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Many studies have shown that redox regulation is an effective therapeutic strategy for different types of cancer. We have previously demonstrated that combined treatment with dissolved hydrogen molecule (H2) and platinum nanocolloid (Pt-nc) has carcinostatic effects and that increased intracellular reactive oxygen species (ROS) levels were closely associated with carcinostatic effects in Ehrlich mouse ascites tumor cells. However, it is unknown whether combined treatment-induced ROS generation can occur in human cancer cells. Therefore, this study aimed to examine the carcinostatic effect of the combined treatment in human cells and investigate the relationship between treatment efficacy and ROS generation. H2 and Pt-nc treatment could exert cytostatic action by inhibiting the growth of human promyelocytic leukemia HL60 and human gastric adenocarcinoma-derived NUGC-4 cells; however, no effect was observed in normal human embryo fibroblast OUMS-36 cells by the temporary exposure. These findings indicate that combined treatment with H2 and Pt-nc may act selectively in tumor cells compared with normal cells. Additionally, combined treatment with H2 and Pt-nc resulted in an approximately 200-fold increase in intracellular ROS levels compared with the control, whereas the suppressive effect of tumor cell growth was abrogated entirely by catalase treatment in NUGC-4 cells. Furthermore, combined treatment with H2 and Pt-nc induced hydrogen peroxide generation, cellular morphological changes, cell death, and a decline in DNA synthesis-positive cells. In conclusion, combined treatment with H2 and Pt-nc can induce carcinostatic/carcinocidal effects through intracellular ROS increase, morphological changes, cell death, and DNA synthesis suppression in the human tumor cell line.

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The present study investigated the association between fibre degradation and the concentration of dissolved molecular hydrogen (H2) in the rumen. Napier grass (NG) silage and corn stover (CS) silage were compared as forages with contrasting structures and degradation patterns. In the first experiment, CS silage had greater 48-h DM, neutral-detergent fibre (NDF) and acid-detergent fibre degradation, and total gas and methane (CH4) volumes, and lower 48-h H2 volume than NG silage in 48-h in vitro incubations. In the second experiment, twenty-four growing beef bulls were fed diets including 55 % (DM basis) NG or CS silages. Bulls fed the CS diet had greater DM intake (DMI), average daily gain, total-tract digestibility of OM and NDF, ruminal dissolved methane (dCH4) concentration and gene copies of protozoa, methanogens, Ruminococcus albus and R. flavefaciens, and had lower ruminal dH2 concentration, and molar proportions of valerate and isovalerate, in comparison with those fed the NG diet. There was a negative correlation between dH2 concentration and NDF digestibility in bulls fed the CS diet, and a lack of relationship between dH2 concentration and NDF digestibility with the NG diet. In summary, the fibre of CS silage was more easily degraded by rumen microorganisms than that of NG silage. Increased dCH4 concentration with the CS diet presumably led to the decreased ruminal dH2 concentration, which may be helpful for fibre degradation and growth of fibrolytic micro-organisms in the rumen.

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The control of dissolved methane (CH4) and hydrogen sulfide (H2S) emissions in anaerobic effluents is essential for minimizing the environmental implications of greenhouse gases, odor, and carbon footprint, as well as for preventing energy loss in the form of unrecovered dissolved methane. This study assessed the feasibility of a vacuum degasifier for the removal of CH4 and H2S from staged anaerobic fluidized membrane bioreactor (SAF-MBR) effluent. The optimization results showed that the efficiency of the nozzle fitted degasifiers were superior to the media packed ones. In three-stage vacuum degasifiers at a -0.8 bar vacuum pressure, H2S removal was mostly pH dependent and 88% removal efficiency was achieved with an initial concentration of 13.6 mg/L. Methane removal was dependent primarily on the number of degasifier units, and approximately 94% efficiency was achieved in a three-stage degasifier. Energy balance analysis showed that energy production exceeded the system energy requirements with 0.05-0.07 kWh/m3 of surplus energy. These results provide deep insights into this new technology for simultaneous removal of dissolved CH4 and H2S, which can be referred for potential future applications.

