Light Wavelength as a Contributory Factor of Environmental Fitness in the Cyanobacterial Circadian Clock.

A circadian clock is an essential system that drives the 24-h expression rhythms for adaptation to day-night cycles. The molecular mechanism of the circadian clock has been extensively studied in cyanobacteria harboring the KaiC-based timing system. Nevertheless, our understanding of the physiological significance of the cyanobacterial circadian clock is still limited. In this study, we cultured wild-type Synechococcus elongatus PCC 7942 and circadian clock mutants in day-night cycles at different light qualities and found that the growth of the circadian clock mutants was specifically impaired during 12-h blue light/12-h dark (BD) cycles for the first time. The arrhythmic mutant kaiCAA was further analyzed by photosynthetic measurements. Compared with the wild type, the mutant exhibited decreases in the chlorophyll content, the ratio of photosystem I to II, net O2 evolution rate and efficiency of photosystem II photochemistry during BD cycles. These results indicate that the circadian clock is necessary for the growth and the maintenance of the optimum function of the photosynthetic apparatus in cyanobacteria under blue photoperiodic conditions.

Microbial Biomanufacturing Using Chemically Synthesized Non-Natural Sugars as the Substrate.

The bioproduction of valuable materials using biomass sugars is attracting attention as an environmentally friendly technology. However, its ability to fulfil the enormous demand to produce fuels and chemical products is limited. With a view towards the future development of a novel bioproduction process that addresses these concerns, this study investigated the feasibility of bioproduction of valuable substances using Corynebacterium glutamicum (C. glutamicum) with a chemically synthesized non-natural sugar solution. Cells were grown using the synthesized sugar solution as the sole carbon source and they produced lactate under oxygen-limited conditions. It was also found that some of the sugars produced by the series of chemical reactions inhibited cell growth since prior removal of these sugars increased the cell growth rate. The results obtained in this study indicate that chemically synthesized sugars have the potential to resolve the concerns regarding future biomass sugar supply in microbial biomanufacturing.

Positive Feedback Mechanism to Increase the Charging Voltage of Li-O2 Batteries.

The large overpotential of nonaqueous Li-O2 batteries when charging causes low round-trip efficiency and decomposition of the electrode materials and electrolyte. The origins of this overpotential have been enthusiastically explored to date; however, a full understanding has not yet been reached because of the complexity of multistep reaction mechanisms. Here, we applied structural and electrochemical analysis techniques to investigate the reaction step that results in the increase of the overpotential when charging. Rietveld refinement of ex situ powder X-ray diffraction showed that a Li-deficient phase of Li2O2, Li2-xO2, formed when discharging and was present over the course of charging. The galvanostatic intermittent titration technique revealed that the rate-determining process in the first step of charging was a solid-solution type of delithiation. The chemical diffusion coefficient of Li+ ions in Li2-xO2, DLi, decreases as the cell voltage increases, which in turn leads to a decrease in the oxidation rate of Li2-xO2. Under galvanostatic conditions, the deceleration of oxidation induces further increase of the cell voltage; therefore, an intrinsic mechanism of positive feedback to increase the cell voltage occurs in the first step. The results demonstrate that the continuity of the first step can be extended by the suppression of changes in any of the elements of the positive feedback loop, i.e., the oxidation rate, cell voltage, or DLi.

Conserved Two-component Hik2-Rre1 Signaling Is Activated Under Temperature Upshift and Plastoquinone-reducing Conditions in the Cyanobacterium Synechococcus elongatus PCC 7942.

