be used for synthesis of sugar derivatives, anodic catalysis in biofuel cells, multi-sugar analysis using enzyme electrode, and for enzymatic detection of 1,5‑anhydro‑d‑glucitol, a clinical marker for diabetes. However, few studies have focused on the fundamental biochemical properties of G3DH, including its electron transfer pathway. In this study, we isolated the G3DH gene from Rhizobium radiobacter, a homologue of marine bacterial G3DH, and reported that the isolated gene fragment contains the genes encoding the G3DH catalytic subunit (subunit I), G3DH hitch-hiker subunit (subunit II), and cytochrome c-like molecule (CYTc). Furthermore, we report the recombinant expression of G3DH from R. radiobacter in Escherichia coli, the characterization of recombinant G3DH and the investigation of the molecular electron pathway of G3DH. We first prepared the G3DH subunit I-II complex using a co-expression vector for both subunits. The G3DH subunit I-II complex showed dye-mediated G3DH activity toward methyl‑α‑d‑glucoside (MαG). Electron paramagnetic resonance (EPR) and inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses revealed that subunit I contains an iron-sulfur cluster. We, then, prepared recombinant CYTc and revealed that it is capable of accepting electrons from the catalytic subunit of G3DH by absorption spectrum analysis. These results suggested that R. radiobacter G3DH possesses an iron‑sulfur cluster that may play an important role in the electron transfer from FAD to cytochrome c like molecule, which is an external electron acceptor of G3DH. Furthermore, we demonstrated that CYTc mediate the electron transfer from G3DH to electrode without the artificial electron mediator.

Denitrifying nirK-containing alphaproteobacteria exhibit different electrode driven nitrite reduction capacities

Biocathodic electrode-driven denitrification has been proved experimentally in complex biofilms. However, experimentation with isolated bacteria in pure culture is still limited. In this paper, six Alphaproteobacteria (Rhizobiales), found to be dominant in a denitrifying biocathode, have been characterized bioelectrochemically. Bacteria were isolated using strict autotrophic conditions in the presence of nitrate or nitrite. Six representative isolates were selected and proven able to denitrify under autotrophic and heterotrophic conditions in liquid media. Bioelectrochemical reduction of nitrate, nitrite and nitrous oxide was tested using cyclic voltammetry. Electrode-driven nitrite reduction was only detected in four of the six isolates. However, no electrode-driven nitrate or nitrous oxide reduction could be detected for any of them. In the presence of nitrite, estimated midpoint potentials for bioelectrocatalyzed reactions ranged from −500 to −534 mV vs. SHE. Two of the isolates exhibited midpoint potentials at −450 and −486 mV vs. SHE when incubated in the absence of any external nitrogenous electron acceptor. These redox peaks were attributed to electrode-driven hydrogen production in the biofilm. We have proven that electrode-driven nitrite reduction is feasible in monospecific biofilms. However, significant variability in relation to electrode-driven nitrogen reduction processes was observed in closely related species, confirming a strain-specific behavior.

Effect of shocked nitrobenzene concentrations on the operational performance of anaerobic sequential batch reactor

Two lab‐scale anaerobic sequence batch reactors (ASBRs), respectively fed with 5 mg L−1 (R1) and 200 mg L−1 (R2) of sulfate were set up in this study. A mixture of the volatile fatty acids (VFAs) containing acetate and propionate was provided as electron donors. The effects of the influent nitrobenzene (NB) concentrations and the external electron acceptor of sulfate on the operational performance of ASBRs were investigated. The results indicated that NB reduction, sulfate reduction and propionate degradation were inhibited or further terminated in response to a shocked influent NB concentration of 50 mg L−1, i.e., 32 g m−3 day−1. However, these biological activities in ASBRs could be reversibly resumed so long as the influent NB regained below 30 mg L−1. The insufficient propionate was the main electron donor for the simultaneous reductions of NB and sulfate. Sulfate can energetically compete with NB for propionate as a co‐substrate, resulting in the better performance of reactor R1 than that of R2. In engineering applications, the influent loading rates of pollutants should be safely declined below toxic thresholds to avoid the failure of system operation. © 2017 American Institute of Chemical Engineers Environ Prog, 37: 712–718, 2018

Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis

When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD+, and a high titer of 371mM (32g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361mM (32g/L) R-BDO with a yield of 81% or 365mM (33g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production.

Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes.

Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a "closed" conformation, suitable for internal electron transfer, and an "open" conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.

Close to the Edge: Growth Restrained by the NAD(P)H/ATP Formation Flux Ratio.

Most fermentative microorganisms grow well-under anaerobic conditions managing a balanced redox and appropriate energy metabolism, but a few species do exist in which cells have to cope with inadequate energy recovery or capture and/or redox balancing. Two cases of these species, i.e., the metabolically engineered Saccharomyces cerevisiae enabling it to ferment xylose and Lactobacillus reuteri fermenting glucose via the phosphoketolase pathway, are here used to introduce a quantification parameter to capture what limits the growth rate of these microorganisms under anaerobic conditions. This dimensionless parameter, the cofactor formation flux ratio (RJ), is the ratio between the redox formation flux (JNADH+NADPH), and the energy carrier formation flux (JATP), which are mainly connected to the central carbon pathways. Data from metabolic flux analyses performed in previous and present studies were used to estimate the RJ-values. Even though both microorganisms possess different central pathways, a similar relationship between RJ and the specific growth rate (μ) was found. Furthermore, for both microorganisms external electron acceptors moderately reduced the RJ-value, thereby raising the μ accordingly. Based on the emerging profile of this relationship an interpretation is presented suggesting that this quantitative analysis can be applied beyond the two microbial species experimentally investigated in the current study to provide data for future targeted strain development strategies.

Photosynthetic Synergism for Sustained Power Production with Microalgae and Photobacteria in a Biophotovoltaic Cell

This article documents the synergetic interaction between oxygenic photosynthesis (microalgae; cathode) and anoxygenic photosynthesis (photosynthetic bacteria; anode) toward bioenergy and biomass generation apart from wastewater treatment using a dual chambered biophotovoltaic cell. Microalgae as the biocathode negate the requirement of an external terminal electron acceptor as they can be efficient in situ oxygenators that facilitate the oxygen reduction reactions. Experiments were performed in a fed batch mode using designed synthetic wastewater at variable organic loading rates in the anodic chamber and domestic sewage as feed in the cathodic chamber with a retention time of 72 h. Operation at OL3 (1500 mg/L) showed maximum power output (0.7 V; 103 mW/m2). Anodic chamber showed a maximum substrate (COD) degradation efficiency of 81%, while the cathode showed a constant COD removal efficiency of 90%. Maximum microalgal lipid productivity of 20% was observed at OL4 and OL5. The designed biophotovoltaic c...

Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR-1 through Coupling Improved Flavin Synthesis and Metal-Reducing Conduit for Pollutant Degradation.

Dissimilatory metal reducing bacteria (DMRB) are capable of extracellular electron transfer (EET) to insoluble metal oxides, which are used as external electron acceptors by DMRB for their anaerobic respiration. The EET process has important contribution to environmental remediation mineral cycling, and bioelectrochemical systems. However, the low EET efficiency remains to be one of the major bottlenecks for its practical applications for pollutant degradation. In this work, Shewanella oneidensis MR-1, a model DMRB, was used to examine the feasibility of enhancing the EET and its biodegradation capacity through genetic engineering. A flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB were coexpressed in S. oneidensis MR-1. Compared to the control strain, the engineered strain was found to exhibit an improved EET capacity in microbial fuel cells and potentiostat-controlled electrochemical cells, with an increase in maximum current density by approximate 110% and 87%, respectively. The electrochemical impedance spectroscopy (EIS) analysis showed that the current increase correlated with the lower interfacial charge-transfer resistance of the engineered strain. Meanwhile, a three times more rapid removal rate of methyl orange by the engineered strain confirmed the improvement of its EET and biodegradation ability. Our results demonstrate that coupling of improved synthesis of mediators and metal-reducing conduits could be an efficient strategy to enhance EET in S. oneidensis MR-1, which is essential to the applications of DMRB for environmental remediation, wastewater treatment, and bioenergy recovery from wastes.

Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras.

Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from Bacillus megaterium BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a Vmax increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome c reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis.

Comment on “Thermodynamically enhancing propionic acid degradation by using sulfate as an external electron acceptor in a thermophilic anaerobic membrane reactor” by Qiao, W., Takayanagi, K., Li, Q., Shofie, M., Gao, F., Dong, R., Li, Y-Y. Water Res. (2016)

Comment on “Thermodynamically enhancing propionic acid degradation by using sulfate as an external electron acceptor in a thermophilic anaerobic membrane reactor” by Qiao, W., Takayanagi, K., Li, Q., Shofie, M., Gao, F., Dong, R., Li, Y-Y. Water Res. (2016)

Acetoin production via unbalanced fermentation in Shewanella oneidensis.

This study describes the realization of an anoxic acetoin production process using the proteobacterium Shewanella oneidensis. Fermentative processes are of high biotechnological relevance since they offer high productivity and a low percentage of substrate consumption for anabolic processes. Nevertheless, the range of compounds that can be produced as sole end product of a fermentative process is limited, since the average oxidation state of substrate and products has to be identical in the absence of an external electron acceptor. This limitation could be overcome by the transfer of the surplus of electrons to a poised electrode surface, which of note is the only known anaerobic electron acceptor that cannot be depleted. In the first genetic engineering step, deletion mutants were developed that are devoid of either one, two, or all three prophages in their genome with the aim to construct a more stable chassis strain for microbe-electrode interaction, due to less prophage induced cell lysis (Gödeke et al., 2011). Current production in a bioelectrochemical system together with the analysis of cells on the anode surface were used as surrogate for the stability assessment. The λ-prophage deletion mutant produced overall 1.34fold more current (6.7 μA cm-2 ) than the wild type and all other constructed strains and showed with 1.1 × 1011 cells the highest cell density on the anode surface (2.3fold more than the wild type). The strain was further modified to contain codon optimized versions of acetolactate synthase and acetolactate decarboxylase derived from Bacillus subtilis. This allowed for the production of a mixture of acetoin and acetate from lactate in an almost 0.4:1 ratio. Further process improvement was reached by the deletion of the acetate kinase and phosphotransacetylase genes ackA/pta. The acetoin yield increased in this mutant from 40 to 86% of the theoretical maximum and acetoin was the only detectable end product. Biotechnol. Bioeng. 2017;114: 1283-1289. © 2017 Wiley Periodicals, Inc.

Electronic characterization of Geobacter sulfurreducens pilins in self-assembled monolayers unmasks tunnelling and hopping conduction pathways.

The metal-reducing bacterium Geobacter sulfurreducens produces protein nanowires (pili) for fast discharge of respiratory electrons to extracellular electron acceptors such as iron oxides and uranium. Charge transport along the pili requires aromatic residues, which cluster once the peptide subunits (pilins) assemble keeping inter-aromic distances and geometries optimal for multistep hopping. The presence of intramolecular aromatic contacts and the predominantly α-helical conformation of the pilins has been proposed to contribute to charge transport and rectification. To test this, we self-assembled recombinant, thiolated pilins as a monolayer on gold electrodes and demonstrated their conductivity by conductive probe atomic force microscopy. The studies unmasked a crossover from exponential to weak distance dependence of conductivity and shifts in the mechanical properties of the film that are consistent with a transition from interchain tunneling in the upper, aromatic-free regions of the helices to intramolecular hopping via aromatic residues at the amino terminus. Furthermore, the mechanistic stratification effectively "doped" the pilins at the amino terminus, favoring electron flow in the direction opposite to the helix dipole. However, the effect of aromatic dopants on rectification is voltage-dependent and observed only at the low (100 mV) voltages that operate in biological systems. The results thus provide evidence for a peptide environment optimized for electron transfer at biological voltages and in the direction needed for the respiration of external electron acceptors. The implications of these results for the development of hybrid devices that harness the natural abilities of the pilins to bind and reduce metals are discussed.