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Widely distributed electrolytic-generators for hydrogen-water are not fully considered for the dependencies of post-electrolytic values of the dissolved hydrogen concentration (DH) and the oxidation-reduction potential (ORP) on the properties of the pre-electrolytic water. We investigated the dependencies of DH and ORP on mineral-based hardness, temperatures and the container materials, and effects on the oral cavity by oral washing or drinking. Along with an increase in mineral-based water-hardness, DH decreased from 960 to 870 µg/L and the ORP unexpectedly increased from -460 to -320 mV. Purified water of almost zero hardness, however, caused a post-electrolytic DH as low as 80 µg/L and an ORP as high as +20 mV. Post-electrolytic DHs were not significantly changed (780-900 µg/L) upon electrolysis at 1.5-30°C and decreased at 40-50°C. The diffusion of hydrogen from the inside to the outside of the container was extremely small even after 12 hours for an aluminum- or stainless steel-made container, but not for containers made of diverse plastics. The ORP of the intact saliva was +136 mV, and decreased to +90 mV at 20 minutes after 1-minute oral-cramming of hydrogen-water, but returned to +135 mV after 60-minute leaving, showing a transient ORP-decrease in the saliva. Drinking-pause for 4 weeks after drinking hydrogen-water, however, saliva ORP, gradually but not instantly, increased to +60 to +80 mV, but upon drinking-resumption and 2 weeks thereafter, decreased again to -100 to -110 mV, suggesting that several-week hydrogen-water drinking caused a certain decrease in the saliva ORP. Thus, the present study provided the appropriate conditions such as hardness and temperatures for hydrogen-water production by the electrolytic generator, and the container materials suitable for hydrogen-water preservation. Furthermore, we clarified ORP changes of human saliva, being an indicator for human oxidative stress. The study was approved by the Medical Ethics Committee of the NPO (Non-Profitable Organization)-Corporate Japanese Center for Anti-Aging Medical Sciences (approval No. 09S02) on May 2, 2012.

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Enteric methane (CH4) emissions are not only an important source of greenhouse gases but also a loss of dietary energy in livestock. Corn oil (CO) is rich in unsaturated fatty acid with >50% PUFA, which may enhance ruminal biohydrogenation of unsaturated fatty acids, leading to changes in ruminal H2 metabolism and methanogenesis. The objective of this study was to investigate the effect of CO supplementation of a diet on CH4 emissions, nutrient digestibility, ruminal dissolved gases, fermentation, and microbiota in goats. Six female goats were used in a crossover design with two dietary treatments, which included control and CO supplementation (30 g/kg DM basis). CO supplementation did not alter total-tract organic matter digestibility or populations of predominant ruminal fibrolytic microorganisms (protozoa, fungi, Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes), but reduced enteric CH4 emissions (g/kg DMI, -15.1%, P = 0.003). CO supplementation decreased ruminal dissolved hydrogen (dH2, P < 0.001) and dissolved CH4 (P < 0.001) concentrations, proportions of total unsaturated fatty acids (P < 0.001) and propionate (P = 0.015), and increased proportions of total SFAs (P < 0.001) and acetate (P < 0.001), and acetate to propionate ratio (P = 0.038) in rumen fluid. CO supplementation decreased relative abundance of family Bacteroidales_BS11_gut_group (P = 0.032), increased relative abundance of family Rikenellaceae (P = 0.021) and Lachnospiraceae (P = 0.025), and tended to increase relative abundance of genus Butyrivibrio_2 (P = 0.06). Relative abundance (P = 0.09) and 16S rRNA gene copies (P = 0.043) of order Methanomicrobiales, and relative abundance of genus Methanomicrobium (P = 0.09) also decreased with CO supplementation, but relative abundance (P = 0.012) and 16S rRNA gene copies (P = 0.08) of genus Methanobrevibacter increased. In summary, CO supplementation increased rumen biohydrogenatation by facilitating growth of biohydrogenating bacteria of family Lachnospiraceae and genus Butyrivibrio_2 and may have enhanced reductive acetogenesis by facilitating growth of family Lachnospiraceae. In conclusion, dietary supplementation of CO led to a shift of fermentation pathways that enhanced acetate production and decreased rumen dH2 concentration and CH4 emissions.