The highly conserved Hik2-Rre1 two-component system is a multi-stress responsive signal-transducing module that controls the expression of hsp and other genes in cyanobacteria. Previously, we found in Synechococcus elongatus PCC 7942 that the heat-inducible phosphorylation of Rre1 was alleviated in a hik34 mutant, suggesting that Hik34 positively regulates signaling. In this study, we examined the growth of the hik34 deletion mutant in detail, and newly identified suppressor mutations located in rre1 or sasA gene negating the phenotype. Subsequent analyses indicated that heat-inducible Rre1 phosphorylation is dependent on Hik2 and that Hik34 modulates this Hik2-dependent response. In the following part of this study, we focused on the mechanism to control the Hik2 activity. Other recent studies reported that Hik2 activity is regulated by the redox status of plastoquinone (PQ) through the 3Fe-4S cluster attached to the cyclic GMP, adenylyl cyclase, FhlA (GAF) domain. Consistent with this, Rre1 phosphorylation occurred after the addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone but not after the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea to the culture medium, which corresponded to PQ-reducing or -oxidizing conditions, respectively, suggesting that the Hik2-to-Rre1 phosphotransfer was activated under PQ-reducing conditions. However, there was no correlation between the measured PQ redox status and Rre1 phosphorylation during the temperature upshift. Therefore, changes in the PQ redox status are not the direct reason for the heat-inducible Rre1 phosphorylation, while some redox regulation is likely involved as oxidation events dependent on 2,6-dichloro-1,4-benzoquinone prevented heat-inducible Rre1 phosphorylation. On the basis of these results, we propose a model for the control of Hik2-dependent Rre1 phosphorylation.

A Tin Oxide-Coated Copper Foam Hybridized with a Gas Diffusion Electrode for Efficient CO2 Reduction to Formate with a Current Density Exceeding 1 A cm-2.

The electrochemical CO2 reduction reaction (CO2 RR) is a promising strategy for closing the carbon cycle. Increasing the current density ( J) for CO2 RR products is a critical requirement for the social implementation of this technology. Herein, nanoscale tin-oxide-modified copper-oxide foam is hybridized with a carbon-based gas-diffusion electrode (GDE). Using the resultant electrode, the Jformate is increased to -1152 mA cm-2 at -1.2 V versus RHE in 1 m KOH, which is the highest value for CO2 -to-formate electrolysis. The formate faradaic efficiency (FEformate ) reaches ≈99% at -0.6 V versus RHE. The achievement of ultra-high-rate formate production is attributable to the following factors: i) homogeneously-modified Sn atoms suppressing H2 evolution and ii) the hydrophobic carbon nanoparticles on GDEs penetrating the macroporous structure of the foam causing the increase in the thickness of triple-phase interface. Additionally, the FEformate remains at ≈70% under a high J of -1.0 A cm-2 for more than 20 h.

NADPH production in dark stages is critical for cyanobacterial photocurrent generation: a study using mutants deficient in oxidative pentose phosphate pathway.

Live cyanobacteria and algae integrated onto an extracellular electrode can generate a light-induced current (i.e., a photocurrent). Although the photocurrent is expected to be correlated with the redox environment of the photosynthetic cells, the relationship between the photocurrent and the cellular redox state is poorly understood. Here, we investigated the effect of the reduced nicotinamide adenine dinucleotide phosphate [NADP(H)] redox level of cyanobacterial cells (before light exposure) on the photocurrent using several mutants (Δzwf, Δgnd, and ΔglgP) deficient in the oxidative pentose phosphate (OPP) pathway, which is the metabolic pathway that produces NADPH in darkness. The NAD(P)H redox level and photocurrent in the cyanobacterium Synechocystis sp. PCC 6803 were measured noninvasively. Dysfunction of the OPP pathway led to oxidation of the photosynthetic NADPH pool in darkness. In addition, photocurrent induction was retarded and the current density was lower in Δzwf, Δgnd, and ΔglgP than in wild-type cells. Exogenously added glucose compensated the phenotype of ΔglgP and drove the OPP pathway in the mutant, resulting in an increase in the photocurrent. The results indicated that NADPH accumulated by the OPP pathway before illumination is a key factor for the generation of a photocurrent. In addition, measuring the photocurrent can be a non-invasive approach to estimate the cellular redox level related to NADP(H) pool in cyanobacteria.