Metagenomic Characterization of Candidatus Smithella cisternae Strain M82_1, a Syntrophic Alkane-Degrading Bacteria, Enriched from the Shengli Oil Field.

The methanogenic degradation of hydrocarbons plays an important role in hydrocarbon-contaminated environments in the absence of an external electron acceptor. Members of Syntrophaceae sublineages were previously reported to be responsible for syntrophic alkane degradation. However, limited information is currently available on their physiological capabilities in nature because it is very challenging to cultivate these as-yet uncultured microbes. We herein performed metagenomic sequencing of the methanogenic hexadecane-degrading culture M82 and recovered a nearly complete genome (2.75 Mb, estimated completeness ≥97%) belonging to Syntrophaceae sublineage II. The assembly genome was tentatively named “Candidatus Smithella cisternae strain M82_1”. Genes encoding alkylsuccinate synthase for alkane activation were identified, suggesting that this organism is capable of oxidizing alkanes through fumarate addition. This capability was further supported by the detection of methyl pentadecyl succinic acid and methyl tetradecyl succinic acid in cultures amended with hexadecane and pentadecane, respectively. Genes encoding enzymes for the β-oxidation of long-chain fatty acids and butyrate were also identified. The electron transfer flavoprotein/DUF224 complex is presumed to link electron flow from acyl-CoA dehydrogenase to a membrane hydrogenase or formate dehydrogenase. Although no indications of Rnf complexes were detected, genes encoding electron-confurcating hydrogenase and formate dehydrogenase were proposed to couple the thermodynamically favorable oxidation of ferredoxin to generate H2 and formate from NADH. Strain M82_1 synthesized ATP from acetyl-CoA by substrate-level phosphorylation or F1F0-ATP synthases. These results provide an insight into the potential metabolic traits and ecophysiological roles of the syntrophic alkane degrader Syntrophaceae.

Reverse Methanogenesis and Respiration in Methanotrophic Archaea.

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., “trace methane oxidation”). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.

Thermodynamically enhancing propionic acid degradation by using sulfate as an external electron acceptor in a thermophilic anaerobic membrane reactor

In this study, sulfate was employed as an external electron acceptor for enhancing the degradation of propionate in a thermophilic anaerobic membrane reactor (AnMBR). The organic loading rate (OLR) was increased gradually from the initial 3.9 kg-COD/m3d to the inhibiting OLR of 14.6 kg-COD/m3d. Feeding was stopped for 98 days but the process did not recover until 500 mg/L of sulfate was added into the AnMBR. After that, the enhanced propionate degradation was achieved up to an OLR of 15 kg-COD/m3d with a reduced sulfate addition of 300 mg/L. However, the thermodynamic calculation indicated that the syntrophic propionic acid degradation, coupled with methanogenesis, was unfavorable with a △G of +3 kJ/mol under the enhanced conditions. Conversely, the utilization of propionic acid by sulfate reduction bacterial (SRB) would be more favourable by having a much lower △G of −180 kJ/mol. The hydrogen conversion was presumed to go through the methanogenesis pathway according to the thermodynamic results. The mechanism of the propionic and hydrogen metabolism was supported as well by comparing the microbial communities with and without sulfate addition. As a result, the role of the sulfate enhancing propionic degradation can be concluded by combining the process performance, thermodynamic, and microbiology results.

