Low O2 Concentrations Research Articles

P-35 - Free-radical fragmentation reactions of biomolecules involving C-C, C-O and C-N bond cleavage

Among a wide variety of homolytic transformations taking place in biomolecules, the oxidation processes occurring in the cell membrane lipids have been the most thoroughly studied ones. For such processes to be realized, the presence of oxygen is necessary. At low O2 concentrations the probability of oxidation processes decreases, enabling other types of free-radical reactions to occur in biomolecules. In our studies, a concept is being developed focusing on the important contribution of free-radical fragmentation reactions involving C-C, C-O and C-N bond rupture in organic molecules to the biosystem damage. Such processes can take place without participation of oxygen and may lead not only to destruction and modification of biologically relevant compounds but to accumulation of physiologically active products as well. The reactions resulting in the C-C, C-O or C-N bond ruptures occurring in an organism are known to be catalyzed by lyases. Many of these reactions proceed according to a free-radical mechanism. The report will be focused on discussion of the above-named fragmentation reactions of biomolecules and the possible consequences of their realization in an organism.

Gene expression of terminal oxidases in two marine bacterial strains exposed to nanomolar oxygen concentrations.

The final step of aerobic respiration is carried out by a terminal oxidase transporting electrons to oxygen (O2). Prokaryotes harbor diverse terminal oxidases that differ in phylogenetic origin, structure, biochemical function, and affinity for O2. Here we report on the expression of high-affinity (cytochrome cbb3 oxidase), low-affinity (cytochrome aa3 oxidase), and putative low-affinity (cyanide-insensitive oxidase (CIO)) terminal oxidases in the marine bacteria Idiomarina loihiensis L2-TR and Marinobacter daepoensis SW-156 upon transition to very low O2 concentrations (<200 nM), measured by RT-qPCR. In both strains, high-affinity cytochrome cbb3 oxidase showed the highest expression levels and was significantly up-regulated upon transition to low O2 concentrations. Low-affinity cytochrome aa3 oxidase showed very low transcription levels throughout the incubation. Surprisingly, however, it was also up-regulated upon transition to low O2 concentrations. In contrast, putative low-affinity CIO had much lower expression levels and markedly different regulation patterns between the two strains. These results demonstrate that exposure to low O2 concentrations regulates the gene expression of different types of terminal oxidases, but also that the type and magnitude of transcriptional response is species-dependent. Therefore, in situ transcriptome data cannot, without detailed knowledge of the transcriptional regulation of the species involved, be translated into relative respiratory activity.

Effects of Electrode Structure and Electron Energy on Abatement of NO in Dielectric Barrier Discharge Reactor

Electrode structure and electron energy effects on NO abatement were studied in two different structure DBD reactors. Final product analysis of NO abatement in coaxial cylinder dielectric barrier discharge (CC-DBD) and tubular multilayer dielectric barrier discharge (TM-DBD) reactors indicated that the electrode structure of TM-DBD was better under low O2 concentration conditions, but the result was opposite because the new NOx was produced in TM-DBD when O2 concentration was increasing. In addition, results of particle-in-cell with Monte Carlo collisions (PIC-MCC) simulation manifested that the largest and the average electron energy were 12.09 eV and 3.35 eV in TM-DBD reactor, respectively, while they were 5.25 eV and 2.96 eV in CC-DBD reactor, respectively. CC-DBD electrode structures are preferable for better NO abatement and no new NOx under oxygen-containing condition.

(Invited) Nitrogenase Electrochemistry for Ammonia Production

The Haber-Bosch process, responsible for producing NH3 from H2 and N2, ranks as one of the most important discoveries in the history of chemistry. Its industrial application in synthetic fertilizers contributed to the continuously increasing population and their quality of life. The Haber-Bosch process, however, is a very energy-intensive process with high temperature (500 °C) and pressure (20 MPa) required for efficient NH3 production. Over 1 % of the world’s energy sources were consumed in order to produce ~140 megatons of NH3 in 2015. Additionally, 3 % of global CO2 emissions are due to the Haber-Bosch related technology. A renewable strategy for ammonia production would therefore be valuable. In nature, the ability to reduce N2 to NH3 is limited to a group of bacteria and archaea classified as diazotrophs, all of which share an enzyme called nitrogenase. Using methylviologen (N,N’-dimethyl-4,4’-bipyridinium) to shuttle electrons to nitrogenase, N2 reduction to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode which utilizes the enzyme hydrogenase to oxidize molecular hydrogen (H2) results in an enzymatic fuel cell (EFC) that is able to produce NH3 from H2 and N2, while simultaneously producing an electrical current. To demonstrate this, 60 mC of charge was passed across H2/N2 EFCs, which resulted in the formation of 286 nmol NH3 mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 %. Importantly, this EFC produces NH3 and electrical energy in a carbon-neutral manner. A protective FeSII protein, which can reversibly lock nitrogenase into a multicomponent protective complex upon exposure to low concentrations of O2, was incorporated into a nitrogenase bioelectrosynthetic cell whereby NH3 was produced using air as a substrate. This marks a significant step forward in overcoming the crippling limitation of nitrogenase’s sensitivity toward O2.