Coral symbionts evolved a functional polycistronic flavodiiron gene.

Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O2. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O2 photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O2-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803.

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b6/f complex, plastocyanin, and cytochrome c6, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700+) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700+ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700+ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

Overlooked Factors Required for Electrolyte Solvents in Li-O2 Batteries: Capabilities of Quenching 1 O2 and Forming Highly-Decomposable Li2 O2.

Although sufficient tolerance against attack by superoxide radicals (O2 - ) has been mainly recognized as an important property for Li-O2 battery (LOB) electrolytes, recent evidence has revealed that other critical factors also govern the cyclability, prompting a reconsideration of the basic design guidelines of LOB electrolytes. Here, we found that LOBs equipped with a N,N-dimethylacetamide (DMA)-based electrolyte exhibited better cyclability compared with other standard LOB electrolytes. This superior cyclability is attributable to the capabilities of quenching 1 O2 and forming highly decomposable Li2 O2 . The 1 O2 quenching capability is equivalent to that of a tetraglyme-based electrolyte containing a several millimolar concentration of a typical chemical quencher. Based on these overlooked factors, the DMA-based electrolyte led to superior cyclability despite its lower O2 - tolerance. Thus, the present work provides a novel design guideline for the development of LOB electrolytes.

Isotopic Depth Profiling of Discharge Products Identifies Reactive Interfaces in an Aprotic Li-O2 Battery with a Redox Mediator.

Prior to the practical application of rechargeable aprotic Li-O2 batteries, the high charging overpotentials of these devices (which inevitably cause irreversible parasitic reactions) must be addressed. The use of redox mediators (RMs) that oxidatively decompose the discharge product, Li2O2, is one promising solution to this problem. However, the mitigating effect of RMs is currently insufficient, and so it would be beneficial to clarify the Li2O2 reductive growth and oxidative decomposition mechanisms. In the present work, Nanoscale secondary ion mass spectrometry (Nano-SIMS) isotopic three-dimensional imaging and differential electrochemical mass spectrometry (DEMS) analyses of individual Li2O2 particles established that both growth and decomposition proceeded at the Li2O2/electrolyte interface in a system containing the Br-/Br3- redox couple as the RM. The results of this study also indicated that the degree of oxidative decomposition of Li2O2 was highly dependent on the cell voltage. These data show that increasing the RM reaction rate at the Li2O2/electrolyte interface is critical to improve the cycle life of Li-O2 batteries.

Quantification of NAD(P)H in cyanobacterial cells by a phenol extraction method.

In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP+)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light-dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.

Mediator-Microorganism Interaction in Microbial Solar Cell: a Fluo-Electrochemical Insight.

Microbial solar cells that mainly rely on the use of photosynthesic organisms are a promising alternative to photovoltaics for solar electricity production. In that way, we propose a new approach involving electrochemistry and fluorescence techniques. The coupled setup Electro-Pulse-Amplitude-Modulation ("e-PAM") enables the simultaneous recording of the produced photocurrent and fluorescence signals from the photosynthetic chain. This methodology was validated with a suspension of green alga Chlamydomonas reinhardtii in interaction with an exogenous redox mediator (2,6-dichlorobenzoquinone; DCBQ). The balance between photosynthetic chain events (PSII photochemical yield, quenching) and the extracted electricity can be monitored overtime. More particularly, the nonphotochemical quenching induced by DCBQ mirrors the photocurrent. This setup thus helps to distinguish the electron harvesting from some side effects due to quinones in real time. It therefore paves the way for future analyses devoted to the choice of the experimental conditions (redox mediator, photosynthetic organisms, and so on) to find the best electron extraction.

Redox-Active Polymers Connecting Living Microbial Cells to an Extracellular Electrical Circuit.

