O2-limited Conditions Research Articles

Coconut shell-based biochars produced by an innovative thermochemical process for obtaining improved lignocellulose-based adsorbents

The urgent need for a simple and cost-effective thermochemical process to produce biochar has prompted this study. The aim was to develop a straightforward thermochemical process under O2-limited conditions for the production of coconut-based biochar (CBB) and to assess its ability to remove methylene blue (MB) through adsorption, comparing it with CBB produced by slow pyrolysis. CBBs were obtained under different atmospheric conditions (O2-limited, muffle furnace biochar (MFB); and inert, pyrolytic reactor biochar (PRB)), at 350, 500, and 700°C, and for 30 and 90'. MFB and PRB were characterized using FTIR, RAMAN, SEM, EDS, and XRD analyses. Adsorption tests were conducted using 1.0gL-1 of MFB and PRB, 10mgL-1 of MB at 25°C for 48h. Characterization revealed that atmospheric conditions significantly influenced the yield and structural features of the materials. PRB exhibited higher yields and larger cavities than MFB, but quite similar spectral features. Adsorption tests indicated that MFB and PRB had qt values of 33.1 and 9.2mgg-1, respectively, which were obtained at 700°C and 90', and 700°C and 30', respectively. This alternative method produced an innovative and promising lignocellulose-based material with great potential to be used as a biosorbent.

Role of Nitric Oxide in Hydroxylamine Oxidation by Ammonia-Oxidizing Bacteria.

An important role of nitric oxide (NO) as either a free intermediate in the NH3 oxidation pathway or a potential oxidant for NH3 or NH2OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO2- production, whereas N. europaea grown under O2 limitation produced NO equivalent to up to 40% of NO2- production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O2 limitation abolished N2O production. These results indicate different turnover rates of NO during NH3 oxidation under O2-replete and O2-limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH3 saturation and adaptation to O2 limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N2O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O2-limited growth conditions.

In Situ Tracking of Crystal-Surface-Dependent Cu2O Nanoparticle Dissolution in an Aqueous Environment.

Metal-oxide-based nanoparticles (MONPs) such as Cu2O NPs have attracted growing attention, but the potential discharges of MONPs have raised considerable concern of their environmental fate including their dissolution behavior. The impacts of morphology on MONP dissolution are largely uncertain due to the lack of in situ tracking techniques. In this study, we combined a series of in situ technologies including liquid-cell transmission electron microscopy and fluorescence probes to reveal the in situ dissolution process of Cu2O NPs in freshwater. Our results suggest that cubic Cu2O NPs exhibit a higher dissolution quantity compared with spherical NPs of the same surface area. The difference was mainly related to the crystal surface, while other factors such as particle size or aggregation status showed minor effects. Importantly, we demonstrated the simultaneous growth of new small NPs and the dissolution of pristine Cu2O NPs during the dissolution of Cu2O NPs. Cubic Cu2O NPs became much less soluble under O2-limited conditions, suggesting that O2 concentration largely affected the dependence of dissolution on the NP morphology. Our findings highlight the potential application of in situ techniques to track the environmental fates of MONPs, which would provide important information for assessing the ecological risks of engineered NPs.

Nitrate-Nitrite-Nitric Oxide Pathway: A Mechanism of Hypoxia and Anoxia Tolerance in Plants.

Oxygen (O2) is the most crucial substrate for numerous biochemical processes in plants. Its deprivation is a critical factor that affects plant growth and may lead to death if it lasts for a long time. However, various biotic and abiotic factors cause O2 deprivation, leading to hypoxia and anoxia in plant tissues. To survive under hypoxia and/or anoxia, plants deploy various mechanisms such as fermentation paths, reactive oxygen species (ROS), reactive nitrogen species (RNS), antioxidant enzymes, aerenchyma, and adventitious root formation, while nitrate (NO3−), nitrite (NO2−), and nitric oxide (NO) have shown numerous beneficial roles through modulating these mechanisms. Therefore, in this review, we highlight the role of reductive pathways of NO formation which lessen the deleterious effects of oxidative damages and increase the adaptation capacity of plants during hypoxia and anoxia. Meanwhile, the overproduction of NO through reductive pathways during hypoxia and anoxia leads to cellular dysfunction and cell death. Thus, its scavenging or inhibition is equally important for plant survival. As plants are also reported to produce a potent greenhouse gas nitrous oxide (N2O) when supplied with NO3− and NO2−, resembling bacterial denitrification, its role during hypoxia and anoxia tolerance is discussed here. We point out that NO reduction to N2O along with the phytoglobin-NO cycle could be the most important NO-scavenging mechanism that would reduce nitro-oxidative stress, thus enhancing plants’ survival during O2-limited conditions. Hence, understanding the molecular mechanisms involved in reducing NO toxicity would not only provide insight into its role in plant physiology, but also address the uncertainties seen in the global N2O budget.

