In order to identify aqueous species formed in Plasma activated media (PAM), quantitative investigations of reactive oxygen and nitrogen species  were performed and compared to Milli-Q water and culture media without and with Fetal Calf Serum. Electron paramagnetic resonance, fluorometric and colorimetric analysis were used to identify and quantify free radicals generated by helium plasma jet in these liquids. Results clearly show the formation of ROS such as hydroxyl radical, superoxide anion radical and singlet oxygen in order of the micromolar range of concentrations. Nitric oxide, hydrogen peroxide and nitrite-nitrate anions  are the major species observed in PAM. The composition of the medium has a major impact on the pH of the solution during plasma treatment, on the stability of the different RONS that are produced and on their reactivity with biomolecules. To emphasize the interactions of plasma with a complex medium, amino acid degradation by means of mass spectrometry was also investigated using methionine, tyrosine, tryptophan and arginine. All of these components such as long lifetime RONS and oxidized biological compounds may contribute to the cytotoxic effect of PAM. This study provides mechanistic insights into the mechanisms involved in cell death after treatment with PAM. It is noteworthy that plasmas can induce either cell proliferation for low doses or cell death by apoptosis for high doses of exposure5. Low temperature plasmas have therefore been studied intensively for wound healing6, sterilization7, blood coagulation4, dental treatment9 and also for the inactivation of various cancer cells from breast10, head and neck11, ovarian12, lung13, prostate14 or colorectal tissues17. It has been shown that plasma species can inactivate cancer cells either directly (interactions of gaseous species with cells) or indirectly when using a previously-prepared plasma-activated liquid media (PAM). In addition, such plasma treatments can selectively inactivate cancer cells without really affecting normal cells20. Interestingly, PAM have numerous advantages: i) they allow selective treatment of internal organ cancer tissues which are difficult to reach by the gaseous species and requiring endoscopes or catheters; ii) they present minimal toxicity for normal tissues; and iii) they remain stable several days after their preparation if they are stored at the right temperature21. In PAMs, reactive oxygen and nitrogen species (RONS) have been shown to induce cancer cell apoptosis12, although the cell death pathways at a molecular level have not yet been clearly elucidated, some studies have suggested mitochondrial dysfunction22. Identification and quantification of the aqueous RONS generated in PAMs could therefore shed light on the mechanisms of action of PAM with regard to tumor eradication and wound healing. We have already described the genotoxic and cytotoxic effects of PAMs on colon adenocarcinoma multicellular tumor spheroids and its selective action on cancer cells elsewhere20.Low temperature plasmas generated at atmospheric pressure have been studied in the past few years for their applications in the field of medicine and biomedicine. Short and long lived reactive oxygen (ROS) and nitrogen species (RNS) can be generated by non-thermal plasmas in either gaseous or aqueous forms when primary plasma species  interact with a liquid phase16, helium with oxygen14 or argon13. The nature and quantity of the plasma species generated depend on the type of plasma device used and the carrier gas composition. Hence, there is a real demand for the identification and quantification of the aqueous RONS  generated when the gaseous plasma species impact the liquid media. Several techniques are currently used to identify and quantify the gaseous plasma products, including optical emission spectroscopy (OES) or laser induced fluorescence25. However, aqueous plasma by-products with a short lifetime are difficult to quantify using OES26, and other methods including chemical dosimetry or fluorescent probes28 electron paramagnetic resonance (EPR) spectroscopy33 appear more appropriate. The later technique requires specific spin traps to allow detection of some aqueous plasma by-products31.Furthermore, most of plasma devices described in the literature which are used to generate PAMs are based on dielectric barrier discharge setups (DBD) using RF, AC or pulsed power supplies with different carrier gases such as helium26. These different liquid media are activated by the plasma jet using different exposure times. The RONS investigated are hydrogen peroxide (H2O2), hydroxyl radical (•OH), singlet oxygen (1O2), superoxide radical (O2•−), nitric oxide (NO•) and nitrite/nitrate anions (NO2/NO3). H2O2 was measured using a fluorometric kit and NO2/NO3 with a colorimetric kit, while the three other free radicals were quantified using EPR spectroscopy and spin traps due to their short life spans.The overall goal of this study is the analysis of several plasma-induced free radicals in three liquid media, i.e. Milli-Q water and a cell culture medium, DMEM, with and without fetal calf serum (FCS), which are exposed to a low temperature plasma jet generated by a DBD setup using helium carrier gas at atmospheric pressureN = aH = 15 G, g = 2.0056 and peak intensity ratio of 1:2:2:1  (data not shown). Under similar experimental conditions, this second signal was also been observed by Tresp et al.34 and was assigned to DMPO-CH3 adduct. DMPO-CH3 spin adduct can only originate from the spin trap itself since no carbon-based molecules are present in Milli-Q water. This assumption seems to be confirmed by the absence of correlation between the signal intensity and the exposure time of the media to the plasma jet.In order to characterize the formation of reactive oxygen species in different media such as Milli-Q water, a cell culture medium, DMEM, without or with fetal calf serum (FCS) upon exposure to cold plasma, spin trapping experiments were carried out using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as spin trap. Figure •OH radical can be formed by the reaction of an oxygen atom with an H2O molecule at the liquid surface39 or from the solvation of gaseous •OH produced in the gas phase of plasma giving rise to the production of aqueous •OH radicals. In a previous work, the presence of oxygen atom and OH radical species was validated by observing their specific radiation using emission spectroscopy15 This indicates that both pathways may be involved in our experimental conditions. This assumption is supported by the data published by Gorbanev et al.40 showing that with dry feed He plasma, OH radical may be produced both in the gas phase and in the sample.water = 0.99, R²DMEM = 0.98, R²DMEM + FCS = 0.92) indicating an increase of OH radical production. Kanazawa et al.27 have already shown similar linear relationship between the amount of OH radical in water and the time of exposure to He plasma jet. For this purpose, they used chemical dosimetry based on the reaction of terephthalic acid with OH radical to generate a fluorescent molecule27.The concentration of OH radical was assessed by exposing the different media with DMPO spin trap to cold plasma. As shown in Fig. 41. These oxidation reactions will reduce the quantities of OH radical trapped by DMPO. Chemical reactions between amino acids and OH radicals were predicted by reactive classical MD simulations42 and observed by high-resolution mass spectrometry43.The concentration of DMPO-OH is evaluated around 4.12 µM, 3 µM and 0.4 µM after 150 s exposure to He plasma in water and DMEM +/− FCS, respectively. The amount of DMPO-OH detected in water is higher than in both biological media. Indeed, OH radicals generated in water are surrounded only by water molecules and other types of radicals. In cell culture media, OH radicals can oxidize organic components such as amino acids, vitamins and proteins31. In DMEM +/−  FCS the half-life was about 1043 s and 442 s, respectively. The stability of the adduct DMPO-OH is affected by the change in pH and/or the presence of nitric acid44. The results suggest that DMEM makes it possible to maintain the pH constant at around 7.4 after exposure to plasma leading to an increase in the stability of the DMPO-OH adduct. In the presence of FCS, its stability is around 2 or 2.3 times lower than in water and DMEM respectively. This might be explained by the fact that the adduct can bind to proteins such as albumin which have documented anti-oxidant properties and/or by the presence of higher quantities of nitric acid than in water and DMEM as we will show in the section nitrite/nitrate anions of manuscript. Nitrite may react with OH radical to form peroxynitrite.We have estimated the half-life of DMPO-OH in the three media considered Milli-Q water and DMEM +/− FCS. To estimate the half-life of DMPO-OH, the signal decay of DMPO-OH was studied from 120 s to 2000 s after plasma exposure. The data were fitted with a polynomial profile of the 3rd order. The results summarized in Table OH > 109 M−1s−1 34) but can also scavenge superoxide anion to form the spin adduct DMPO-OOH which has a much lower reaction rate (kO2− < 102 M−1s−1 34). The difficulty lies in the fact that adduct DMPO-OOH tends to decompose rapidly in DMPO-OH, making it difficult to detect.DMPO used as a spin trap in EPR experiments can react with many ROS under different rate coefficients and trapping times. The EPR signal obtained after plasma exposure Fig.  of the d31 when exposed to plasma. In water, the addition of SOD leads to a decrease of about 10.2 ± 3.34% in the concentration of DMPO-OH adduct  and DBD plasma generated in humid ambient air. Superoxide anion is detected in water but in a much lower concentration than OH radical.To highlight the presence of superoxide anion in the media after plasma treatment, superoxide dismutase (SOD) was added before plasma exposure. Found in almost all aerobic organisms this enzyme is a metalloprotein that catalyzes the dismutation of superoxide anion radical into molecular oxygen and hydrogen peroxide. The addition of SOD (150 units of SOD per well) to the media leads to the dismutation of the superoxide anion and thus to inhibition of DMPO-OOH spin adduct formation2O2 is one of the main ROS produced by cold plasmas45. As proposed by Gorbanev et al., H2O2 is generated inside the plasma and is delivered into the medium40. In our previous work20, we showed that hydrogen peroxide produced in PAM remains stable when PAM is stored at 4 °C for at least 