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This study investigated the hydrogen mass transfer limitations in a biotrickling filter inoculated with hydrogenotrophic methanogens for biogas upgrading. A highly sensitive dissolved hydrogen probe allowed measuring concentrations in real-time. Experiments were conducted to test the mass transfer resistance in the gas and liquid films. Results demonstrated that the main resistance resides in the trickling liquid film and that promoting direct gas-biofilm mass transfer could improve upgrading performance by about 20%. Increasing the gas velocity (keeping a constant gas contact time) lowered the upgrading capacity. This was explained by the lowering of the concentration to the average concentration throughout the bed, which resulted a lower reaction rate. At extended gas contact times, the bioreactor shifted from microbial to diffusion limitation, causing lower upgrading capacities. Methane-containing biogas mimics (H2/CH4/CO2) were successfully upgraded to natural gas pipeline standards (>97% methane) with only minor performance reduction compared to upgrading just a H2/CO2 mixture.

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The reactivity of H2 pre-reduced acceptor-doped ceria materials Gd0.10 Ce0.90 O2-δ (GDC10) and Sm0.15 Ce0.85 O2-δ (SDC15) was tested with respect to the reduction of CO2 to CO in the context of the reverse water-gas shift reaction. It was demonstrated that not only oxygen vacancies, but also dissolved hydrogen is a reactive species for the reduction of CO2 . Dissolved hydrogen must be considered upon discussion of the mechanism of the reverse water-gas shift reaction on ceria-derived materials apart from oxygen vacancies and formates. The reduction of CO2 is preceded by the formation of carbonate species of different thermal stability and reactivity. The stability of these carbonates was directly demonstrated by in situ infrared spectroscopy and revealed the largely reversible nature of CO2 ad- and desorption. In comparison to pre-reduced samples, decreased carbonate coverage is obtained after oxidative treatments of GDC10 and SDC15. No significant effect of the sample treatment (O2 oxidation or H2 reduction) on the surface carbonate stability was noticed. Mono-dentate carbonates and carboxylates appear to be more easily formed on pre-reduced (i. e. defective) samples. Ce4+ reduction to Ce3+ (by H2 ) and re-oxidation to Ce4+ (by CO2 ) on GDC10/SDC15 were directly monitored by infrared spectroscopic analysis of a distinct, IR-active electronic transition of Ce3+ . These results show the complex interplay of oxygen vacancy/dissolved hydrogen reactivity and surface chemical aspects in acceptor-doped ceria materials.

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Hydrogen is a key metabolite that connects microbial fermentation and methanogenesis in the rumen. This study was to investigate the effects of elevated H2 produced by elemental Mg on rumen fermentation and methanogenesis in dairy cows. Four nonlactating Chinese Holstein dairy cows were employed for this experiment in a replicated crossover design. The 2 dietary treatments included a basal diet supplemented with Mg(OH)2 (14.5 g/kg of feed dry matter) or elemental Mg (6.00 g/kg of feed dry matter). When compared with Mg(OH)2 treatment, cows fed diet with elemental Mg had similar rumen Mg2+ concentration, but higher rumen dissolved H2 and methane concentrations at 2.5 h after morning feeding. Also, elemental Mg supplementation decreased feed digestibility, rumen volatile fatty acid concentration, and relative abundance of group Ruminococcaceae_UCG-014, genus Bifidobacterium, and group Mollicutes_RF9, increased acetate to propionate ratio, succinate concentration, and abundance of family Christensenellaceae. Elemental Mg supplementation increased enteric CH4 emission, altered methanogen community with increased abundance of order Methanomassiliicoccales, 16S ribosomal RNA gene copies of methanogens, and order Methanobacteriales. In summary, the pulse of elevated dissolved H2 after feeding produced by elemental Mg inhibited rumen fermentation and feed digestibility by decreasing the abundance of carbohydrate-degrading bacteria, promoted H2 incorporation into succinate by increasing family Christensenellaceae and genus Bacteroidales_BS11, and increased H2 utilization for methanogenesis by favoring growth of methanogens.