Genome-wide transcriptional regulation in Saccharomyces cerevisiae in response to carbon dioxide

Sugar metabolism by Saccharomyces cerevisiae produces ample amounts of CO2 under both aerobic and anaerobic conditions. High solubility of CO2 in fermentation media, contributing to enjoyable sensory properties of sparkling wine and beers by S. cerevisiae, might affect yeast metabolism. To elucidate the overlooked effects of CO2 on yeast metabolism, we examined glucose fermentation by S. cerevisiae under CO2 as compared to N2 and O2 limited conditions. While both CO2 and N2 conditions are considered anaerobic, less glycerol and acetate but more ethanol were produced under CO2 condition. Transcriptomic analysis revealed that significantly decreased mRNA levels of GPP1 coding for glycerol-3-phosphate phosphatase in glycerol synthesis explained the reduced glycerol production under CO2 condition. Besides, transcriptional regulations in signal transduction, carbohydrate synthesis, heme synthesis, membrane and cell wall metabolism, and respiration were detected in response to CO2. Interestingly, signal transduction was uniquely regulated under CO2 condition, where upregulated genes (STE3, MSB2, WSC3, STE12, and TEC1) in the signal sensors and transcriptional factors suggested that MAPK signaling pathway plays a critical role in CO2 sensing and CO2-induced metabolisms in yeast. Our study identifies CO2 as an external stimulus for modulating metabolic activities in yeast and a transcriptional effector for diverse applications.

Effect of respiratory inhibitors and quinone analogues on the aerobic electron transport system of Eikenella corrodens

The effects of respiratory inhibitors, quinone analogues and artificial substrates on the membrane-bound electron transport system of the fastidious β-proteobacterium Eikenella corrodens grown under O2-limited conditions were studied. NADH respiration in isolated membrane particles were partially inhibited by rotenone, dicoumarol, quinacrine, flavone, and capsaicin. A similar response was obtained when succinate oxidation was performed in the presence of thenoyltrifluoroacetone and N,N’-dicyclohexylcarbodiimide. NADH respiration was resistant to site II inhibitors and cyanide, indicating that a percentage of the electrons transported can reach O2 without the bc1 complex. Succinate respiration was sensitive to myxothiazol, antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Juglone, plumbagin and menadione had higher reactivity with NADH dehydrogenase. The membrane particles showed the highest oxidase activities with ascorbate-TCHQ (tetrachlorohydroquinone), TCHQ alone, and NADH-TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine), and minor activity levels with ascorbate-DCPIP (2,6-dichloro-phenolindophenol) and NADH-DCPIP. The substrates NADH-DCPIP, NADH-TMPD and TCHQ were electron donors to cyanide-sensitive cbb' cytochrome c oxidase. The presence of dissimilatory nitrate reductase in the aerobic respiratory system of E. corrodens ATCC 23834 was demonstrated by first time. Our results indicate that complexes I and II have resistance to their classic inhibitors, that the oxidation of NADH is stimulated by juglone, plumbagin and menadione, and that sensitivity to KCN is stimulated by the substrates TCHQ, NADH-DCPIP and NADH-TMPD.

Hydrogen utilization by Methylocystis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs

Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H2 to the culture headspace had the most significant positive effect on the growth yield under CH4 (6%) and O2 (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (±0.52) to 13.82 (±0.69) mg cell dry weight per mmol CH4, while CH4 consumption was significantly reduced. Regardless of H2 addition, CH4 utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O2/CH4 mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H2 addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H2 (37d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H2 may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH4, such as the production of high-quality feed protein.