7 days. H2O2 may be considered to be a central genotoxic agent produced during the exposure of media to a He plasma jet. This activity may be attributed to its ability to diffuse through cell membrane and to generate OH radical through a Fenton reaction catalyzed by iron containing proteins. Hydroxyl radical is known for its high reactivity with cellular components, its potent ability to induce DNA damage such as guanine oxidations, DNA cleavage and to cause cell death.H2O2 by He plasma jet was characterized and quantified using a fluorometric Hydrogen Peroxidase Assay kit .The formation of H2O2 concentration in Milli-Q water and cell culture media increases linearly with the exposure time to the plasma jet . The linear increase in hydrogen peroxide concentration versus exposure time was also reported by Adachi et al. using an Argon plasma jet and a DMEM cell culture medium13. In the three different media, the hydrogen peroxide concentration can reach 1.60 mM ± 0.12 mM for 150 s of plasma exposure time. This is also very close to the concentration determined in our previous studies20. The H2O2 quantities found are similar to those obtained by Tresp et al.using argon plasma to treat saline solution, buffered saline solution and cell culture medium33. In contrast to OH radical and superoxide anion radical, the quantities of hydrogen peroxide produced do not depend on the type of the medium treated. This result suggests that H2O2 will not be consumed via a Fenton reaction in DMEM +/− FCS.As shown in Fig. 45. Wu et al. suggested that singlet oxygen was produced in plasma and diffused into the solvent31.It has been shown that singlet oxygen may be generated in water or cell culture media after the treatment of these liquid media by He plasma jet and Air DBD plasmaN = aH = 16 G, g = 2.0059. This signal, detected in the three media, is attributed to the TEMPO spin adducts indicating the formation of singlet oxygen or ozone upon exposure to the He plasma jet. In water, the amount of TEMPO increases linearly with the exposure time  , a selective scavenger of singlet oxyimen48 in water before plasma exposure induced a decrease of around 80% and 51% of the formation of TEMPO after 30 s and 150 s of exposure. This data clearly shows that the formation of TEMPO results from the oxidation of TEMP by singlet oxygen. Moreover, in our plasma jet configuration, with pure He, ozone cannot be detected along the plasma jet by using the UV absorption method at 253.7 nm. This indicates that ozone concentration is very low or negligible. This observation is also in good agreement with the investigations of Kawasaki et al.49. Both data show that involvement of ozone to the oxidation of TEMPO is negligible in our experimental conditions. The concentration of TEMPO produced in water was evaluated to be about 13.85 µM after 150 s exposure to the plasma jet. The intensity of the TEMPO spin adduct signal in both DMEM and DMEM + FCS is negligible when compared to the case of Milli-Q water. This may be explained by the fact the singlet oxygen is a very high reactive oxygen species known to oxidize cysteine, methionine and tryptophan free amino acids and proteins50. Singlet oxygen is consumed in both DMEM and DMEM with FCS. It is worth noting that singlet oxygen contributes to the formation of oxide radicals in PAM leading to a depletion of cellular nutrients. The formation of these oxidized by-products in DMEM +/− FCS may also contribute to the cytotoxic effects of PAM.In order to characterize the formation of singlet oxygen in the different media after plasma exposure, EPR experiments were performed using 2,2,6,6-tetramethylpiperidine (TEMP) as the spin trap. As shown in Fig. 88) Fig. . The addN = 16.2 G, aH = 10.3 G, g = 2.0776. It may be attributed to the PBN-H spin adduct. The PBN spin adduct of the •H atom was also observed with cold atmospheric dry-helium40 and argon32 plasma.Hydrogen radical was detected by EPR spectroscopy using α-phenyl-N-tert-butylnitrone (PBN) spin trap. The EPR signal obtained during plasma treatment of DMEM is shown in Fig. DMEM = 0.96, R²DMEM + FCS = 0.97). These results suggest that the He plasma jet produces a small quantity of hydrogen radical. This is in total agreement with the spectroscopy studies showing that the He plasma jet generate very small amounts of gaseous H15.Moreover, a small magnitude signal associated with PBN-H is also observed in Milli-Q water and cell culture media not exposed to the low temperature plasma jet . Figure N = 9.8 G, aH = 4.4 G, g = 2.0068. The presence of C-PTI in water after plasma exposure confirms the generation of nitric oxide.Nitric oxide was detected by indirect EPR spectroscopy using the Carboxy-PTIO spin trap that can react with nitric oxide to produce Carboxy-PTI and •NO cannot be estimated from the amount of C-PTI formed because C-PTI may be reduced by both hydroxyl and hydrogen radicals to the parent C-PTIO55. Nitric oxide in liquids arises after solvation of gaseous nitric oxide produced in the plasma plume observed using emission spectroscopy in our earlier study15. Production of nitric oxide in liquids has already been observed in Milli-Q water after its exposure to helium56 or argon plasma35 and also in buffered saline solution treated with air plasma58. In aqueous solution, nitric oxide is a highly reactive species that may react with oxygen to produce nitrite (NO2−) (Reaction 1)59. Formation of NO2− in media is known to induce a decrease in the pH of the solution.As shown in Fig. 62. Nitrous acid, which is in acidic equilibrium with nitrite, may decompose in acidic medium into nitric oxide and nitrogen dioxide (Reactions 2 and 3)58. It is also known that nitrite is not stable under acidic conditions.As shown in Fig. via reaction 3. In contrast, in cell culture media the amount of NO radical detected is very low. Due to the presence of a buffer, for example phosphate buffer at pH 7.4 in these media, the change of pH will be very slight upon plasma exposure. In phosphate buffer, the pH varies by less than one pH unit62 At physiological pH conditions, nitrite will be more stable and subsequently reactions 2 and 3 will not occur or at least be limited in DMEM +/− FCS. This may explain the low concentration of NO in both cell culture media compared to water.In water, nitric oxide may be regenerated 63. NO radicals and related species are able to modify proteins through chemical reactions without involving enzymes. Nitric Oxide groups can bind to a transition metal found in protein or thiol residues of amino acids such as cysteine64. Tyrosine is one of the main target of RONS65. Nitric oxide can also provide nitrogen sources for the formation of the nitro group (NO2). The presence of proteins and amino acids in culture medium can explain the low nitric oxide concentration detected in DMEM +/− FCS.Another possible explanation for the low concentration of NO in biological liquids could be the high reactivity of this radical with bio-macromoleculeswater = 0.92, R²DMEM = 0.97, R²DMEM+FCS = 0.97). The nitrite concentration in DMEM with fetal calf serum is 2.87 ± 0.47 and 18.77 ± 2.26 times higher than in DMEM and in Milli-Q water respectively.The formation of nitrite and anions during exposure of the different media to a plasma jet was investigated and quantified. The concentration of nitrite in Milli-Q water and culture media Fig.  increaseIt is more difficult to quantify the nitrate anion concentration. Figure Moreover, the negligible values obtained during anion quantification may be due to the non-linearity of the calibration curves  that increased significantly due to the dilution factor.2−) is not stable. It will decompose rapidly into nitrogen dioxide which may subsequently react with hydroxyl radicals produced by the He plasma jet. This chemical reaction leads to the formation of peroxynitrous acid which is not stable in acidic pH and converts into stable nitrate NO3−. Lukes et al.59 have shown that, under acidic conditions, nitrite will react with hydrogen peroxide to generate peroxynitrite and subsequently nitrate anion. Girard et al.62 have clearly identified the formation of peroxynitrite anion in physiological pH under cold atmospheric plasma exposure. This cascade of chemical reactions may explain both the higher level of nitric oxide and the lower quantity of NO2− in water than in DMEM +/−  FCS. These reactions will be less efficient in buffered media with a pH of around 7. Hence, the nitrite concentration in DMEM +/−  FCS will be higher than in water. Moreover nitrate/nitrite anions can be the targets of short lifetime ROS such as hydroxyl radical66 leading to the formation of peroxynitrite. In complex media such as DMEM +/− FCS, this reaction will compete with the oxidation of biomolecules by ROS. However, the production of peroxynitrite in biological media via this pathway will be minor. Also, the higher concentration of nitrite observed in DMEM + FCS than in DMEM may be explained by the presence of copper proteins such as cytochrome c in FCS that can contribute to the oxidation of nitric oxide into nitrite68.Nitrite and anions concentrations in PAM seem to be affected by the composition of the different media. Both nitrogen species are formed in plasma treated media through the dissolution of nitrogen oxides formed in plasma jet. Their formation and their stability in water and DMEM +/−  FCS will depend on different parameters such as the pH of the solution, the presence of amino acids, metallo-proteins and the production of ROS by the He plasma jet. As mentioned above, we have observed in water a drastic decrease in pH, about 2 units, after 150 s exposure to He plasma. In these acidic conditions, nitrous acid  are known to have both deleterious and beneficial effects on cell dynamics70. Interestingly, the RNS produced by cold atmospheric plasma jets and the quantity produced seem to induce the death of cancer cells in particular offering interesting selectivity between healthy and cancerous cells70.It has been shown that nitrate and nitrite anions can be recycled in NO in cellset al.43. Figure Oxidation of aqueous solutions of tyrosine, tryptophan, methionine and arginine after He plasma treatment were studied. The oxidized products were analyzed by HPLC coupled with mass spectrometry (HPLC-QTrap 4500-MS). In addition to the peaks corresponding to the reactants, the mass chromatograms of solutions exposed to plasma showed peaks corresponding to hydroxylation and nitration of tyrosine and tryptophane, sulfoxidation of methionine and hydroxylation