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Rumen cannulation is a widely employed technique in ruminant nutrition research. However, the gap between skin and rumen cannula can cause leakage of fermentation gases and influx of atmospheric air, which may adversely affect the anaerobic environment in the rumen. The present study was designed to investigate the effects of rumen cannulation on headspace gases, dissolved gases, fermentation end products, and methanogen community in the rumen of dairy cows. Eight Holstein cows were used in the experiment. Four cows were surgically fitted with rumen cannulas, whereas the other 4 intact cows were used as control. Rumen cannulation decreased gaseous hydrogen and methane concentrations, dissolved carbon dioxide concentration, and relative abundances of Methanosphaera, and increased the saturation factor of dissolved hydrogen and dissolved methane, dissolved methane concentration, volatile fatty acid concentration, 16S ribosomal RNA gene copies of methanogens, and Simpson index of methanogen community. In summary, rumen cannulation causes a reduction in headspace gaseous hydrogen and gaseous methane, which may not decrease dissolved gas concentrations due to an increase in saturation factors. Furthermore, rumen cannulation alters methanogen community with increased methanogen population and decreased relative abundances of Methanosphaera.

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BACKGROUND: Urea pretreatment is an efficient strategy to improve fiber digestibility of low quality roughages for ruminants. Nitrate and oil are usually used to inhibit enteric methane (CH4) emissions from ruminants. The objective of this study was to examine the combined effects of urea plus nitrate pretreated rice straw and corn oil supplementation to the diet on nutrient digestibility, nitrogen (N) balance, CH4 emissions, ruminal fermentation characteristics and microbiota in goats. Nine female goats were used in a triple 3 × 3 Latin Square design (27 d periods). The treatments were: control (untreated rice straw, no added corn oil), rice straw pretreated with urea and nitrate (34 and 4.7 g/kg of rice straw on a dry matter [DM] basis, respectively, UN), and UN diet supplemented with corn oil (15 g/kg soybean and 15 g/kg corn were replaced by 30 g/kg corn oil, DM basis, UNCO). RESULTS: Compared with control, UN increased neutral detergent fiber (NDF) digestibility (P < 0.001) and copies of protozoa (P < 0.001) and R. albus (P < 0.05) in the rumen, but decreased N retention (-21.2%, P < 0.001), dissolved hydrogen concentration (-22.8%, P < 0.001), molar proportion of butyrate (-18.2%, P < 0.05), (acetate + butyrate) to propionate ratio (P < 0.05) and enteric CH4 emissions (-10.2%, P < 0.05). In comparison with UN, UNCO increased N retention (+34.9%, P < 0.001) and decreased copies of protozoa (P < 0.001) and methanogens (P < 0.001). Compared with control, UNCO increased NDF digestibility (+8.3%, P < 0.001), reduced ruminal dissolved CH4 concentration (-24.4%, P < 0.001) and enteric CH4 emissions (-12.6%, P < 0.05). CONCLUSIONS: A combination of rice straw pretreated with urea plus nitrate and corn oil supplementation of the diet improved fiber digestibility and lowered enteric CH4 emissions without negative effects on N retention. These strategies improved the utilization of rice straw by goats.