Process intensification for cytochrome P450 BM3-catalyzed oxy-functionalization of dodecanoic acid.

Selective oxy‐functionalization of nonactivated C‐H bonds is a long‐standing “dream reaction” of organic synthesis for which chemical methodology is not well developed. Mono‐oxygenase enzymes are promising catalysts for such oxy‐functionalization to establish. Limitation on their applicability arises from low reaction output. Here, we showed an integrated approach of process engineering to the intensification of the cytochrome P450 BM3‐catalyzed hydroxylation of dodecanoic acid (C12:0). Using P450 BM3 together with glucose dehydrogenase for regeneration of nicotinamide adenine dinucleotide phosphate (NADPH), we compared soluble and co‐immobilized enzymes in O2‐gassed and pH‐controlled conversions at high final substrate concentrations (≥40mM). We identified the main engineering parameters of process output (i.e., O2 supply; mixing correlated with immobilized enzyme stability; foam control correlated with product isolation; substrate solubilization) and succeeded in disentangling their complex interrelationship for systematic process optimization. Running the reaction at O2‐limited conditions at up to 500‐ml scale (10% dimethyl sulfoxide; silicone antifoam), we developed a substrate feeding strategy based on O2 feedback control. Thus, we achieved high reaction rates of 1.86g·L−1·hr−1 and near complete conversion (≥90%) of 80mM (16g/L) C12:0 with good selectivity (≤5% overoxidation). We showed that “uncoupled reaction” of the P450 BM3 (~95% utilization of NADPH and O2 not leading to hydroxylation) with the C12:0 hydroxylated product limited the process efficiency at high product concentration. Hydroxylated product (~7g; ≥92% purity) was recovered from 500ml reaction in 82% yield using ethyl‐acetate extraction. Collectively, these results demonstrate key engineering parameters for the biocatalytic oxy‐functionalization and show their integration into a coherent strategy for process intensification.

Metabolic characterisation of Magnetospirillum gryphiswaldense MSR-1 using LC-MS-based metabolite profiling.

Magnetosomes are nano-sized magnetic nanoparticles with exquisite properties that can be used in a wide range of healthcare and biotechnological applications. They are biosynthesised by magnetotactic bacteria (MTB), such as Magnetospirillum gryphiswaldense MSR-1 (Mgryph). However, magnetosome bioprocessing yields low quantities compared to chemical synthesis of magnetic nanoparticles. Therefore, an understanding of the intracellular metabolites and metabolic networks related to Mgryph growth and magnetosome formation are vital to unlock the potential of this organism to develop improved bioprocesses. In this work, we investigated the metabolism of Mgryph using untargeted metabolomics. Liquid chromatography-mass spectrometry (LC-MS) was performed to profile spent medium samples of Mgryph cells grown under O2-limited (n = 6) and O2-rich conditions (n = 6) corresponding to magnetosome- and non-magnetosome producing cells, respectively. Multivariate, univariate and pathway enrichment analyses were conducted to identify significantly altered metabolites and pathways. Rigorous metabolite identification was carried out using authentic standards, the Mgryph-specific metabolite database and MS/MS mzCloud database. PCA and OPLS-DA showed clear separation and clustering of sample groups with cross-validation values of R2X = 0.76, R2Y = 0.99 and Q2 = 0.98 in OPLS-DA. As a result, 50 metabolites linked to 45 metabolic pathways were found to be significantly altered in the tested conditions, including: glycine, serine and threonine; butanoate; alanine, aspartate and glutamate metabolism; aminoacyl-tRNA biosynthesis and; pyruvate and citric acid cycle (TCA) metabolisms. Our findings demonstrate the potential of LC-MS to characterise key metabolites in Mgryph and will contribute to further understanding the metabolic mechanisms that affect Mgryph growth and magnetosome formation.