of arginine  exposed to a DBD plasma jet using helium at atmospheric pressure as a carrier gas. We have shown that the composition of the medium has a major impact on the pH of the solution during plasma treatment, on the stability of the different RONS that are produced and on their reactivity with biomolecules. Reactions of RONS with DMEM +/− FCS generate oxidized products which may be toxic for cells. Our data indicate that beside the production of long lifetime RONS, oxidized biological compounds form and accumulate in PAM leading to a decrease in essential nutrients for cell growth. All of these components such as long lifetime RONS and oxidized biological compounds may contribute to the cytotoxic effect of PAM previously observed on HCT116 spheroids20. Moreover, this suggests that the cytotoxicity of OH radical produced by He cold plasma jet can be mainly due to the production of cytotoxic chemicals in DMEM +/− FCS rather than a direct effect on cellular constituents. Moreover, long lifetime species (H2O2 and nitrites/nitrates) can penetrate into cells and can be potential precursors of intracellular reactive oxygen species. Indeed, these species can lead in turn to the formation of •OH radical via a Fenton reaction involving H2O2 and NO• synthesis via recycling of nitrites/nitrates anions by the cells56. •OH radical and NO• are highly cytotoxic and genotoxic for cells. Therefore, under the present plasma exposure conditions, the obtained PAM can necessarily generate many potential cytotoxic and genotoxic by-products which have interesting applications, particularly for cancerous cell inactivation.We have characterized and quantified the formation of radical species such as hydroxyl radical, superoxide anion, singlet oxygen, nitric oxide and long lifetime RONS such as H71. In short, two aluminum tape electrodes are wrapped around a quartz tube and connected to a High-voltage mono-polar square pulses generator. A power supply with the following characteristics is applied: 10 kV voltage, 9.69 kHz frequency and 1 µs pulse duration. Helium gas flows through the quartz tube at a flow rate of 3 L min−1.Figure Plasma activated medium (PAM), was produced by exposing 100 µL of water or cell culture medium DMEM with or without fetal to the He plasma jet. These different media were exposed for up to 150 s in 96 well plate, leading to a 20 µl decrease in volume after plasma exposure (data not shown). Plasma exposures were performed under the same experimental conditions  and at the same distance of 2 cm between plasma jet tube output and the upper-surface of the liquid medium.40.Electron paramagnetic resonance spectroscopy (EPR) is a technique based on the magnetic resonance between an unpaired electron and an external magnetic field. Due to the very short lifetime of radicals, EPR measurement is often used with spin-trap reagents. The reaction of a radical with the spin-trap reagent leads to the formation of a longer-lived spin adduct. EPR spectra were recorded with a Bruker ESP 500E spectrometer at room temperature. The following instrumental settings were employed for the measurements: central field: 3516 G; sweep width: 100 G; microwave frequency: 9.87 GHz; modulation frequency: 100 kHz; microwave power: 5.15 mW; scanning time: 84 s; number of scans: 6. Figure 3) were purchased from Sigma-Aldrich.The spin trapping reagents, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 2,2,6,6-tetramethylpiperidine (TEMP), α-phenyl-N-tert-butylnitrone (PBN), 2-(4-Carboxyphenyl)−4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide potassium salt (C-PTIO), D-Mannitol (OH* scavenger), and superoxide dismutase (superoxide anion scavenger) Sodium azide (NaN•OH) superoxide anion (02•−), singlet oxygen (1O2), nitric oxide radical (NO•) and others including hydrogen radical (H•) are presumed to be produced in the different media upon their exposure to plasma. To characterize their formation, the spin trapping reagents DMPO (252 mM), TEMP (2 mM), PBN (21.6 mM) and C-PTIO (833 µM) were added to the different samples immediately before plasma treatment. All samples were transferred in to glass capillary tube (50 µL) immediately after plasma exposure.Reactive oxygen species such as hydroxyl radical , enzyme catalyzing the conversion of O2•− into H2O2 and O2 and directly related to the intensity of the DMPO-OH EPR signal. The DMPO-OH intensity was estimated directly from the half-height of the second peak of the quartet EPR spectra obtained for different media.To confirm the formation of O72 and WinSim2002 software were used for EPR spectra simulations [available online https://www.niehs.nih.gov/research/resources/software/tox-pharm/tools].All measurements were performed in triplicate for each sample and the changes in the EPR signal were monitored for 2000 seconds after plasma exposure. Easyspin (MATLAB library)2O2 concentrations in PAM were quantified using a fluorometric Hydrogen Peroxidase Assay kit . This kit uses horseradish peroxidase and a red fluorescent peroxidase substrate  and allows quantification of H2O2 in a range of concentrations between 0 and 10 µM. PAM were diluted 1/50 before each measurement in order to achieve an adequate concentration of H2O2. Calibration curves  using Griess reagent and nitrite reductase. The kit was initially planned for measurements in a range of concentrations between 0 and 100 µM of NO2− and NO3−. PAM were diluted 1/20 before each measurement in order to have an adequate concentration. Calibration curves  at room temperature.NOsee Fig.  were plo−1 rising at 15 min a plateau corresponding to 50% acetonitrile and 50% ammonium acetate for 5 minutes. The mass spectrometer was equipped with an electrospray ion (ESI) source (turbo ion spray (TIS) and was operated in positive mode. Nitrogen served as auxiliary, collision gas, and nebulizer gas. The detection was scan mode with a step size of 0.1 atomic mass unit (amu) and a scan range of 50–500 amu. Mass chromatograms, i.e., representations of mass spectrometry data as chromatograms , were registered using different scan ranges.The LC/MS system was equipped with an HPLC chromatograph (HPLC Agilent 1100 series) and a triple quadrupole mass spectrometer (QTRAP Applied Biosystems). HPLC analyses were performed using a Waters X Bridge C18 (3.5 µm) column (2.1 × 150 mm), and a gradient elution starting with 5% acetonitrile and 95% ammonium acetate at a flow rate of 0.6 mL minThe prepared concentration of each amino acids  was 0.2 mM in MilliQwater. Each solution was treated by He plasma at various time of exposure (0–240 s).Supplementary Data


Thiothrix species is responsible for sludge washout and loss of performance in dairy wastewater treatment plants. A long-term study was conducted over 1.5 years to test three different mitigation strategies in a full-scale plant composed of two parallel sequential batch reactors (SBR1 and 2). Strategies based on polyaluminium chloride addition and volatile fatty acids reduction were ineffective to permanently solve the problem. On the contrary, modification of the reactor cycle based on the implementation of a periodic starvation proved efficient to solve the biomass wash-out and drastically reduce the sludge volume index in both reactors. Bacterial diversity analysis using 16S amplicon sequencing and quantitative PCR indicated a reduction of Thiothrix abundance from 51.9 to 1.0% in SBR1 and from 71.8 to 0.6% in SBR2. Simultaneously, the abundance of the glycogen-accumulating bacterium Candidatus Competibacter increased in both reactors. Microscopy analysis confirmed the transition between a bulking sludge towards a granular-like sludge. This study confirms the applicability of a periodic starvation to (1) solve recurring Thiothrix bulking, (2) convert loose aggregates into dense and compact granular-like structures and (3) considerably reduce energy demand for aeration.Filamentous bulking caused by  Thiothrix thrive in wastewaters characterized by high organic loading rates, high concentrations of low molecular weight fatty acids and reduced sulfur compounds, low dissolved oxygen (DO) concentrations and nutrient deficits3.Filamentous bulking is a major cause of activated sludge process deficiency. It arises from an overgrowth of filamentous organisms and induces important losses of biomass that considerably reduce the removal efficiency of suspended solids, chemical oxygen demand (COD) and nutrients. The challenge in preventing filamentous bulking resides in the large diversity of filamentous organisms and their specific ecology. In particular, filamentous bacteria phylogenetically related to the gamma-proteobacterium 4. Butter production releases wastewaters with particularly high concentrations of fats, contributing to a high COD content5 while ice-cream wastewaters tend to have a lower, more biodegradable, COD content6. Since oxygen consumption in the treatment plant is often kept to a minimum by most industrials to reduce energy costs, COD-rich dairy wastewaters frequently lead to the growth of filamentous bacteria7. Additionally, unbalanced ratio of nitrogen, phosphorus and readily biodegradable COD (rbCOD) is susceptible to favor the overgrowth of Thiothrix species.Dairy effluents are characterized by high concentrations of lactose, fats and proteins that contribute to high COD contents. Wastewaters from cheese manufacturing factories have the highest COD concentrations and often present a deficit in nitrogen2O2, iron or aluminium salts) to kill indistinctively microorganisms protruding from the flocs, including filamentous microbes. They often prove unsatisfactory when the bulking episodes are recurring due to their transient effect on the activated sludge ecosystem. Specific methods include procedures that modify the conditions encountered in the reactor by the biomass to create a hostile environment for the targeted filamentous organisms. Although these strategies require a preliminary identification of the microbial species involved in the bulking event, they are usually preferable as they can permanently solve the process dysfunction. A common setup in wastewater treatment plants consists in installing an anaerobic plug-flow selector ahead of the biological treatment to provide the biomass with a high food-to-microorganisms ratio. This system is meant to favor microorganisms having high rate kinetics for substrate uptake and a metabolic ability to accumulate internal storage products8. Ideally, the anaerobic selector must be followed by a fully aerated reactor performing a complete