Key role of hydrogen peroxide in antimicrobial activity of spring, Honeydew maquis and chestnut grove Corsican honeys on Pseudomonas aeruginosa DNA.

We started to determine the antibacterial efficiency of Corsican chestnut grove and honeydew maquis honeys on Pseudomonas aeruginosa. No morphological alteration of the bacterial surface was observed. Antimicrobial action seems to be related to the synergy between hydrogen peroxide and phenolic compounds. The exerted pro-oxidant activity leads to a degradation of P. aeruginosa plasmidic DNA. This is the first study that investigate the primary antibacterial mechanism of Corsican honeys.

Mimicking physiological O2 tension in the female reproductive tract improves assisted reproduction outcomes in pig.

Is O2 tension in the pig oviduct and uterus affected by the estrous cycle stage and the animal's age, and can the outcome of in vitro embryo development be improved by mimicking these physiological values? O2 tension within the pig reproductive organs is affected by the animal's age, and values close to those measured in vivo have a positive impact on embryo development and quality when used during IVF and embryo culture (EC). To obtain a healthy embryo in vitro, it is necessary to adopt a culture microenvironment that approximates physiological conditions. Despite advances in surgical procedures and sensitive probes that allow accurate assessment of in vivo O2 tension, few such studies have been conducted recently in mammals. In addition, no reference values of physiological O2 tension in the reproductive tract exist for large animal models such as pig, and the effect of O2 tension on ART outcomes is unknown. This study was conducted in pigs. We measured oviductal and uterine O2 tension (n = 29 and 13, respectively) and then examined how the use of the physiological values in pig IVF and EC affected pig ART output (n = 1447 oocytes). The oviductal and uterine O2 tension at the different stages of the estrous cycle was monitored using a laparo-endoscopic single-site surgery (LESS) assisted approach along with a flexible and thin miniaturized luminescent probe. Two groups of pigs, Large-white × Landrace breed, were used: for the first group, 16 pre-pubertal gilts (5 months old and 95 kg) were induced to ovulate with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); in the second group 13 mature sows (24-48 months and 185 kg) were used. IVF and EC were performed at two different O2 tensions: Atmospheric O2 (20%) and the mean in vivo value measured (7%). At 18-20 h post-insemination (hpi), a small sample of presumptive zygotes were fixed, stained and examined under epifluorescence microscopy to assess the fertilization rates. At 48 hpi, cleavage was evaluated under the stereomicroscope. Finally, at 180 hpi, development to the blastocyst stage was quantified, blastocyst morphology was assessed, and embryos were fixed and stained to count the mean cell number per blastocyst. The mean O2 content within the pig oviduct and uterus was always lower than in ambient air. The average O2 percentage was higher in gilts (10.0%) than in sows (7.6%) (P < 0.0001). The cleavage rate of porcine in vitro fertilized embryos maintained under 7% O2 during IVF and EC was significantly higher (60.0 ± 2.3) compared with those cultured under 20% O2 (32.0 ± 2.2) (P < 0.05). An increase in the number of cells in embryos cultured under the low O2 concentration (88.9 ± 5.9) was observed compared to those cultured under 20% O2 (59.0 ± 5.0) (P < 0.05). None. Although minimally invasive surgery was used the effect of anesthesia and manipulations on O2 tension within the organs are unknown. Using physiological oxygen concentrations in IVF/EC could improve ART outcomes. This study was funded by Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER). Grants AGL2012-40180-C03-01 and AGL2015-66341-R. The authors declare no conflict of interest.