Improvement of bioelectricity generation and microalgal productivity with concomitant wastewater treatment in flat-plate microbial carbon capture cell

Abstract The unprecedented increase in global population has triggered the energy crisis, climate change and waste disposal issues which are necessitated the advancements in waste to energy technologies. The present study is directed towards evaluating the potential of microbial carbon capture cells (MCC) for simultaneous power generation, wastewater treatment and microalgal biomass production. Chlorella sorokiniana was used to establish the biocathode using anodic effluent as feed. The developed MCC was affected by physico-chemical parameters such as inlet pH, light intensity and photoperiod. The inoculum age of 12 h, inoculum size of 20%v/v, inlet pH 7.5, light intensity of 140 µmol m−2 s−1 and photoperiod of 12:12 (Light:dark) were asserted as most suitable conditions for achieving high performance of biocathode. The electricity generation was dependent upon the O2 availability at cathode and a drastic drop in voltage was observed under O2 limited conditions. Supplementation of anodic off-gas alone to cathode was not sufficient to sustain the growth of microalgae. However, combining internal anodic CO2 channelling with external CO2 pumping at cathode enhanced the performance of MCC. The experimental results revealed maximum open circuit voltage of 637 mV with a maximum power density of 2.32 W m−3. The maximum microalgal dry cell mass of 812 mg L−1 was achieved with an overall COD removal efficiency and energy recovery of 92–95% and 59%, respectively. These sustainability studies show that such a strategy can be applied for real time industrial flue gas treatment along with wastewater treatment in the future.

Development of homeothermic endothermy is delayed in high-altitude native deer mice (Peromyscus maniculatus).

Altricial mammals begin to independently thermoregulate during the first few weeks of postnatal development. In wild rodent populations, this is also a time of high mortality (50-95%), making the physiological systems that mature during this period potential targets for selection. High altitude (HA) is a particularly challenging environment for small endotherms owing to unremitting low O2 and ambient temperatures. While superior thermogenic capacities have been demonstrated in adults of some HA species, it is unclear if selection has occurred to survive these unique challenges early in development. We used deer mice (Peromyscus maniculatus) native to high and low altitude (LA), and a strictly LA species (Peromyscus leucopus), raised under common garden conditions, to determine if postnatal onset of endothermy and maturation of brown adipose tissue (BAT) is affected by altitude ancestry. We found that the onset of endothermy corresponds with the maturation and activation of BAT at an equivalent age in LA natives, with 10-day-old pups able to thermoregulate in response to acute cold in both species. However, the onset of endothermy in HA pups was substantially delayed (by approx. 2 days), possibly driven by delayed sympathetic regulation of BAT. We suggest that this delay may be part of an evolved cost-saving measure to allow pups to maintain growth rates under the O2-limited conditions at HA.

Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane

Methane is considered a next-generation feedstock, and methanotrophic cell-based biorefinery is attractive for production of a variety of high-value compounds from methane. In this work, we have metabolically engineered Methylomicrobium alcaliphilum 20Z for 2,3-butanediol (2,3-BDO) production from methane. The engineered strain 20Z/pBudK.p, harboring the 2,3-BDO synthesis gene cluster (budABC) from Klebsiella pneumoniae, accumulated 2,3-BDO in methane-fed shake flask cultures with a titer of 35.66 mg/L. Expression of the most efficient gene cluster was optimized using selection of promoters, translation initiation rates (TIR), and the combination of 2,3-BDO synthesis genes from different sources. A higher 2,3-BDO titer of 57.7 mg/L was measured in the 20Z/pNBM-Re strain with budA of K. pneumoniae and budB of Bacillus subtilis under the control of the Tac promoter. The genome-scale metabolic network reconstruction of M. alcaliphilum 20Z enabled in silico gene knockout predictions using an evolutionary programming method to couple growth and 2,3-BDO production. The ldh, ack, and mdh genes in M. alcaliphilum 20Z were identified as potential knockout targets. Pursuing these targets, a triple-mutant strain ∆ldh ∆ack ∆mdh was constructed, resulting in a further increase of the 2,3-BDO titer to 68.8 mg/L. The productivity of this optimized strain was then tested in a fed-batch stirred tank bioreactor, where final product concentrations of up to 86.2 mg/L with a yield of 0.0318 g-(2,3-BDO) /g-CH4 were obtained under O2-limited conditions. This study first demonstrates the strategy of in silico simulation-guided metabolic engineering and represents a proof-of-concept for the production of value-added compounds using systematic approaches from engineered methanotrophs.