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Generation of ammonia from nitrate reduction is slower compared with urea hydrolysis and may be more efficiently incorporated into ruminal microbial protein. We hypothesized that nitrate supplementation could increase ammonia incorporation into microbial protein in the rumen compared with urea supplementation of a low-protein diet fed to lactating dairy cows. Eight multiparous Chinese Holstein dairy cows were used in a crossover design to investigate the effect of nitrate or an isonitrogenous urea inclusion in the basal low-protein diet on rumen fermentation, milk yield, and ruminal microbial community in dairy cows fed a low-protein diet in comparison with an isonitrogenous urea control. Eight lactating cows were blocked in 4 pairs according to days in milk, parity, and milk yield and allocated to urea (7.0 g urea/kg of dry matter of basal diet) or nitrate (14.6 g of NO3-/kg of dry matter of basal diet, supplemented as sodium nitrate) treatments, which were formulated on 75% of metabolizable protein requirements. Nitrate supplementation decreased ammonia concentration in the rumen liquids (-33.1%) and plasma (-30.6%) as well as methane emissions (-15.0%) and increased dissolved hydrogen concentration (102%), microbial N (22.8%), propionate molar percentage, milk yield, and 16S rRNA gene copies of Selenomonas ruminantium. Ruminal dissolved hydrogen was positively correlated with the molar proportion of propionate (r = 0.57), and negatively correlated with acetate-to-propionate ratio (r = -0.57) and estimated net metabolic hydrogen production relative to total VFA produced (r = -0.58). Nitrate reduction to ammonia redirected metabolic hydrogen away from methanogenesis, enhanced ammonia incorporation into rumen microbial protein, and shifted fermentation from acetate to propionate, along with increasing S. ruminantium 16S rRNA gene copies, likely leading to the increased milk yield.

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The activity of the pre-reduced perovskites La0.6 Sr0.4 FeO3-δ (LSF64) and SrTi0.7 Fe0.3 O3-δ (STF73) for the CO2 reduction to CO was investigated with special focus on the reactivity of oxide-dissolved hydrogen. This is of particular interest in hydrogen solid-oxide electrolysis cell (H-SOEC) technology, where proton-conducting ceramics are used and the reaction 2e- +2H+ +CO2 →CO+H2 O is of central importance. To clarify if hydrogen dissolved in LSF64 and STF73 partakes in the CO2 reduction, temperature-programmed reduction (TPR) in H2 , followed by temperature-programmed reoxidation (TPO) in CO2 and, moreover, temperature-programmed desorption (TPD) of ad- and absorbed species were utilized. The experiments reveal that 50 mol % of the CO2 is converted by hydrogen dissolved in STF73 and reacts quantitatively. On the other hand, LSF64 converts less than 20 mol % of CO2 via dissolved hydrogen and a residual of bulk OH is still detectable after CO2 -TPO.

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Objective To measure the catalytic effect of ammonium salt solution on hydrogen production , and analyze the relationships between hydrogen concentration and oxygen concentration , oxidation-reduction potential , and pH in solution . Methods Magnesium and water reacted at different concentrations of NH4Cl, (NH4)2SO4, NH4HCO3,(NH4)2CO3, NaHCO3 and Na2SO3 in 40℃water bath for 0, 2, 4, 6, 8 and 10 hours, and above-mentioned four indicators of ammonium salt solution were measured after these reactions .Results The hydrogen concentration in the solution increased with the reaction time and the concentration of the ammonium salt solution .Oxygen concentration and oxidation-reduction potential decreased .The hydrogen concentration in the solution was significantly negatively correlated with the oxygen concentration (r=-0.984).pH increased with the hydrogen concentration in the ammonium salt solution .Conclusion The ammonium salt solution has a good catalytic effect on magnesium and water reaction .NH4Cl has the strongest catalytic effect under the same reaction conditions,followed by (NH4)2SO4, NH4HCO3 and (NH4)2CO3.Thus, this study can provide detailed data on hydrogen production in different solutions .