substrate oxidation to give an ecological advantage to microorganisms holding storage products. The presence of an anaerobic tank ahead of a completely mixed aerobic reactor was previously reported to limit/suppress Thiothrix-caused bulking in dairy wastewater treatment plants9. While Thiothrix remained physiologically active under prolonged anaerobic conditions, it seemed that its growth was limited in such environments10.Strategies to control filamentous bulking can be either unspecific or specific. Unspecific methods involve the use of chemicals  Candidatus Accumulibacter and the glycogen accumulating organism (GAO) Candidatus Competibacter. These accumulating bacteria are particularly interesting since they contribute to increase the overall density of the sludge  by building dense internal inclusions of glycogen, polyhydroxyalcanoate (PHA) or polyphosphate13.Process configurations involving successive anaerobic and aerobic reactors are restricted to continuous flow systems but the operational scheme can be implemented in sequential batch systems by alternating anaerobic and aerobic phases. In sequential batch reactors (SBRs), this strategy is commonly named the “feast and famine” regime or “periodic starvation” and is specifically used in aerobic granulationThiothrix-based filamentous bulking. The plant was composed of two parallel SBRs fed by an influent pre-treated in a dissolved air flotation (DAF) unit. The sludge densification was achieved by applying a feast-and-famine regime in both SBRs. Thiothrix abundance was followed by molecular tools and the overall performances of the plant were followed over a period of 1.5 years.Based on these previous lab-scale experiments, the present study aimed to implement a sludge densification strategy in an industrial wastewater treatment plant treating dairy wastewaters and experiencing a recurring Thiothrix species was experienced by a wastewater treatment plant (WWTP) treating the process water from the dairy factory Luxlait . These events were responsible for an increase of the total suspended solids (TSS) in the effluent. The operator adapted the plant with a drum filter downstream from the biological stage  and total phosphorus (TP), respectively  around day 60 but the initial settleability of the sludge was regained at around day 100. A microscopic observation of the sludge revealed a lesser amount of interconnecting filaments and the presence of small floccular aggregates  on both SBRs and lasted until day 63. During the whole period, the SBRs were fed aerobically during 2 h  combined with a high aerobic fill time ratio (FTR) and a relatively low DO (1.4 to 4.0 mg L−1). The possible ways of improvement were limited: the influent composition could not be easily adapted, a significantly lower aerobic FTR would involve costly capital expenditure  and the DO in the SBRs had to remain low to avoid excessive operational expenditure. VFA were hypothesized to arise from a hydraulic retention time (HRT) of around 15 h in the buffer tanks upstream from the biological treatment that caused the development of acidogenic bacteria. Consequently, the strategy adopted was the installation of an aerator in the buffer tanks upstream from the SBRs to slightly increase the DO. This modification was applied from day 166 until day 384. Although the average VFA load decreased from 0.144 to 0.092 kg CODVFA m−3 d−1, it remained sufficient to favor the overgrowth of Thiothrix, which uses readily biodegradable substrates14. According to the “kinetic selection” theory15, filamentous microorganisms are characterized by a maximum growth rate (μmax) and affinity constant (Ks) lower than the floc-forming microorganisms. Consequently, the low CODVFA concentration (10 ± 8 mg O2 L−1) during the aerobic feeding phase led to a higher specific growth rate for Thiothrix compared to floc-forming bacteria. The sludge SVI from SBR2 increased steadily over the first 116 days of operation until it reached 950 ml g−1 . At day 282, the sludge was dominated by filamentous organisms that reduced the bulk density  under anaerobic conditions16.It was therefore decided to change the approach by modifying SBR2 cycle while maintaining the aeration in the buffer tanks. The feed was provided under unaerated conditions (feast) during 95 min followed by an extended aeration period of 210 min (famine). This procedure was intended to avoid VFA consumption by −1 and the same cycle modification was also applied to SBR1. After two weeks, this feast and famine regime was able to maintain the residual concentration of CODVFA and soluble COD (mainly soluble substrates such as lactose) at the end of the anaerobic phase below 1 mg O2 L−1 and 40 mg O2 L−1, respectively. This feeding pattern improved the SVI of SBR2 from 950 to 200 mL g−1 in 90 days and the biomass concentration stabilized around 3.5 g L−1  while the TSS slightly increased to reach 3.4 g L−1. The settleability of the sludge from both reactors was maintained and SVI peaks were no longer observed until the end of the experiment. The aeration in the buffer tanks was stopped at day 385 to verify the stability of the sludge despite a potential installation of acidogenic metabolisms upstream from the biological treatment (Strategy IIIb). The SVI and biomass concentration remained unchanged in both reactors, which proved that the feast-famine regime was sufficient by itself to solve the bulking event. To highlight additional benefits of the densification strategy, the drum filter unit was by-passed from day 418 to day 461. Interestingly, the removal efficiencies for total COD, TN and TP remained stable at 96 ± 3%, 88 ± 7% and 93 ± 7%, respectively . Although fed with wastewater coming from the same buffer tank, the distribution of the bacterial phyla in the reactors was different . The overrepresentation of this phylum was mostly due to the abundance of Thiothrix-related OTUs (71.8%). More generally, the variation of Proteobacteria abundance over time in both reactors was mainly driven by modifications of Thiothrix abundance , the relative gene frequency of both species reached a total of 71.8% in SBR2  was observed around day 349 but its concentration quickly regained a low and stable value until the end of the experiment. The densification strategy had a similar effect on SBR1. The relative gene frequency of genus Thiothrix steadily declined shortly after the beginning of Strategy III from 51.9 to 1.0% between days 362 and 413 20. Most common GAO reported in the literature were the gamma-proteobacterium Candidatus Competibacter and the alpha-proteobacterium Defluviicoccus22. Although Defluviicoccus was initially present with a similar gene frequency as Ca. Competibacter on day 278 in SBR1 (0.5% for both genera) and SBR2 (0.3 and 0.5% for Ca. Competibacter and Defluviicoccus respectively), the gene frequency of Ca. Competibacter became dominant soon after the beginning of the densification strategy  to 4.4% (day 376). In SBR2, an increase from 0.7 to 5.7%  to 0.1% (day 313) despite a status quo in GAO abundance. It should be noted that the drop of Ca. Accumulibacter abundance was observed at the same time in both reactors  convert loose aggregates into dense and compact granular-like structures and (3) considerably reduce energy demand for aeration.In conclusion, this long-term full-scale study confirms the applicability of a periodic starvation to (1) solve recurring 3). During production, a second buffer tank (200 m3) received a continuous flow (~50 m3 h−1) from a DAF unit that removed about half of the COD content. The stream was then treated with two conventional SBRs operated in parallel . The pre-treated wastewater characteristics are given in Table −1. The designed sludge loading rate was 0.2 kg COD kg−1 TSS d−1. Before being discharged in the receiving watercourse, the biologically treated wastewater was post-treated with a drum filter to keep the TSS concentration below the required limits.The investigated full-scale WWTP treated the process water of a dairy factory Fig. . The ran12, the other specific strategy (Strategy III) investigated the impact of the SBR feeding pattern by implementing either an aerated fill or an anaerobic fill. This approach explored the feasibility to select floc-forming microorganisms able to convert VFA into storage biopolymers, notably phosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) by applying an extended anaerobic feeding mode, which enabled lysis and fermentation of particulate organic substrates into VFA.In 2015, both SBRs frequently experienced losses of activated sludge due to filamentous bulking sludge phenomena. An experimental approach was set-up and divided into three mitigation strategies (Table 30) were determined by reading the volume of the settled bed in a column after 30 min settling and calculated from the dry weight in mixed liquor suspended solids (MLSS). An additional dilution step of MLSS was performed prior to settling if necessary. Microscopic observations were performed with a Leica photonic microscope. The morphology of filaments and flocs was evaluated on a week-to-week basis.Reactor performances were recorded on a daily (working day) basis by sampling the wastewater after the DAF treatment and after the drum filter polishing unit Fig. . Phospha® DNA isolation kit (MoBio Laboratories) following manufacturer’s instructions. qPCR analysis were performed using primers targeting 16S rRNA gene from Ca. Accumulibacter (517 F and PAO846)26 and Thiothrix eikelboomi (21Nf and 21Nr)27. For each analysis, a 7-point decimal calibration curve was established with solutions containing a known concentration of the amplicon generated by end-point PCR with the same primer pair. DNA concentration of the purified PCR product was measured with the QuantiFluor® dsDNA System (Promega) and was subsequently converted in quantity of fragments per unit of volume. Samples were measured in duplicates. qPCR was conducted on a CFX Connect Real-Time PCR Detection System (BioRad). Volumes of 20 μl of reaction mix were prepared with 10 μl GoTaq® qPCR Master Mix (Promega), 0.3 μM of both primers and 1 μl of DNA extract. The qPCR program was constructed with an activation step of 2 min at 95 °C, followed by 40 cycles of denaturation for 15 s at 95 °C and annealing/extension for 1 min at 60 °C. A melt curve was performed immediately after the last amplification cycle by increasing the temperature by 0.5 °C every 5 s up to 95 °C. Data processing was performed on CFX Manager 3.0 software (BioRad). Target-specific 16S rRNA