Influence of organic carbon and nitrate loading on partitioning between dissimilatory nitrate reduction to ammonium (DNRA) and N2 production

Biologically available nitrogen is removed from ecosystems through the microbial processes of anaerobic ammonium oxidation (anammox) or denitrification, while dissimilatory nitrate reduction to ammonium (DNRA) retains it. A mechanistic understanding of controls on partitioning among these pathways is currently lacking. The objective of this study was to conduct a manipulative experiment to determine the influence of organic C and NO 3 − loading on partitioning. Sediment was collected from a location on the southern New England shelf (78 m water depth) and sieved. Half of the sediment was mixed with freeze-dried phytoplankton and the other half was not. Sediment was then spread into 1.5 mm, “thin discs” closed at the bottom and placed in large aquarium tanks with filtered, N 2 /CO 2 sparged seawater to maintain O 2 limited conditions. Half of the discs received high NO 3 − loading, while the other half received low NO 3 − loading, resulting in a multifactorial design with four treatments: no C addition, low NO 3 − (−C−N); C addition, low NO 3 − (+C−N); no C addition, high NO 3 − (−C+N); and C addition, high NO 3 − (+C+N). Sediment discs were incubated in the tanks for 7 weeks, during which time inorganic N (NH 4 + , NO 3 − , and NO 2 − ) was monitored, and sediment discs were periodically removed from the tanks to conduct 15 N isotope labeling experiments in vials to measure potential rates of anammox, denitrification, and DNRA. Temporal dynamics of inorganic N concentrations in the tanks were indicative of anoxic N metabolism, with strong response of the build up or consumption of the intermediate NO 2 − , depending on treatments. Vial incubation experiments with added 15 NO 2 − + 14 NH 4 + indicated significant denitrification and DNRA activity in sediment thin discs, but incubations with added 15 NH 4 + + 14 NO 2 − indicated anammox was not at all significant. Inorganic N concentrations in the tanks were fit to a reactive transport model assuming different N transformations. Organic C decomposition rates were inferred based on modeled rates as well as stoichiometric conversions of NH 4 + production in pre-incubated vials. Based on model results, partitioning between DNRA and N 2 production was positively linearly related to the ratio of C decomposition to NO 3 − reduction rates (C/NO 3 − ) but not C decomposition alone. Based on vial results, partitioning was significantly related to C decomposition. Overall, this study supports the hypothesis that high organic C loading is a prerequisite for DNRA to be favored over denitrification but that N 2 production may still be significant when organic C is high depending on NO 3 − availability.

Deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) induces simultaneous utilization of xylose and glucose in xylose-utilizing Saccharomyces cerevisiae.

Simultaneous co-utilization of xylose and glucose is a key issue in engineering microbes for cellulosic ethanol production. We coupled xylose utilization with glucose metabolism by deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) through pentose phosphate pathway flux. Simultaneous utilization of xylose and glucose then occurred in the engineered Saccharomyces cerevisiae strain with the xylose utilization pathway. Xylose consumption occurred at the beginning of glucose consumption by the engineered yeast without RPE1 in a mixed sugar fermentation. About 3.2 g xylose l(-1) was utilized simultaneously with consumption of 40.2 g glucose l(-1) under O2-limited conditions. In addition, an approximate ratio (~1:10) for xylose and glucose consumption was observed in the fermentation with different sugar concentration by the engineered strain without RPE1. Simultaneous utilization of xylose is realized by the coupling of glucose metabolism and xylose utilization through RPE1 deletion in xylose-utilizing S. cerevisiae.

Linking oxygen availability with membrane potential maintenance and K+ retention of barley roots: implications for waterlogging stress tolerance.