Metabolism of Fructophilic Lactic Acid Bacteria Isolated from the Apis mellifera L. Bee Gut: Phenolic Acids as External Electron Acceptors

Fructophilic lactic acid bacteria (FLAB) are strongly associated with the gastrointestinal tracts (GITs) of Apis mellifera L. worker bees due to the consumption of fructose as a major carbohydrate. Seventy-seven presumptive lactic acid bacteria (LAB) were isolated from GITs of healthy A. mellifera L. adults, which were collected from 5 different geographical locations of the Apulia region of Italy. Almost all of the isolates showed fructophilic tendencies: these isolates were identified as Lactobacillus kunkeei (69%) or Fructobacillus fructosus (31%). A high-throughput phenotypic microarray targeting 190 carbon sources was used to determine that 83 compounds were differentially consumed. Phenotyping grouped the strains into two clusters, reflecting growth performance. The utilization of phenolic acids, such as p-coumaric, caffeic, syringic, or gallic acids, as electron acceptors was investigated in fructose-based medium. Almost all FLAB strains showed tolerance to high phenolic acid concentrations. p-Coumaric acid and caffeic acid were consumed by all FLAB strains through reductases or decarboxylases. Syringic and gallic acids were partially metabolized. The data collected suggest that FLAB require external electron acceptors to regenerate NADH. The use of phenolic acids as external electron acceptors by the 4 FLAB showing the highest phenolic acid reductase activity was investigated in glucose-based medium supplemented with p-coumaric acid. Metabolic responses observed through a phenotypic microarray suggested that FLAB may use p-coumaric acid as an external electron acceptor, enhancing glucose dissimilation but less efficiently than other external acceptors such as fructose or pyruvic acid.IMPORTANCE Fructophilic lactic acid bacteria (FLAB) remain to be fully explored. This study intends to link unique biochemical features of FLAB with their habitat. The quite unique FLAB phenome within the group lactic acid bacteria (LAB) may have practical relevance in food fermentations. The FLAB phenome may have implications for the levels of hexose metabolism products in fermented foods, as well as food probiotication. Due to the harsh conditions of honeybees' GITs, these bacteria had to develop specific physiological and biochemical characteristics, such as tolerance to phenolic acids. The screening of FLAB strains based on metabolic pathways involving phenolic acids may allow the selection of starter cultures with both technological and functional beneficial attributes. Bioconversion of phenolic compounds may contribute to the aroma attributes and biofunctionality of fermented foods. Thus, the selection of FLAB strains as starter cultures with specific enzymatic activities involving phenolic acids may have a promising role in food fermentations.

Accelerated photocatalytic degradation of organic pollutant over metal-organic framework MIL-53(Fe) under visible LED light mediated by persulfate

Photocatalysis based on metal-organic frameworks (MOFs) is being actively investigated as a potential technology in visible light harvesting and utilizing processes. Herein we report that MIL-53(Fe), an earth-abundant Fe-containing MOF material, shows photocatalytic activity for the degradation of Acid Orange 7 (AO7) from aqueous solution under visible LED light irradiation, yet the photocatalytic performance of bare MIL-53(Fe) was not satisfactory due to the fast recombination of photoinduced electron-hole pairs. This can be effectively overcome by adding the external electron acceptor (e.g., persulfate, PS) to the catalytic process. The accelerated photocatalytic degradation of AO7 is demonstrated by the result that the degradation efficiency of AO7 in the MIL-53(Fe)/PS/Vis process reached almost 100% within 90min as compared to only 24% under the identical experimental conditions for the MIL-53(Fe)/Vis process. To investigate the mechanism of the MIL-53(Fe)/PS/Vis process, photoluminescence (PL) spectra, electrochemical measurements and electron paramagnetic resonance (EPR) analysis were performed. It was concluded that the efficient separation of photogenerated electrons and holes by the introduced PS and the subsequent formation of reactive radicals resulting from the activation of PS by photogenerated electrons accounted for the accelerated photocatalytic degradation of AO7 in the MIL-53(Fe)/PS/Vis process. Furthermore, the applicability of MIL-53(Fe) used in the persulfate-mediated photocatalytic process was systematically investigated in terms of the identification of reactive radicals, the reusability and stability of the photocatalyst, as well as the effect of operating parameters. The findings of this work highlighted the great potential of MOFs as photocatalysts and elucidated a new opportunity for persulfate remediation of contaminated water.