gene quantification was expressed as number of copies of 16S rRNA operon per mg TSS.Quantitative PCR (qPCR) were performed on sludge collected in SBR1 and SBR2 on a regular basis . The number of reads per sample ranged from 16 570 to 61 540. The forward primer was constructed with the Illumina i5 adapter, an 8–10 bp barcode and primer 28 F (5′-AGTTTGATCNTGGCTCAG-3′). The reverse primer was constructed with the Illumina i7 adapter, an 8–10 bp barcode and primer 388 R (5′-TGCTGCCTCCCGTAGGAGT-3′). Amplifications were performed with Qiagen HotStar Taq master mix and 0.25 μM of each primer. Reactions were performed on ABI Veriti thermocyclers (Applied Biosytems) under the following thermal profile: 95 °C for 5 min, then 35 cycles of 94 °C for 30 sec, 54 °C for 40 sec, 72 °C for 1 min, followed by one cycle of 72 °C for 10 min and 4 °C hold. Amplification products were pooled equimolar. Each pool was size selected using Agencourt AMPure XP (BeckmanCoulter) in a 0.7 ratio for both rounds. Size selected pools were quantified using the Qubit 2.0 Fluorometer (Life Technologies) and loaded on an Illumina MiSeq 2 × 250 flow cell at 10 pM.28. Reads were trimmed and sorted from longest to shortest. Then, a prefix de-replication was performed using the USEARCH algorithm29. A clustering at a 4% divergence was performed using USEARCH clustering algorithm. Singleton clusters were removed at this stage. Operational Taxonomic Unit (OTU) selection was performed with the UPARSE OTU selection algorithm30 to classify clusters into OTUs. Chimera checking was performed using the UCHIME chimera detection software31 executed in de novo mode. Each centroid was mapped to its corresponding OTU and tagged as chimeric or non-chimeric. USEARCH global alignment algorithm29 was then used to map each read to its corresponding non-chimeric cluster. Finally, a correction step was performed between each sequence of a cluster and the consensus sequence of that cluster. Sequences were then matched against a database of high quality sequences derived from NCBI. The sequence data have been submitted to the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) under the accession number SRP095156. The abundance of taxa was expressed as “relative gene frequency”. This parameter corresponded to the number of reads of a given taxon in a sludge sample divided by the total number of reads in the same sludge sample (expressed in percent).Forward and reverse reads were merged together using the PEAR Illumina paired-end read mergerSupplementary material


Here we apply for the first time wavelet analysis to the PSII RC 2DES data to obtain time-resolved 2D frequency maps. These maps allow us to demonstrate that (i) coherence between the excitons initiating the two different charge separation pathways is active for more than 500 fs, and (ii) coherence between exciton and charge-transfer states, the reactant and product of the charge separation reaction, respectively; is active for at least 1 ps. These findings imply that the PSII RC employs coherence (i) to sample competing electron transfer pathways, and ii) to perform directed, ultrafast and efficient electron transfer.Experimental/theoretical evidence for sustained vibration-assisted electronic (vibronic) coherence in the Photosystem II Reaction Center (PSII RC) indicates that photosynthetic solar-energy conversion might be optimized through the interplay of electronic and vibrational quantum dynamics. This evidence has been obtained by investigating the primary charge separation process in the PSII RC by two-dimensional electronic spectroscopy (2DES) and Redfield modeling of the experimental data. However, while conventional Fourier transform analysis of the 2DES data allows oscillatory signatures of vibronic coherence to be identified in the frequency domain in the form of  The energetic disorder arises from fast nuclear motions of all atoms in the system (dynamic disorder) as well as from slow collective protein motions (static disorder). The static disorder plays a central role in the PSII RC dynamics because it generates a variety of different protein conformations within the sample ensemble, that is, a collection of energetically different RC complexes. From all possible realizations of the disorder (protein conformations) one may expect realizations optimal for ultrafast and efficient charge separation as well as non-optimal realizations that may lead to unwanted energy losses. Nevertheless, the near-unity charge separation quantum efficiency in the PSII RC implies that the system is able to overcome energetic disorder.In oxygenic photosynthesis the site of solar-energy conversion, the photosystem II reaction center (PSII RC), is a membrane-bound pigment-protein complex that converts solar photons into a stable charge-separated state which, in turn, creates an electrochemical potential across the membrane. Remarkably, charge separation in the PSII RC takes place with near-unity quantum efficiency despite the intrinsically highly disordered energy landscape of this complexa (Chl) and two pheophytin a (Phe) molecules arranged in two quasi-symmetric branches (D1 and D2) in the center of the complex as well as two chlorophylls and two β-carotenes located at its periphery4 . In this scenario, charge separation proceeds via two different routes, the ChlD1 and PD1 pathways9, which provide functional flexibility to the complex. The two pathways are:The PSII RC contains four chlorophyll ranches D and D2 iD1δ+PheD1δ−)*≈681nm and (PD2δ+PD1δ−ChlD1)*≈675nm/(PD2+PD1−)δ*≈690nm, via the ChlD1 and PD1 paths, respectively10 *≈675nm is small, in the following we refer to (PD2δ+PD1δ−)*≈675nm for simplicity. Note that (PD2δ+PD1δ−)*≈675nm has a high (PD2PD1≈660nm and a low (PD2δ+PD1δ−≈675nm energy component and that (PD2+PD1−)δ*≈690nm is a CT state with exciton character (CT-exciton)7  26 and Fourier transform (FT) analysis allowed to visualize the coherence between electronic states in 2D frequency maps [obtained by Fourier transformation of the real rephasing 2D spectra along the population time (T)]. These maps display features corresponding only to the states oscillating at a certain frequency and, therefore, are more selective than the 2D spectra. Moreover, two main 2D frequency maps characteristics: (i) the oscillation frequency (in or out of resonance with the electronic states energy gaps), and (ii) the position of the most intense bands (corresponding or not to the states in resonance with the oscillation frequency); provide an invaluable tool to classify the observed coherences as mainly electronic, mixed electronic-vibrational (vibronic) or mainly vibrational28. We found that three main frequency modes, 120, 340 and 730 cm−1, representative of mainly electronic, mixed electronic-vibrational (vibronic) and mainly vibrational coherence, respectively, dominate the oscillatory dynamics13. The first two types of coherences have functional significance and will be analyzed and discussed below, the non-functional vibrational coherence will be analyzed and discussed in the Supplementary Information *≈675nm and (ChlD1δ+PheD1δ−)*≈681nm] δ*≈690nm] δ*≈690nm, strongly suggesting that charge separation in the PSII RC proceeds via a coherent mechanism12.Recently we have demonstrated that vibration-assisted electronic (vibronic) coherence is at play in the PSII RC charge separation process at physiological temperatureion Figs  and S2. T. For that reason, here we investigate the dynamics of coherence during the process of charge separation in the PSII RC by applying a novel analysis of 2DES data whose advantages have only very recently been demonstrated30: Wavelet Transform (WT)32. The WT technique is a powerful tool to investigate coherent dynamics in multichromophoric systems due to the fact that it combines the frequency and time information available from 2DES.However, even though the 2D frequency maps provide crucial information about coherences between electronic states, these maps are a static representation due to the fact that the time information has been exchanged by frequency information after FT of the 2D spectral traces along 26 experiment is a tool to visualize the existence of strongly coupled pigment chains by the coherent excitation of the corresponding electronic states. If any pair of states contains a mixing of pigments, such as the exciton-CT states presented above, their coherent excitation will generate dynamic coherence visualized as amplitude oscillations in the 2D traces. In this manner the dynamic coherence between the states created by the coherent broadband and ultrashort laser excitation employed in the 2DES experiment highlights the inner coherence (that is the coherent mixing of the pigments within states)13. It is important to realize that the inner coherence is an intrinsic property of the system, and therefore, its presence is consistent with directed and efficient energy and electron transfer also upon non-coherent excitation, specifically in natural photosynthesis under sunlight illumination33.At this point, it is important to emphasize that the 2DESMethods section). Briefly, CWT allows to extract both the frequency and time information contained in 2DES experimental data, however, a compromise between frequency and time resolution must be found; extremely high frequency resolution will result in poor time resolution and vice versa . The 2Dion Fig. . Accordi−1time-resolved 2D frequency maps at 80 K obtained by CWT are shown in Fig. D2PD1≈660nm and  nm cross-peak [coherence between (PD2δ+PD1δ−≈675nm and (PD2+PD1−)δ*≈690nm]12  for both the  nm and  nm cross-peaks. Therefore, the obtained interfering frequencies for the below diagonal cross-peaks are: 340–70 = 270 cm−1 and 340 + 70 = 410 cm−1. Not surprisingly, the calculated interfering frequencies, that is: 270 and 410 cm−1, are close to some of the closely spaced beating frequencies experimentally observed and present in the 2D spectral data12: 265 and 440 cm−1. This correspondence indicates that, at least in part, the observed dynamics may arise from interference effects rather than entirely from vibronic dynamics.The 340 ± 20 cm]12 Fig. . The cro−1 2D frequency map at 80 K  nm 2D trace in the 340 cmiod Fig. . These rIn relation to the CWT analysis, the windowed Fourier transform analysis shows that the interference artefact is not introduced by the CWT analysis, the interference artefact is a consequence of the compromise between frequency and time resolution that needs to be made when performing the CWT analysis (for details see the