Oxygen deprivation is a key determinant of root growth and functioning under waterlogging. In this work, changes in net K(+) flux and membrane potential (MP) of root cells were measured from elongation and mature zones of two barley varieties under hypoxia and anoxia conditions in the medium, and as influenced by ability to transport O2 from the shoot. We show that O2 deprivation results in an immediate K(+) loss from roots, in a tissue- and time-specific manner, affecting root K(+) homeostasis. Both anoxia and hypoxia induced transient membrane depolarization; the extent of this depolarization varied depending on severity of O2 stress and was less pronounced in a waterlogging-tolerant variety. Intact roots of barley were capable of maintaining H(+) -pumping activity under hypoxic conditions while disrupting O2 transport from shoot to root resulted in more pronounced membrane depolarization under O2 -limited conditions and in anoxia a rapid loss of the cell viability. It is concluded that the ability of root cells to maintain MP and cytosolic K(+) homeostasis is central to plant performance under waterlogging, and efficient O2 transport from the shoot may enable operation of the plasma membrane H(+) -ATPase in roots even under conditions of severe O2 limitation in the soil solution.

Transformation and removal of arsenic in groundwater by sequential anodic oxidation and electrocoagulation

Oxidation of As(III) to As(V) is generally essential for the efficient remediation of As(III)-contaminated groundwater. The performance and mechanisms of As(III) oxidation by an as-synthesized active anode, SnO2 loaded onto Ti-based TiO2 nanotubes (Ti/TiO2NTs/Sb-SnO2), were investigated. The subsequent removal of total arsenic by electrocoagulation (EC) was further tested. The Ti/TiO2NTs/Sb-SnO2 anode showed a high and lasting electrochemical activity for As(III) oxidation. 6.67μM As(III) in synthetic groundwater was completely oxidized to As(V) within 60min at 50mA. Direct electron transfer was mainly responsible at the current below 30mA, while hydroxyl radicals contributed increasingly with the increase in the current above 30mA. As(III) oxidation was moderately inhibited by the presence of bicarbonate (20mM), while was dramatically increased with increasing the concentration of chloride (0-10mM). After the complete oxidation of As(III) to As(V), total arsenic was efficiently removed by EC in the same reactor by reversing electrode polarity. The removal efficiency increased with increasing the current but decreased by the presence of phosphate and silica. Anodic oxidation represents an effective pretreatment approach to increasing EC removal of As(III) in groundwater under O2-limited conditions.

Nudix Hydrolase Controls Nucleotides and Glycolytic Mechanisms in HypoxicAspergillus nidulans

Nucleoside diphosphates linked to moiety X (Nudix) hydrolase functions were investigated in hypoxic Aspergillus nidulans cells. Among three nudix hydrolase isozymes, NdxA transcription was up-regulated under oxygen (O2)-limited conditions. A gene disruptant of the NdxA-encoding gene (NdxAΔ) accumulated more NADH and ADP-ribose than the wild type (WT) under the same conditions. These results indicate that NdxA hydrolyzes these nucleotides in hypoxic fungal cells, which accords with the thesis that NdxA hydrolyzes NADH and ADP-ribose. Under O2-limited conditions, NdxAΔ decreased glucose consumption, the production of ethanol and lactate, cellular ATP levels, and growth as compared with WT. WT cultured under hypoxia converted exogenously added fructose 1,6-bisphophate, a glycolytic intermediate, to glyceraldehyde 3-phosphate (GAP). The hypoxic NdxAΔ cells accumulated 3.0- to 4.2-fold more GAP than WT under the same conditions, indicating that NdxA increased GAP oxidation by a glycolytic mechanism. Steady-state kinetics indicated that NADH and ADP-ribose competitively inhibited fungal GAP dehydrogenase (GAPDH) with Ki values of 34- and 55-µM, respectively. These results indicate that NdxA hydrolyzes cellular NADH- and ADP-ribose, derepresses GAPDH activity, and hence up-regulates glycolysis in hypoxic A. nidulans cells. That NdxAΔ consumed less pyruvate and tricarboxylate cycle intermediates than WT suggests that NdxA-dependent hydrolysis of nucleotides controls the catabolism of these carbon sources under O2-limited conditions.