Investigation of Intra- or Inter-Electron Transfer Pathway of Multiheme Electron Transfer Subunit of Iron Sulfur Flavo Cytochrome Type Glucose Dehydrogenase Complex

【Introduction】 With the increase demand for more accurate and precise monitoring of blood glucose, further development of novel principles and biosensing molecules are expected, to be dedicated for the diabetes patient care. Especially, the development of novel continuous glucose monitoring (CGM) system is inevitable in order to realize the future artificial pancreas. We have been engaged in the study and application of the bacterial FAD dependent glucose dehydrogenase (FADGDH) complex isolated from Burkholderia cepacia. FADGDH complex is composed of three subunits: a catalytic subunit (α-subunit), a hitch hiker protein of α-subunit (γ-subunit), and an electron transfer subunit (β-subunit).1-4 FADGDH complex represents several bacterial FAD dependent dehydrogenase complexes, which are also composed of the three subunits. These enzyme complexes are capable of direct electron transfer to an electrode because of the presence of electron transfer multiheme cytochrome c subunit. Thanks to this property, direct electron transfer principle based CGM is being developed using FADGDH complex. Recently, we have reported that catalytic subunit of such FAD dehydrogenase complexes harbor an Fe-S cluster, and the Fe-S cluster is involved in the intra electron transfer from FAD in the catalytic subunit, and also in the inter- electron transfer between catalytic subunit and electron transfer subunit5. These enzyme complexes are now categorized as the iron sulfur flavo cytochrome type dehydrogenase complexes However, the electron transfer pathway in the electron transfer subunit, as well as the the mechanisms of the electron transfer to the external electron acceptors are yet to be elucidated. In this paper, we report the site direct mutagenesis studies of the heme ligands of electron transfer subunit of FADGDH complex, the β-subunit, and attempt to elucidate the intra- and inter-electron transfer pathway of β-subunit. 【Materials and Methods】 The recombinant FADGDH complex wild type and those harboring mutant β-subunit were prepared using Escherichia coliBL21(DE3). The recombinant FADGDH complexes were recovered from the membrane fraction. The purification of FADGDH complexes was carried out by the combination of hydrophobic interaction chromatography. Dehydrogenase activity was measured using either Hexaammine ruthenium(III)chloride or phenazine methosulfate as an electron acceptor. Also, wild type FADGDH complex or mutant complex was immobilized on the working electrode of a glassy carbon electrode, and investigated direct electron transfer properties. 【Results and Discussion】 The results of recombinant expression of wild type FADGDH complex and all mutant complexes were successful, and the soluble enzyme complexes were prepared. The dye mediated dehydrogenase activity of mutant complexes showed different preference of electron acceptors compared with those of wild type complex. Also, the chrono-amperometric measurement of mutant FADGDH complexes showed different properties, depend on introduced mutation. Considering the our previous results of estimation of redox potential of three hemes of β-subunit, we propose the order of intra-molecular electron transfer in the β-subunit, and also the heme responsible for the electron transfer to the external electron acceptors. (1) K. Sode, et al., Enzyme Microbiol. Technol. 19, 82-85(1996) (2) T. Yamazaki, et al., Appl.Biochm.Biotechnol. 77, 325-335(1999) (3) T. Tsuya, et al., J. Biotechnol. 123, 127-136(2006) (4) H. Yamaoka, et al., Biotechnol Lett. 26, 1757-1761(2004) (5) M. Shiota, et al., Bioelectrochemistry. (2016)