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We tested the hypotheses that supplementation of a diet with elemental Mg increases ruminal dissolved H2 (dH2) in rumen fluid, which in turn alters rumen fermentation and microbial community in goats. In a randomised block design, twenty growing goats were allocated to two treatments fed the same basal diet with 1·45 % Mg(OH)2 or 0·6 % elemental Mg. After 28 d of adaptation, we collected total faeces to measure total tract digestibility, rumen contents to analyse fermentation end products and microbial groups, and measured methane (CH4) emission using respiration chambers. Ruminal Mg2+ concentration was similar in both treatments. Elemental Mg supplementation increased dH2 at 2·5 h post morning feeding (+180 %, P<0·001). Elemental Mg supplementation decreased total volatile fatty acid concentration (-8·6 %, P<0·001), the acetate:propionate ratio (-11·8 %, P<0·03) and fungal copy numbers (-63·6 %, P=0·006), and increased propionate molar percentage (+11·6 %, P<0·001), methanogen copy numbers (+47·9 %, P<0·001), dissolved CH4 (+35·6 %, P<0·001) and CH4 emissions (+11·7 %, P=0·03), compared with Mg(OH)2 supplementation. The bacterial community composition in both treatments was overall similar. Ruminal dH2 was negatively correlated with acetate molar percentage and fungal copy numbers (P<0·05), and positively correlated with propionate molar percentage and methanogen copy numbers (P<0·05). In summary, elemental Mg supplementation increased ruminal dH2 concentration, which inhibited rumen fermentation, enhanced methanogenesis and seemed to shift fermentation pathways from acetate to propionate, and altered microbiota by decreasing fungi and increasing methanogens.

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A hollow fiber degassing membrane (DM) was applied to enhance organic matter degradation and methane gas production of anaerobic granular sludge process by reducing the dissolved hydrogen gas (D-H2) concentration in the liquid phase. DM was installed in the bench-scale anaerobic granular sludge reactors and D-H2 was removed through DM using a vacuum pump. Degasification improved the organic matter degradation efficiency to 79% while the efficiency was 62% without degasification at 12,000mgL-1 of the influent T-COD concentration. Measurement of D-H2 concentrations in the liquid phase confirmed that D-H2 was removed by degasification. Furthermore, the effect of acetate concentrations on the organic matter degradation efficiency was investigated. At acetate concentrations above 3gL-1, organic matter degradation deteriorated. Degasification enhanced the propionate and acetate degradation. These results suggest that degasification reduced D-H2 concentration and volatile fatty acids concentrations, prevented pH drop, and subsequent enhanced organic matter degradation.

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An amperometric sensor based on flow injection analysis (FIA) of dissolved hydrogen molecules was first developed using electrodeposited platinum particles on glassy carbon electrodes modified with nitrogen-containing functional groups (Pt-NGC) as the working electrode. A glassy carbon (GC) electrode was covalently modified by electrochemical oxidation/reduction procedures. The redox waves between hydrogen ions and hydrogen molecules at highly positive potential range in the hydrodynamic voltammogram were obtained by using a Pt-NGC electrode. The specific electrocatalytic activity for the electrode oxidation of hydrogen molecules has successfully been applied to the FIA of dissolved hydrogen. The typical current vs. time curve was obtained by the repetitive measurement of dissolved hydrogen, and the measurement of dissolved hydrogen was fully completed in a short time (∼15 s). A linear relationship was obtained between the oxidation current of hydrogen molecules and dissolved hydrogen concentration. This indicates that our proposed technique can be used for the determination of the dissolved hydrogen concentration. The fabrication method of the present sensor is very simple because the direct modification of the glassy carbon electrode surface can be performed, differing from the tedious fabrication method in which electrocatalytic carbon powder prepared must be immobilized to the surface of the glassy carbon electrode using Nafion coating and high temperature treatment.