Methods section).real dynamics and the interference artefact, we have performed a simulation of the time evolution of the real rephasing 2D spectra for a single optical excited state linearly coupled to modes of frequency 120, 190, 265, 340 and 440 cm−1 12. The 2D signal was generated using the nonlinear response function formalism presented previously41, assuming the impulsive limit for the laser excitation. The microscopic model consists of a single optical transition between ground and excited state with the excited state energy surface displaced relative to the ground state along the vibrational coordinates. An additional overdamped brownian oscillator coordinate has been included to account for the residual dephasing environment42. The influence of the intra-molecular vibrational modes and the protein environment on the 2D signal was accounted for by using the line shape approach42. The environment is characterized by a reorganization energy of 50 cm−1 and a temperature of 80 K. All vibrational modes were coupled to the excited state with a Huang-Rhys factor equal to 0.1 . By including both ground state bleach and stimulated emission (no decay of the excited state is assumed), the 3rd order response function can be written down analytically in the time domain41, and the 340 cm−1 2D frequency map can be obtained by numerical Fourier transform , as observed in the experimental PSII RC 2D spectral data .Applying the CWT to the simulated real rephasing 2D spectra at the peaks (A-D) highlighted in Fig. ata Figs  and 3c. We also note that several other minor features in the theoretical scalograms are rather different from the experimentally observed wavelet scalograms. For example, the theoretical scalograms in Fig. time-resolved 2D frequency maps contains real information which we interpret and discuss in the Discussion section.However, excluding the slow recurrences and the minor differences discussed above, there is a remarkable and clear difference between simulated and experimental dynamics: the simulated data shows no decay (since the underlying model does not include any electronic dynamics) whereas the experimental data shows a different decay pattern for the different cross-peaks highlighted in Fig. −1time-resolved 2D frequency map at both 80 K and 277 K demonstrates that the coherence between the precursors of the two different charge separation pathways  is maintained in a significant fraction of PSII RC complexes for more than 500 fs following photo-excitation, allowing the system to coherently sample its energy landscape and therefore, to choose the most optimal pathway according to each specific realization of the disorder.On the one hand, the interpretation of the 120 cm−1time-resolved 2D frequency map obtained by CWT suffer from interference effects revealed by an amplitude beating with a 500 fs period by three independent methods: (i) the calculation of the expected interfering frequencies based on the observed period of rise/decay features and the fact that those frequencies correspond to the experimentally observed frequencies, (ii) the windowed Fourier transform analysis that shows that the rise/decay features are a consequence of interference between the 340 cm−1 frequency and other frequencies present in the 2D data, and (iii) the simulation of the dynamics including the experimentally observed frequencies that display the same rise/decay features as the ones present after the CWT analysis. However, even in the presence of interference effects, the CWT analysis of the 2D experimental data at 80 K reveals a remarkable fact: the  and  nm cross-peaks [coherence between (PD2PD1≈660nm and (PD2δ+PD1δ−≈675nm and coherence between (PD2PD1≈660nm and (PD2+PD1−)δ*≈690nm] display a considerable decay from 300 to 700 fs  whereas the  nm cross-peak [coherence between (PD2δ+PD1δ−≈675nm and (PD2+PD1−)δ*≈690nm] shows a minor decay (1.4-fold decay) δ*≈690nm on the ps time range at cryogenic (80 K) and room temperature (277 K).On the other hand, we have unequivocally shown that all the cross-peak features in the 340 cmay) Figs  (similaray) Figs . In othetime-resolved 2D frequency maps. Therefore, the CWT analysis allows a consistent physical interpretation of coherent energy and electron transfer processes and provides an unprecedented detailed view of the charge separation events in photosynthesis: we have shown that the functional vibronic coherence between the electronic states involved in the charge separation process in the PSII RC persists during the time scales of energy and electron transfer allowing the system to perform ultrafast and highly efficient solar to electrochemical energy conversion at both cryogenic and physiologically relevant temperatures.In conclusion, our results show that the CWT analysis allows us to extract the full potential of the 2DES technique by retaining both the frequency and time information contained in the experimental 2D data by means of τ and λt. A detailed description of the 2D data processing procedure is presented in ref. The details of sample preparation, experimental setup and data acquisition for the experimental data presented here can be found in ref. 32 that have in common the utilization of a zero mean and short-time oscillating function Ψ (T) called mother wavelet. This function can be adapted to the specific characteristics of the temporal signal in order to optimize the analysis. In the analysis presented in this work we use the complex Morlet wavelet function43:b is a bandwidth parameter and Fc is the wavelet center frequency. The Morlet wavelet is a complex exponential centred at frequency Fc, windowed by a zero-mean gaussian function, with σ = (Fb/2)0.5. These two parameters have to be defined in order to get a good compromise between the frequency and time resolution. A large Fb value (for instance Fb = 10) produces high frequency resolution at the expense of low time resolution. On the contrary, a small Fb value (Fb = 0.5) produces the opposite effect. Therefore, a range of Fb values has to be explored in order to select the Fb value to obtain optimal frequency and time resolution. In the present work we have chosen Fb = 2, and Fc has been fixed to 1. The effect of the Fb in the frequency/time wavelet resolution relation is shown in the Supplemental Information  nm corresponding to the 340 cm−1 2D frequency map. By comparing the two limiting cases [top (Fb = 10) and bottom (Fb = 0.5) panels] with the optimal case [middle (Fb = 2) panels], it is clear that increasing excessively the time resolution results in low frequency resolution and vice versa.The WT analysis refers to several time-frequency transformations and signal processing techniquesion Fig.  for two T in the mother wavelet function is translated by a factor u and dilated by a factor s called scale, giving the wavelet atom function Ψ*us(T), which provides the effective basis for the transformation:The variable T [2Dspectra(T)] we expand the temporal signal 2Dspectra(T) in the time-frequency domain via the inner product of this input signal with a wavelet atom function Ψ*us(T), giving rise to the Continuous WT (CWT) as it is shown in Eq. In the analysis of the 2D spectra as a function of population time T, by scanning u over the full temporal domain of the 2Dspectra(T) for a fixed scale s. The operation is repeated, when necessary, with a different scale to obtain the CWT for the whole temporal axis and a chosen interval of s values.The main advantage of WT with respect to the simpler windowed Fourier transform analysis, is that WT realizes a multi-resolution analysis by using different time intervals  for different frequencies. For low frequencies, the WT utilizes long timescales which yields optimal frequency resolution whereas for high frequencies the WT employs short timescales thus achieving better temporal localization. Therefore, the WT is optimally adapted to each of the frequencies involved in the signal. Usually, the CWT is obtained over the entire temporal axis, in this case the population time 44 function cwt ). This function computes the continuous wavelet transform (CWT) of the real-valued signal x. The wavelet transform is computed for the specified scales using the analyzing wavelet function wname. In our study we have used the complex Morlet wavelet (cmor) function as a mother wavelet  *Fc./(frequencies) where 20 fs is the time step of the experimental 2D data analyzed; and the frequencies are the input parameters of the frequencies under study.The wavelet analysis has been performed to each 2D trace independently using the MATLABelet Eq. . This mos(T) Eq. , which pSupplementary Information


Innovations in nanostructuring of inorganic crystalline solids are often limited by prerequisite critical nucleation energy and solute supersaturation for formation of a phase. This research provides direct evidence supporting the viability of an unconventional irradiation-induced nanostructuring process, via transmission electron microscopy, that circumvents these preconditions. Using polymorphic silicon carbide (SiC) as a prototype, a surprising two-step nucleation route is demonstrated through which nanoscale distribution of the second phase is achieved by reaction of solutes with neutron irradiation-induced precursors. In the first step, nanoscale α–SiC precipitates in a β–SiC matrix unexpectedly nucleate heterogeneously at structural defects. This occurs at significantly lower temperatures compared with the usual β→α transition temperature. Subsequently, α–SiC precipitate acts as a surrogate template for its structural and compositional transition into a fission product precipitate, palladium silicide. These discoveries provide a modern view of irradiation engineering in polymorphic ceramics for advanced applications. It is well known that this activation energy is partially abated in metals and ceramics by heterogeneous precipitation of the second phase at structural defects such as grain boundaries2 or interphase interfaces3. Exploiting metallurgical principles for controlled distribution of intragranular nanoscale precipitates, particularly in ceramics, is an attractive prospect4.Diverse aspects of science, ranging from organic to inorganic to mineral crystallization, have examined the nucleation process. Classical nucleation theory explains that the emergence of a stable cluster of a new, distinct second phase is dependent on overcoming critical nucleation energy5. Apart from electronic applications6, it is being considered as a radiation-tolerant material in nuclear fission