Effect of CO2 and H2O on the combustion characteristics and ash formation of pulverized coal in oxy-fuel conditions

The combustion characteristics and ash formation of pulverized coal under different atmospheres (O2/N2, O2/CO2 and O2/H2O/CO2) were investigated using an isothermal thermal analysis system. The burning rate in O2/N2 was faster than that in O2/CO2 due to differences in the physical properties between N2 and CO2 at 800 °C. The effect of CO2 gasification was enhanced with increasing temperature; as a result, the overall reaction rate in O2/CO2 approached and exceeded the combustion rate in O2/N2. However, the effect of gasification was weakened by increasing O2 concentration, and the overall reaction rate in O2/CO2 decreased and eventually lagged the combustion rate in O2/N2. Because of the distinct physical properties of H2O, the burning rate in O2/CO2/H2O was lower than that in O2/N2 but higher than that in O2/CO2 at 800 °C. At a low O2 concentration, the influence of CO2 and H2O gasification was enhanced with increasing temperature; thus, the overall reaction rate in O2/H2O/CO2 was higher than that in O2/CO2. Moreover, the burning rate in O2/H2O/CO2 increased slightly with increasing H2O concentration. However, higher O2 concentrations weakened the effect of gasification, leading to similar burning processes under different atmospheres. The XRD analysis of the ash samples showed that CO2 and H2O had no significant effect on the transformation of main minerals.

Effect of H2O on the NO emission characteristics of pulverized coal during oxy-fuel combustion.

The NO emission characteristics of Datong bituminous coal and Yangquan anthracite in O2/H2O/CO2 atmospheres were investigated by using a fixed-bed reactor system, and the emission characteristics were compared with the experimental results from O2/N2 and O2/CO2 atmospheres, especially at low O2 concentrations and high temperatures. The results showed that NO emissions of pulverized coal in O2/CO2 environments were less than those in the O2/N2 environments, regardless of the O2 concentration and the furnace temperature. Adding H2O decreased the possibility of reactions between the reductive groups (NH) and the oxygen radical during devolatilization, which led to a decrease in NO emissions at 1000°C. However, as the furnace temperature increased, "additional" nitrogen precursors (HCN and NH3) generated by enhanced char-H2O gasification were quickly oxidized to generate a large amount of NO during char oxidation that exceeded the amount of NO reduced by NH during devolatilization. Thus, the NO emissions in O2/CO2/H2O atmosphere were higher than those in O2/CO2 atmosphere at a low O2 concentration. However, as the O2 concentration increased, the NO emissions in O2/CO2/H2O atmosphere became lower than those in O2/CO2 atmosphere because the effect of H2O gasification became weaker. The NO emissions of Yangquan anthracite (YQ) were higher than those of DT, but the changing trend of YQ was similar to that of DT.

Neuronal-like differentiated SH-SY5Y cells adaptation to a mild and transient H2 O2 -induced oxidative stress.

Neuronal-like differentiated SH-SY5Y cells are a suitable model to investigate PC, an endogenous neuroprotective response to a mild and transient H2 O2 -induced oxidative stress, elicited by 24-hour exposure to very low H2 O2 concentrations and mediated by both apoptotic and inflammatory pathways. This model reflects in vivo PC mechanisms occurring after brain trauma and provides novel information about pathways and time of protection useful for an appropriate pharmacological intervention.

Methane oxidation in industrial biogas plants—Insights in a novel methanotrophic environment evidenced by pmoA gene analyses and stable isotope labelling studies

A broad methanotrophic community consisting of 16 different operational taxonomic units (OTUs) was detected by particulate methane monooxygenase A (pmoA) gene analyses of reactor sludge samples obtained from an industrial biogas plant. Using a cloning-sequencing approach, 75% of the OTUs were affiliated to the group of type I methanotrophs (γ-Proteobacteria) and 25% to type II methanotrophs (α-Proteobacteria) with a distinct predominance of the genus Methylobacter. By database matching, half of the total OTUs may constitute entirely novel species. For evaluation of process conditions that support growth of methanotrophic bacteria, qPCR analyses of pmoA gene copy numbers were performed during a sampling period of 70 days at varying reactor feeding scenarios. During the investigation period, methanotrophic cell counts estimated by qPCR fluctuated between 3.4 × 104 and 2 × 105 cells/mL with no distinct correlation to the organic loading rate, the amount of CH4, O2 and NH4-N. Methanotrophic activity was proofed even at low O2 levels (1%) by using stable carbon isotope labelling experiments of CH4 in batch experiments inoculated with reactor sludge. Supplementation of 13C labelled CH4 in the headspace of the reaction vials unambiguously confirmed the formation of 13C labelled CO2. Thus, industrial biogas reactors can be considered as a further methanotrophic habitat that exhibits a unique methanotrophic community which is specifically adapted to high CH4 and low O2 concentrations. To the best of our knowledge, our study is the first accurate detection and quantification of methanotrophic bacteria in industrial biogas reactors.