Plant material addition affects soil nitrous oxide production differently between aerobic and oxygen-limited conditions

Abstract It is a common agricultural practice for crop residues to be plowed into the soil or left on the soil surface. Soil addition of crop residues can considerably modify soil microbial activity and net N mineralization, and in general such modifications are negatively related to the C:N ratios of crop residues. Yet, little is known on the impacts of crop residues of different C:N ratios on soil nitrous oxide (N 2 O) production under different aeration conditions via nitrification and denitrification. In this study, an 84-day laboratory incubation was conducted under aerobic and O 2 -limited conditions and soil N 2 O production was measured every 3 days after the addition of plant materials with a wide range of C:N ratios from 14 to 297. Two aerobic conditions were created by adjusting the water content of soil at a bulk density of 1.1 g cm −3 to 30% water-filled pore space (WFPS) and 60% WFPS, and two O 2 -limited conditions were made by 90% WFPS and fluctuation between 90% and 30% WFPS. Each fluctuation cycle lasted 9 days and soil water content was readjusted to 90% WFPS at the end of each cycle. We also measured microbial respiration activity and net N mineralization periodically (i.e., 3, 7, 14, 28, 42, 56, 70, and 84 days) during the incubation and microbial biomass C at the end of incubation. At aerobic conditions, soil amendments of plant materials, regardless of their C:N ratios, all enhanced soil N 2 O production. However, net N mineralization was dependent on plant material C:N ratios, being significantly higher or lower than the control for C:N ratios ∼15 and C:N ratios ≥44, respectively. Such inconsistent responses indicated that nitrifiers mediating nitrification and therefore byproduct N 2 O production could strongly compete with heterotrophic microbes for NH 4 + and therefore net N mineralization was not a good predictor for nitrification-associated N 2 O production. Interestingly, plant material additions reduced soil N 2 O production by up to ∼95% at O 2 -limited conditions, perhaps due to NO 3 − limitation. Soil NO 3 − production via nitrification could be low at O 2 -limited conditions, and soil NO 3 − availability could be further reduced due to increases in microbial biomass and thus microbial N assimilation after plant material additions. This NO 3 − limitation might enhance N 2 O reduction to N 2 , by which denitrifiers could harvest more energy from the consumption of limited NO 3 − . Nonetheless, our results revealed contrasting differences in N 2 O production between aerobic and O 2 -limited conditions following soil amendments of plant materials.

Antitubercular pharmacodynamics of phenothiazines

Phenothiazines have been shown to exhibit in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb) and multidrug-resistant Mtb. They are predicted to target the genetically validated respiratory chain component type II NADH:quinone oxidoreductase (Ndh). Using a set of compounds containing the phenothiazine pharmacophore, we have (i) investigated whether chemical validation data support the molecular target and (ii) evaluated pharmacophore tractability for further drug development. Recombinant Mtb Ndh was generated and its functionality confirmed by steady-state kinetics. Pharmacodynamic profiling of the phenothiazines, including antitubercular efficacy in aerobic and O2-limited conditions, time-kill assays and isobole analyses against first-line antituberculars, was performed. Potential mitochondrial toxicity was assessed in a modified HepG2 cell-line assay and against bovine cytochrome bc1. Steady-state kinetic analyses revealed a substrate preference for coenzyme Q2 and an inability to utilize NADPH. A positive correlation between recombinant Ndh inhibition and kill of aerobically cultured Mtb was observed, whilst enhanced potency was demonstrated in a hypoxic model. Time-kill studies revealed the phenothiazines to be bactericidal whilst isobolograms exposed antagonism with isoniazid, indicative of intracellular NADH/NAD(+) couple perturbation. At therapeutic levels, phenothiazine-mediated toxicity was appreciable; however, specific mitochondrial targeting was excluded. Data generated support the hypothesis that Ndh is the molecular target of phenothiazines. The favourable pharmacodynamic properties of the phenothiazines are consistent with a target product profile that includes activity against dormant/persistent bacilli, rapid bactericidal activity and activity against drug-resistant Mtb by a previously unexploited mode of action. These properties warrant further medicinal chemistry to improve potency and safety.

Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O(2), lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O(2) limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.