and fusion reactors8 due to the isotropic nature of the 3 C β-SiC polytype. The irradiation of materials is widely observed to bring microstructural changes due to unexpected phase transformations14. Such irradiation engineering for nanostructured SiC can be useful for electronic or spintronic applications16.Silicon carbide (SiC)—a polymorphic material—is known to exist in more than 250 polytypes7.This article describes a counterintuitive intragranular nucleation process in SiC that indirectly avoids the critical nucleation energy barrier. To the best of the research team’s knowledge, it cannot be explained completely by any known nucleation mechanism. This process provides a means for precipitating out a second phase in a rather incompliant matrix under neutron irradiation. This phase transition has been observed in neutron irradiated SiC utilized as a fission product barrier in TRISO  coated nuclear fuel particles18, the intragranular nanoscale precipitation of fission products, mainly palladium silicide, has raised questions regarding their transport mechanisms20. The near homogenous distribution of these nanoscale Pd rich precipitates in β-SiC matrix20 has led to obvious conclusion based on classical nucleation theory to date that Pd interacts with β-SiC to form these precipitates. Hence, there have been numerous modeling effort to simulate the intragranular Pd transport in TRISO fuel by studying the interaction of Pd with β-SiC23. However, Gentile et al. has experimentally demonstrated that α-SiC is more susceptible than β-SiC to react with Pd24. The present novel dual-step nucleation mechanism of Pd silicide in SiC certainly gives light to these experimental-theoretical controversies of intragranular transport of fission products and has been demonstrated and explained in rest of the paper.Although transport of fission products along grain boundaries of the β-SiC layer in TRISO coated fuel is most probable from a metallurgical perspective25 about 2–4 nm in diameter are visible in a scanning transmission electron microscopy (STEM) image in Fig. 26.Transmission electron microscopy (TEM) of unirradiated SiC layer of TRISO fuel in Fig. (111) of β-SiC and d0006 of 6 H α-SiC are both 0.25 nm27. The usual transformation of β-SiC into α-SiC takes place at 2,000 °C or higher27. The presence of the α variant of SiC has been observed to nucleate at an irradiation temperature of 1,000–1,200 °C, which is nearly 50 percent lower than the transformation temperature28. This unusual behavior can be attributed to accelerated diffusion in Si and C that facilitates reconstructive transformation of β-SiC into α-SiC heterogeneously at linear and planar defects28. The structural defect assisted reconstructive transformation is expected to lower the nucleation energy barrier associated with α-SiC formation. Figures 2 cubic structure. It has been reported that Pd can react with Si within SiC to form ordered L12 Pd3Si-type structures (space group Pnma)21. There are many other possibilities for structure and stoichiometry29. Previously, similar intergranular precipitates have been observed, but their structural identification has not been done26. Graphite, the byproduct of this solid state reaction, was not observed in this microscopic investigation.The structural analysis by high resolution (HR) TEM imaging further unveils the identities of these precipitates. Figure 3Si is aided by the intragranular transport of Pd via linear or planar defects. However, there is possibility of Pd transport in the bulk β-SiC matrix26. These results lead to the intriguing puzzle of why Pd reacts with the α variant of SiC only. Interestingly, the presence of nanoscale α–SiC variant avails the readiness of Pd reaction with SiC as compared with that with a single phase, β-SiC24. It can be extrapolated that α-SiC precipitates potentially accelerated the Pd silicide formation. It is probably energetically expensive for Pd silicide to create new semi-coherent or incoherent interfaces with the β-SiC phase, together with the fact that it has a lower chemical affinity with β-SiC. This allows Pd3Si precipitates to be metamorphosed upon the surrogate α-SiC phase without movement of any phase boundary. Hence, this process uniquely circumvents the nucleation barrier by adopting the morphology of parent α-SiC rather than creating new interfaces with β-SiC. Also, the accelerated solute diffusion along structural defects does not require a conventional solute supersaturation for second phase precipitation. Though only one type of Pd silicide was found here, there is the possibility of formation of other types depending upon the processing conditions29.Unlike the isolated α-SiC and Pd-rich precipitates presented in Figs β ||{0001}α, is observed. The FFT of the bottom part of the structure shows the superlattice spots, apart from the principal β-SiC spots, corresponding to a primitive L12 cubic structure.The structural analysis of such a semi-transformed region in β-SiC is shown in Fig. et al.30, suggests that the out of pile experiments do not simulate the relevant diffusion coefficient of fission products in SiC. Although Pd silicide formation in SiC has also been observed in other type of irradiations such as swift heavy ions (SHI)29, there is no microscopy data available that can ensure the applicability of SHI to induce such nanostructuring in polymorphic ceramics. The comparison of microstructure under neutron and proton irradiation reports that proton irradiation can likely reproduce the microstructure evolution of neutron irradiation since the average size and number density of dislocation loops and voids are similar31. Hence, similar nanostructuring remains a possibility in various polymorphic ceramics such as ZrO232 and ZrC33 by various irradiation sources.A comparison of neutron irradiated TRISO fuel with out-of-pile experiments, as reported by van Rooyen In conclusion, the present study reports a heterogeneous nucleation process in a simple polymorphic crystalline solid that involves two discrete reconstructive and diffusive transformations in a sequence. The phase formed upon the irradiation-induced reconstructive transformation subsequently acts as a surrogate phase and facilitates its reaction with a fission product that is transported in the microstructure via linear and planar defects. The fission product precipitates adopt the exact morphology of the parent phase without movement of any phase boundary and bypass the activation energy otherwise required for its nucleation. These exemplary results challenge the conventional wisdom of precipitation in a nuclear reactor environment. They provide a new view of irradiation-induced nanostructuring in ceramics for advanced applications.TM energy dispersive spectroscopy (EDS) system. Gatan Digital micrograph and TIATM  software were used for post-processing of TEM data. The simulations of diffraction patterns were carried out using JEMSTM software.TRISO coated nuclear fuel was fabricated under standard conditions and subjected to irradiation under the AGR-1 and AGR-2 experimental programs in the Advanced Test Reactor at Idaho National Laboratory. The irradiation conditions for these two programs are reported in Table The datasets generated during the current study are available from the corresponding author on reasonable request.Supplementary Information


NO) levels than native lowlanders. Since nitrate itself can reduce the oxygen cost of exercise in normoxia it may confer additional benefits at high altitude. Xtreme Alps was a double-blinded randomised placebo-controlled trial to investigate how dietary nitrate supplementation affects physiological responses to hypoxia in 28 healthy adult volunteers resident at 4559 m for 1 week; 14 receiving a beetroot-based high-nitrate supplement and 14 receiving a low-nitrate ‘placebo’ of matching appearance/taste. ENO, vital signs and acute mountain sickness (AMS) severity were recorded at sea level (SL) and daily at altitude. Moreover, standard spirometric values were recorded, and saliva and exhaled breath condensate (EBC) collected. There was no significant difference in resting cardiorespiratory variables, peripheral oxygen saturation or AMS score with nitrate supplementation at SL or altitude. Median ENO levels increased from 1.5/3.0  mPa at SL, to 3.5/7.4 mPa after 5 days at altitude (D5) in the low and high-nitrate groups, respectively (p = 0.02). EBC nitrite also rose significantly with dietary nitrate (p = 0.004), 1.7–5.1  μM at SL and 1.6–6.3 μM at D5, and this rise appeared to be associated with increased levels of ENO. However, no significant changes occurred to levels of EBC nitrate or nitrosation products (RXNO). Median salivary nitrite/nitrate concentrations increased from 56.5/786 μM to 333/5,194  μM  with nitrate supplementation at SL, and changed to 85.6/641 μM and 341/4,553 μM on D5. Salivary RXNO rose markedly with treatment at SL from 0.55 μM to 5.70 μM. At D5 placebo salivary RXNO had increased to 1.90 μM whilst treatment RXNO decreased to 3.26 μM. There was no association with changes in any observation variables or AMS score. In conclusion, dietary nitrate supplementation is well tolerated at altitude and significantly increases pulmonary NO availability and both salivary and EBC NO metabolite concentrations. Surprisingly, this is not associated with changes in hemodynamics, oxygen saturation or AMS development.Nitric oxide (NO) production plays a central role in conferring tolerance to hypoxia. Tibetan highlanders, successful high-altitude dwellers for millennia, have higher circulating nitrate and exhaled NO (E •Xtreme Alps is a randomised controlled field study of dietary nitrate at altitude.•Nitrate increased levels of exhaled NO and salivary nitrite, nitrate & RXNO.•Breath condensate nitrate, oxygen saturation and blood pressure did not change following dietary nitrate supplementation.•Increases in exhaled NO correlated with increases in breath condensate nitrite.