Significant advantages of sulfur-doped graphene in neutral media as electrocatalyst for oxygen reduction comparing with Pt/C

Sulfur-doped graphene (SDG) has been found to be an efficient electrocatalyst for oxygen reduction reaction. However, previous studies on the catalytic activity of SDG have been mainly confined to O2-saturated alkaline media which is a typical alkaline fuel cell environment. Air-cathode microbial fuel cells (ACMFCs), as a novel energy conversion and wastewater treatment technology, use the oxygen from air as cathodic reactant in neutral media with low concentration of O2. Thus, it is meaningful to explore the catalytic performance of SDG in such ACMFC environment. The result showed that in ACMFC environment, the peak current density of SDG in CV test was surprisingly 4.5 times higher than that of Pt/C, indicating a much stronger catalytic activity of SDG. Moreover, SDG exhibited a stronger tolerance against the crossover of glucose (a typical anodic fuel in ACMFC) and better stability than Pt/C in neutral media.

Nitric oxide (NO) in the oxygen minimum zone off Peru

Nitric oxide (NO) is a short-lived compound of the marine nitrogen cycle. However, measurements of NO in seawater are analytically challenging and our knowledge about its oceanic distribution is, therefore, rudimentary. NO was measured in the oxygen minimum zone (OMZ) of the eastern tropical South Pacific Ocean (ETSP) off Peru during R/V Meteor cruise M93 in February/March 2013. NO concentrations ranged from close to or below the detection limit (0.5 nmol L−1) in the surface layer to 9.5 nmol L−1 in the OMZ. NO concentrations increased significantly when oxygen (O2) concentrations dropped below 1–2 µmol L−1. We found positive correlations between NO and NO2- as well as between NO and the abundance of archaeal amoA, a marker gene for archaeal nitrifiers. No trends between NO and nirS and hzo, marker genes for canonical denitrification and anammox, respectively, were found. To this end, we conclude that NO off Peru was mainly produced by archaeal nitrifier-denitrification at low O2 concentrations in the OMZ.

Hypoxia Improves Germination of the Problematic Invader Garlic Mustard (Alliaria petiolata) of North American Forests

Abstract Garlic mustard [Alliaria petiolate (M. Bieb.) Cava & Grande] is a problematic European invader of temperate Eastern North American forests. We studied its germination response at reduced oxygen (O2) concentrations. Seeds of garlic mustard were exposed to different O2 concentrations (20.9, 15, 10, 5, and 2.5 %) in two laboratory experiments. Germination rate increased, and it took fewer days to attain 50 % germination (t50) at 15 % O2 than at 20.9 % O2. Germination declined at 5 % O2, and seedlings became stunted at 2.5 % O2. The ability of garlic mustard to thrive under low O2 concentrations may allow it to invade a wide variety of suboptimal habitats, such as shady understories where leaf litter cover the forest floors and on compressed and compact soils. Its ability to grow at low O2 levels may contribute to its invasiveness.

The reaction of fluorine atoms with methanol: yield of CH3O/CH2OH and rate constant of the reactions CH3O + CH3O and CH3O + HO2.

Xenondifluoride, XeF2, has been photolysed in the presence of methanol, CH3OH. Two reaction pathways are possible: F + CH3OH → CH2OH + HF and F + CH3OH → CH3O + HF. Both products, CH2OH and CH3O, will be converted to HO2 in the presence of O2. The rate constants for the reaction of both radicals with O2 differ by more than 3 orders of magnitude, which allows an unequivocal distinction between the two reactions when measuring HO2 concentrations in the presence of different O2 concentrations. The following yields have then been determined from time-resolved HO2 profiles: φCH2OH = (0.497 ± 0.013) and φCH3O = (0.503 ± 0.013). Experiments under low O2 concentrations lead to reaction mixtures containing nearly equal amounts of HO2 (converted from the first reaction) and CH3O (from the second reaction). The subsequent HO2 decays are very sensitive to the rate constants of the reaction between these two radicals and the following rate constants have been obtained: k(CH3O + CH3O) = (7.0 ± 1.4) × 10-11 cm3 s-1 and k(CH3O + HO2) = (1.1 ± 0.2) × 10-10 cm3 s-1. The latter reaction has also been theoretically investigated on the CCSD(T)//M06-2X/aug-cc-pVTZ level of theory and CH3OH + O2 have been identified as the main products. Using μVTST, a virtually pressure independent rate constant of k(CH3O + HO2) = 4.7 × 10-11 cm3 s-1 has been obtained, in good agreement with the experiment.