Microbial electrochemical systems in which metabolic electrons in living microbes have been extracted to or injected from an extracellular electrical circuit have attracted considerable attention as environmentally-friendly energy conversion systems. Since general microbes cannot exchange electrons with extracellular solids, electron mediators are needed to connect living cells to an extracellular electrode. Although hydrophobic small molecules that can penetrate cell membranes are commonly used as electron mediators, they cannot be dissolved at high concentrations in aqueous media. The use of hydrophobic mediators in combination with small hydrophilic redox molecules can substantially increase the efficiency of the extracellular electron transfer process, but this method has side effects, in some cases, such as cytotoxicity and environmental pollution. In this Review, recently-developed redox-active polymers are highlighted as a new type of electron mediator that has less cytotoxicity than many conventional electron mediators. Owing to the design flexibility of polymer structures, important parameters that affect electron transport properties, such as redox potential, the balance of hydrophobicity and hydrophilicity, and electron conductivity, can be systematically regulated.

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942.

The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global control systems have evolved as systems to adapt to regularly or irregularly changing light environments. Here, we report that the two global regulators mutually interact in cyanobacterium Synechococcus elongatus PCC7942, a model photosynthetic organism whose clock molecular mechanism is well known. Electrochemical assay using a transmembrane electron mediator revealed that intracellular redox of S. elongatus PCC7942 cell exhibited circadian rhythms under constant light conditions. The redox rhythm disappeared when transcription/translation of clock genes is defunctionalized, indicating that the transcription/translation controlled by a core KaiABC oscillator generates the circadian redox rhythm. Importantly, the amplitude of the redox rhythm at a constant light condition was large enough to affect the KaiABC oscillator. The findings indicated that the intracellular redox state is actively controlled to change in a 24-h cycle under constant light conditions by the circadian clock system.

Electrochemical Formation of Fe(IV)=O Derived from H2 O2 on a Hematite Electrode as an Active Catalytic Site for Selective Hydrocarbon Oxidation Reactions.

The high-valence iron species (Fe(IV)=O) in the cytochrome P450 enzyme superfamily is generated via the activation of O2 , and serves as the active center of selective hydrocarbon oxidation reactions. Furthermore, P450 can employ an alternate route to produce Fe(IV)=O, even from H2 O2 without O2 activation. Meanwhile, Fe(IV)=O has recently been revealed to be the reactive intermediate during H2 O oxidation to O2 on hematite electrodes. Herein, we demonstrated the generation of Fe(IV)=O on hematite electrodes during the electrochemical oxidative decomposition of H2 O2 using in situ UV-visible absorption spectra. The generation of Fe(IV)=O on hematite electrodes from H2 O2 exhibited 100 mV lower overpotential than that from H2 O. This is because H2 O2 serves not only as the oxygen source of Fe(IV)=O, but also as the additional oxidant. Finally, we confirmed that the Fe(IV)=O generated on hematite electrodes can serve as the catalytic site for styrene epoxidation reactions.

Cell-Membrane Permeable Redox Phospholipid Polymers Induce Apoptosis in MDA-MB-231 Human Breast Cancer Cells.

Induction of oxidative stress is an effective approach to causing apoptotic death of cancer cells. Since oxidative stress is generally caused by an intracellular redox imbalance, altering the intracellular redox is a promising strategy toward the growth suppression of cancer cells. Here, we attempted to induce apoptosis in MDA-MB-231 human breast cancer cells by adding a cell-membrane permeable redox phospholipid polymer that can alter the intracellular redox. We found that apoptosis and the deactivation of oxidative phosphorylation were induced in the MDA-MB-231 cells in the presence of the oxidized form of the redox polymer. Remarkably, such phenomena were not observed in the presence of the reduced form of the redox polymer that cannot intercept metabolic electrons. These results indicate that the redox polymer that mediates extracellular electron transfer (EET) generates oxidative stress, leading to the apoptosis of the cancer cells.

Photo-induced direct interfacial charge transfer at TiO2 modified with hexacyanoferrate(iii).