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Dissolved hydrogen is a symbol gas decomposed by power transformer oil for electrical faults such as overheat or partial discharges. A novel D-shaped fiber Bragg grating (D-FBG) sensor is herein proposed and was fabricated with magnetron sputtering to measure the dissolved hydrogen concentration in power transformer oil in this paper. Different from the RI (refractive index)-based effect, D-FBG in this case is sensitive to curvature caused by stress from sensing coating, leading to Bragg wavelength shifts accordingly. The relationship between the D-FBG wavelength shift and dissolved hydrogen concentration in oil was measured experimentally in the laboratory. The detected sensitivity could be as high as 1.96 µL/L at every 1-pm wavelength shift. The results proved that a simple, polished FBG-based hydrogen sensor provides a linear measuring characteristic in the range of low hydrogen concentrations in transformer oil. Moreover, the stable hydrogen sensing performance was investigated by X-ray diffraction analysis.

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Acetaldehyde is a detrimental substance produced in alcoholic liquor aging. We assessed an ability of hydrogen-storing microporous silica 'Microcluster' (MC+) to reduce acetaldehyde, as compared with autoclave-dehydrogenated MC+ (MC-). Acetaldehyde was quantified spectrophotometrically by an enzymatic method. Authentic acetaldehyde was treated by MC+ for 20min, and decreased from 43.4ppm to 10.9ppm, but maintained at 49.3ppm by MC-. On the other hand, acetaldehyde contained in a distilled spirit was decreased from 29.5ppm to 3.1ppm at 20min by MC+, but not decreased by MC-. Addition of MC+ or MC- to distilled water without acetaldehyde showed no seeming effect on the quantification used. Accordingly acetaldehyde in a distilled spirit is reduced to ethanol by hydrogen contained in MC+, but not by the silica moiety of MC+. Hydrogen gas of 1.2mL was released for 20min from MC+ of 0.59g in water, resulting in dissolved hydrogen of 1.09ppm and an oxidation- reduction potential of -687.0mV indicative of a marked reducing ability. Thus, MC+ has an ability to reduce acetaldehyde in a distilled spirit due to dissolved hydrogen released from MC+.

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Hydrogen (H2) is an essential substrate for methanogens to produce methane (CH4), and also influences pathways of volatile fatty acids (VFA) production in the rumen. Dissolved H2 (H2 (aq)) is the form of H2 available to microbes, and dissolved CH4 (CH4 (aq)) is important for indicating methanogens activity. Rumen H2 (aq) concentration has been estimated by assuming equilibrium with headspace gaseous H2 (H2 (g)) concentration using Henry's law, and has also been directly measured in the liquid phase in some in vitro and in vivo experiments. In this in vivo study, H2 (aq) and CH4 (aq) concentration measured directly in rumen fluid and their corresponding concentrations estimated from their gaseous phase concentrations, were compared to investigate the existence of equilibrium between the gas and liquid phases. Twenty-four Tibetan sheep were randomly assigned to two mixed diets containing the same concentrate mixed with oat grass (OG diet) or barley straw (BS diet). Rumen gaseous phase and contents were sampled using rumenocentesis and oral stomach tubing, respectively. Rumen H2 (aq) and CH4 (aq) concentration and VFA profile differed between sheep fed OG and BS diets. Measured H2 (aq) and CH4 (aq) concentration were greater than H2 (aq) and CH4 (aq) concentrations estimated using gas concentrations, indicating lack of equilibrium between gas and liquid phase and supersaturation of H2 and CH4 in rumen fluid. As a consequence, Gibbs energy changes (ΔG) estimated for various metabolic pathways were different when calculated using dissolved gases concentrations directly measured and when using dissolved gases concentrations assuming equilibrium with the gaseous phase. Dissolved CH4, but not CH4 (g), was positively correlated with H2 (aq). Both H2 (aq) and H2 (g) concentrations were positively correlated with the molar percentage of butyrate and negatively correlated with the molar percentage of acetate. In summary, rumen fluid was supersaturated with both H2 and CH4, and H2 (aq) was closely associated with the VFA profile and CH4 (aq) concentration. The assumption of equilibrium between dissolved gases and gaseous phase affected ΔG estimation.