Impact of eight closures in controlled industrial conditions on the shelf life of two (red and rosé) wines

Aims: The management of O2, CO2 and SO2 at bottling and the choice of closure are two key factors of the shelf life of bottled wines before bringing them to market. The impact of eight closures (four screw caps, two synthetic stoppers and two technical stoppers) was evaluated on a red Merlot/Tannat wine. The results of a rosé wine are also discussed.Methods and results: Analytical monitoring (O2, CO2, SO2, aphrometric pressure, L*, a*, b*) was carried out over 538 days of storage at 20°C, along with two sensory analyses at 10 and 17 months. The average wine total O2 content at the time of bottling was 2 mg/L. Intra- and inter-procedure variability was controlled, including for dissolved CO2 content.Conclusion: Unlike closures with the highest Oxygen Transmission Rate (OTR), the two technical stoppers and the two screw caps with Saranex seal, harboring the lowest OTR, matched with the wines exhibiting a low total O2 content at equilibrium (from 4 to 18 months after bottling), with more free SO2 and less color change. However, the OTR gradient (5 to 67 µg/d) observed through the physicochemical analyses was not necessarily confirmed by the two sensory analyses.Significance and impact of the study: This study puts into perspective the impact of closure OTR on the sensory characteristics evolution of wine consumed within the first two years, especially when total O2 at bottling exceeds 1.5 mg/L.

A revised photocatalytic transformation mechanism for chlorinated VOCs: Experimental evidence from C2Cl4 in the gas phase

The photodegradation of gaseous perchloroethylene (PCE) was investigated on titanium dioxide under UV light at 365 nm at the gas/solid interface in a CSTR photo-reactor (Continuous Stirred Tank Reactor). The rate and products are strongly affected by oxygen presence. Gaseous products of PCE degradation agree with literature. The production of active chlorine (sum of Cl2, HClO, ClO and ClO2) was investigated both in the presence of oxygen and in anoxic conditions. At low O2 concentration no gaseous active chlorine was determined, while a significant amount was measured in the presence of oxygen. By considering that in the absence of O2 the only possible form of active chlorine is Cl2, this highlights that Cl is not produced, and that chain reactions promoted by the chlorine radical do not occur on the TiO2 surface.The photocatalytic transformation of C2Cl4 was investigated at different concentrations. The rate follows a first order kinetic that is rationalized with a photocatalytic kinetic model in which the substrate is able to react simultaneously with both photogenerated holes and electrons. In anoxic conditions adsorbed halogenated organic compounds with molecular weights higher than that of PCE were produced and chloride ions accumulated at the surface. Reductive pathways have a key role in PCE degradation. The water vapor has a detrimental role on the PCE transformation rate due to the competition with PCE adsorption on reactive sites with 2:1 stoichiometry.The addition of chloride ions on TiO2 surface slows down the PCE degradation rate and the production of gaseous CCl4 but increments that of C2Cl6 in anoxic conditions. This is rationalized by a mechanism in which direct hole transfer to substrate occurs followed by chloride anion addition to the carbocation.

Influence of operating parameters on arsenic transformation during municipal sewage sludge incineration with cotton stalk

Addition of cotton stalk (CS) has been proved to promote dramatically the transformation of toxic As3+ to less toxic As5+ in the fly ash during municipal sewage sludge (MSS) incineration. However, the fate of arsenic during co-firing of MSS and CS in different operating parameters was still unclear. In the present study, the effects of incineration temperatures and O2 content in the flue gas on speciation transformation of arsenic during MSS and 70% MSS/30% CS incineration were investigated in a bubbling fluidized bed. The results show that less arsenic is distributed in bottom ash whereas more arsenic is migrated to the fly ash and flue gas, with the temperature increasing from 800 °C to 950 °C. The arsenic capture in fly ash is facilitated predominantly by the condensation and/or physical adsorption of As2O3(g) at the temperatures from 800 °C to 900 °C. The chemical oxidation of As2O3(g) is favored by forming various arsenates (As5+) at 950 °C. At low O2 content from 1% to 5%, some arsenic compounds in MSS such as As2S3 can react with O2 to produce As2O3(g), and then more As2O3(g) is captured in the fly ash by the inherent mineral compounds like CaO through the condensation and/or physical adsorption. Further increasing O2 content especially to 9% stimulates significantly the oxidation of As3+ to As5+ in the fly ash, which is mainly attributed to the chemical reactions between As2O3(g), various mineral compounds and sufficient O2.