Photo-induced electron-transfer reactions occurring at the interface between titanium dioxide modified with hexacyanoferrate(iii) (Fe(iii)-CN-TiO2) were characterized. After the modification of TiO2 with [Fe(CN)6]3- ions, a new absorption appeared in the visible light region (410 to 700 nm). Absorption spectroscopy measurements showed that Fe(iii)-CN-TiO2 was converted to Fe(ii)-CN-TiO2 under visible light irradiation (>520 nm), which indicates that the new absorption was assignable to direct charge transfer from the valence band to the modified Fe(iii) ions.

Specific Interaction between Redox Phospholipid Polymers and Plastoquinone in Photosynthetic Electron Transport Chain.

Redox phospholipid polymers added in culture media are known to be capable of extracting electrons from living photosynthetic cells across bacterial cell membranes with high cytocompatibility. In the present study, we identify the intracellular redox species that transfers electrons to the polymers. The open-circuit electrochemical potential of an electrolyte containing the redox polymer and extracted thylakoid membranes shift to positive (or negative) under light irradiation, when an electron transport inhibitor specific to plastoquinone is added upstream (or downstream) in the photosynthetic electron transport chain. The same trend is also observed for a medium containing living photosynthetic cells of Synechococcus elongatus PCC7942. These results clearly indicate that the phospholipid redox polymers extract photosynthetic electrons mainly from plastoquinone.

Effects of TiCl4 treatment on the structural and electrochemical properties of a porous TiO2 layer in CH3NH3PbI3 perovskite solar cells.

The effects of surface treatment with TiCl4 on the structural and electrochemical properties of a porous titanium oxide (pTiO2) layer deposited on a fluorine-doped tin oxide (FTO)/glass substrate covered with a dense TiO2 layer (pTiO2/dTiO2/FTO/glass) were systematically investigated in order to obtain an optimum pTiO2 layer for use in CH3NH3PbI3 perovskite solar cells. As confirmed by thermal desorption spectroscopy (TDS) analyses, the amount of surface hydroxyl groups in pTiO2 varied when the pTiO2/dTiO2/FTO/glass sample was treated with solutions with different concentrations of TiCl4 (i.e., 20, 50, 80, and 100 mM). Photoelectrochemical (PEC) analyses of the pTiO2/dTiO2/FTO/glass samples after TiCl4 treatment showed significant increments of photocurrent densities compared to the pTiO2/dTiO2/FTO/glass sample without TiCl4 treatment regardless of the concentration of TiCl4 used in the solution. Electrochemical impedance spectroscopy (EIS) analyses of the TiCl4-treated pTiO2/dTiO2/FTO/glass samples also indicated a lower recombination probability with an increase in TiCl4 concentration. The results suggest that TiCl4 treatment resulted in passivation of defect sites on the surface of the TiO2 nanoparticles as well as improvement of the interconnectivity between the TiO2 nanoparticles in pTiO2. In contrast, the power conversion efficiencies (PCEs) and short circuit current densities of CH3NH3PbI3 perovskite solar cells based on these pTiO2/dTiO2/FTO/glass samples exhibited volcano-like patterns depending on the TiCl4 concentration used for the pTiO2 treatment: the highest PCE was obtained by using pTiO2/dTiO2/FTO/glass treated with 50 mM of TiCl4 solution. Structural analysis of the CH3NH3PbI3 perovskite part performed by X-ray diffraction (XRD) indicated that the formation of CH3NH3PbI3 perovskite was inhibited by the presence of surface hydroxyl groups in the pTiO2 film without TiCl4 treatment. TiCl4 treatment using TiCl4 solutions with concentrations up to 50 mM enhanced the formation of the CH3NH3PbI3 perovskite layer, whereas TiCl4 treatment using TiCl4 solutions with concentrations higher than 50 mM was detrimental due to the formation of nanoparticulate TiO2 aggregates that induce poor porosity and act as recombination sites.