Low oxygen and argon in the Neoproterozoic atmosphere at 815 Ma

The evolution of Earth's atmosphere on >106-yr timescales is tied to that of the deep Earth. Volcanic degassing, weathering, and burial of volatile elements regulates their abundance at the surface, setting a boundary condition for the biogeochemical cycles that modulate Earth's atmosphere and climate. The atmosphere expresses this interaction through its composition; however, direct measurements of the ancient atmosphere's composition more than a million years ago are notoriously difficult to obtain. Gases trapped in ancient minerals represent a potential archive of the ancient atmosphere, but their fidelity has not been thoroughly evaluated. Both trapping and preservation artifacts may be relevant. Here, I use a multi-element approach to reanalyze recently collected fluid-inclusion data from halites purportedly containing snapshots of the ancient atmosphere as old as 815 Ma. I argue that those samples were affected by the concomitant trapping of air dissolved in brines and contaminations associated with modern air. These artifacts lead to an apparent excess in O2 and Ar. The samples may also contain signals of mass-dependent fractionation and biogeochemical cycling within the fluid inclusions. After consideration of these artifacts, this new analysis suggests that the Tonian atmosphere was likely low in O2, containing ≤10% present atmospheric levels (PAL), not ∼50% PAL as the data would suggest at face value. Low concentrations of O2 are consistent with other geochemical constraints for this time period and further imply that the majority of Neoproterozoic atmospheric oxygenation occurred after 815 Ma. In addition, the analysis reveals a surprisingly low Tonian Ar inventory—≤60% PAL—which, if accurate, challenges our understanding of the solid Earth's degassing history. When placed in context with other empirical estimates of paleo-atmospheric Ar, the data imply a period of relatively slow atmospheric Ar accumulation in the Paleo- and Meso-Proterozoic, followed by extensive degassing in the late Neoproterozoic or early Cambrian, before returning to a relatively quiescent state by the Devonian. This two-step structure resembles that for the evolution of atmospheric O2, hinting at a common driving force from the deep Earth. Some caution is warranted, however, because still more enigmatic contaminations than the ones presented here may be relevant. Gases trapped in minerals may offer important constraints on the evolution of Earth's surface, climate, and atmosphere, but potential contaminations and other confounding factors need to be considered carefully before these records can be considered quantitative.

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2

Biocatalysts that mediate the H2-dependent reduction of NAD+ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD+-reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD+-reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H2-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H2-mediated NAD+ reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD+-reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H2-driven cofactor recycling under oxic conditions at elevated temperatures.

Small Purge Method to Sample Vapor from Groundwater Monitoring Wells Screened Across the Water Table

AbstractGroundwater monitoring wells are present at most hydrocarbon release sites that are being assessed for cleanup. If screened across the vadose zone, these wells provide an opportunity to collect vapor samples that can be used in the evaluation of vapor movement and biodegradation processes occurring at such sites. This paper presents a low purge volume method (modified after that developed by the U.S. EPA) for sampling vapor from monitoring wells that is easy to implement and can provide an assessment of the soil gas total petroleum hydrocarbon (TPH) and O2 concentrations at the base of the vadose zone. As a result, the small purge method allows for sampling of vapor from monitoring wells to support petroleum vapor intrusion (PVI) risk assessment. The small purge volume method was field tested at the Hal's service station site in Green River, Utah. This site is well‐known for numerous soil gas measurements containing high O2 and high TPH vapor concentrations in the same samples which is inconsistent with well‐accepted biodegradation models for the vapor pathway. Using the low purge volume method, monitoring wells were sampled over, upgradient, and downgradient of the light nonaqueous phase liquid (LNAPL) footprint. Results from our testing at Hal's show that vapor from monitoring wells over LNAPL contained very low O2 and high TPH concentrations. In contrast, vapor from monitoring wells not over LNAPL contained high O2 and low TPH concentrations. The results of this study show that a low purge volume method is consistent with biodegradation models especially for sampling at sites where low permeability soils exist in and around a LNAPL source zone.