•Dietary nitrate did not alter the incidence of Acute Mountain Sickness at 4559 m. For example, native lowlanders seem to rely heavily on increased erythropoiesis to maintain oxygen content at altitude NO is a ubiquitous signalling and effector molecule, and an important modulator of oxygen delivery through effects on blood pressure and blood flow as well as mitochondrial respiration. As well as these vasoactive properties, it also plays crucial roles in inflammation (including pulmonary and airway inflammation) and in the immune system, as a cytotoxic entity and by abating oxidative stress l-arginine by nitric oxide synthases (NOS) is an oxygen-requiring process, production of NO through serial reduction of inorganic nitrate (NO3−) to nitrite (NO2−) and NO is inhibited by oxygen NO is produced through two main pathways in the body, both regulated by oxygen. While the enzymatic formation of NO from Besides its use as an ergogenic aid in sports medicine The Xtreme Alps expedition was a double-blinded randomised placebo-controlled trial designed to investigate how dietary nitrate supplementation affects performance at altitude 22.1Details of the Xtreme Alps expedition and protocols have been previously described The study took place in August 2010 at the Capanna Regina Margherita (‘Margherita Hut’) on the summit of Monte Rosa , which contains a research laboratory inside, managed by the University of Turin. Baseline measurements were conducted in London  six weeks earlier: mean barometric pressure 100.5 kPa, mean laboratory temperature 24.1 °C and mean oxygen partial pressure 19.7 kPa. Participants were then separated into 2 groups (Trek 1 and 2) based on their availability, with Trek 2 starting their ascent seven days after Trek 1. All investigators and participants initially flew to Milan (102 m) and remained there one night before starting to ascend the following day; by road to Alagna (1205 m), by lift to Punta Indren (3250 m), and then on foot to the Gnifetti Hut (3611 m). Severe weather conditions meant Trek 1 only stayed at 3611 m for two nights before ascending on foot to the Margherita Hut, whilst Trek 2 ascended on foot after three nights at 3611 m. Subjects then remained at 4559 m for the rest of the study duration  see A before 2.2http://www.consort-statement. org/) and accounting for the need for equal numbers of individuals in both groups whilst maintaining gender distribution. Both participants and investigators were blinded to group allocations for the duration of the study, including the analysis of the primary data sets.Following enrolment, participants were randomly allocated to receive either a high nitrate supplement (intervention group) or low nitrate supplement (placebo group) in accordance with the CONSORT 2010 guidelines  – these individuals were never intended to be part of the main study but presented here for comparison to the placebo group. All meals consumed by subjects during the study period were standardised in order to minimise nutritional nitrate intake.The intervention juice was a custom formulated all-natural beetroot/fruit juice blend . The total daily nitrate dose targeted by the high nitrate intervention was similar to that shown by others to be effective in improving exercise performance at sea level (range 0.10–0.18 mmol/kg/day) 2.42), heart rate (HR), respiratory rate (RR) and blood pressure (BP) were measured after five minutes of rest, sitting upright. Finally, each participant also recorded medications taken and their symptoms according to the Lake Louise score – a validated score for diagnosing and grading AMS All participants completed a daily diary first thing each morning before any oral intake . Peripheral oxygen saturation  were measured using an ultrasonic spirometer . Due to a corrupted file all spirometric recordings taken at high altitude were lost; therefore, only sea level data are reported.Breath-to-breath analysis of inhaled and exhaled NO was performed using a spirometric analysis device  connected to a gas phase chemiluminescence analyzer ; two identical units were used in parallel throughout. In accordance with current American Thoracic Society/European Respiratory Society recommendations for exhaled NO measurements NO concentrations are always accurate and do not require correction for altitude. However, criticism has been raised in relation to other devices and measurement principles using e.g. electrochemical cells for NO detection where readings can vary with changes in partial pressure NO) and partial pressures of exhaled NO (PeNO).Both chemiluminescence analysers were equipped with additional stainless-steel bulkhead fittings, which supplied their ozone generators with pure oxygen  from a cylinder. This hardware adjustment ensured adequate ozone generation to maintain optimal performance even at lower barometric pressures. For consistency, medical grade oxygen was also used to supply the ozone generators at sea level. All measurement devices were calibrated daily, both at sea level and at altitude, with NO (4.00 ppm) using 100% nitrogen as zero gas and a calibrated 100 mL volumetric syringe. The CLD88sp devices measure continuously ambient temperature and pressure by internal sensors. Each morning accurate barometric pressure and temperature were entered into the software for verification purposes as part of the calibration procedure. The CLD88sp takes the ambient pressure into account during the gas calibration and compensates automatically for diurnal pressure changes. Thus, displayed FeFor each measurement subjects inspired to total lung capacity before slowly expiring against a defined resistance to ensure proper velum closure. During expiration an electronic visual display helped each subject maintain a steady flow of 50 ml/s in accordance with ATS guidelines - a smiley face against a green background was displayed when the ultrasonically measured flow was within limits (50 ml/s ±5%), with red warning signs flashing when the flow fell below the minimum or exceeded the maximum level. A minimum of 3 successful repeats with stable flow rates for at least 6 s were collected for each measurement.2.6Samples of unstimulated saliva were collected using dedicated collection devices  immediately before participants ate their midday meal. Swabs were placed sublingually for 2 min at sea level or 5 min at altitude  before collection into a sterile container and centrifugation at 3000 rpm for 15 min; 1 ml of saliva was aliquoted and stored in two separate cryovials at −40 °C for later determination of nitrosation products, nitrite and nitrate. Subjects had not consumed their respective supplement, brushed their teeth, or had recent acute hypoxic or hyperoxic exposure (e.g. as part of other research protocols) before sample collection.®, Respiratory Research, Inc., Charlottesville, VA, USA) for 15 min. Two 1 ml EBC aliquots were frozen and stored at −40 °C straight after collection and analysed later in the UK.Exhaled breath condensate (EBC), a readout of alveolar surface chemistry, was collected non-invasively by breathing through a mouthpiece connected to a sampling tube  and dried before use) contained within an insulated pre-cooled aluminium sleeve , frozen at −40 °C straight after homogenisation and analysed later in the UK.NO metabolite concentrations were quantified using reductive gas-phase chemiluminescence  and HPLC (for nitrite and nitrate), as described elsewhere 2.7www.graphpad.com/scientific-software/prism), were used to analyse these variables  as there was no requirement to model for different ascent profiles.Sea level spirometry values were normally distributed when tested. Separate t-tests, performed in GraphPad Prism 6 software  to account for the multiple time points at which measurements were taken as well as the nitrate intervention. All physiological observations were normally distributed, as were all other measured variables after logarithmic transformation. All statistical tests  were performed on parametric log-transformed data. Significance was assumed when p < 0.05. A sensitivity analysis was performed with missing values replaced by mean values to verify the model. Correlation between breath condensate nitrite and ENO was performed on non-parametric untransformed data using Spearman's Rank with a 2-tailed significance test. Physiological observations and AMS scores are reported as means (±standard deviation). For easier reading, all other values are reported in the text as median (interquartile range) of anti-logged/untransformed data (non-parametric) to allow presentation in original units.Statistical analyses for E33.1The nitrate concentration of the placebo supplement was 1.4 (±0.1) mM; consumption of 3 × 200 mL therefore amounted to 11.5 μmoles/kg bw per day (bw = body weight), which is less than the average UK daily intake of around 24.5 μmoles/kg bw per person per day Food provided at altitude varied somewhat from day to day, therefore representative food samples were collected from typical meals . Average ranges per meal for nitrate were as follows: 50–217 μmoles for breakfast, 132–447 μmoles for lunch, and 559–1201 μmoles for dinner. The average ranges per meal for nitrite were considerably smaller, amounting to 0.17–0.46 μmoles for breakfast, 0.64–1.40 μmoles for lunch, and 0.71–2.10 μmoles for dinner. This amounted to an average intake of 18 (±11) μmoles nitrate/kg bw per day and 0.038 (±0.023) μmoles nitrite/kg bw per day, well within normal UK daily intakes 3.22 initially decreased with each incremental rise in altitude, before stabilising as further time was spent at each elevation (see 2 at sea level was 98.3% (±1.1) and 97.7% (±1.5) for Trek 1 and 2 respectively, decreasing to 77.3% (±8.0) and 81.1% (±6.84) on their first day at 4,559 m . Mean RRs for Trek 1 and 2 respectively were 10.6 (±2.9) and 11.8 (±2.6) at sea level, increasing to 16.1 (±5.7) and 14.2 (±4.0) at arriving at 4,559 m, and decreasing to 12.3 (±3.1) and 11.9 (±3.1) by day 10. A similar response pattern was also seen for resting heart rate and blood pressure  between the intervention and placebo groups, p = 0.29 see . Similar3.4All sea level spirometry variables were normally distributed. All subjects (2 in the treatment group smoked) had normal lung function and there was no significant difference seen between the placebo and treatment groups for any sea level spirometry variable when tested see .Table 1SNO levels, and this result remained significant after adjusting for multiple comparisons. Nitrate supplementation also significantly increased nitrite concentrations in EBC.All measured breathing and salivary variables were normally distributed after log transformation. As shown in NO levels (p = 0.02), with median values from 1.5 mPA (IQR 1.2–2.6) to 2.99 mPa (IQR 1.98–4.44) at sea level and from 3.4 mPa (IQR 1.8–4.6) to 7.4 mPA (IQR 3.9–10.4) by day 5 at altitude. In the same model, treatment with high nitrate supplementation also significantly increased sea level nitrite concentrations in EBC (p = 0.004), with median values increasing from 1.66 μM (IQR 1.29–1.80) to 5.05 μM (IQR 1.22–9.95) at sea level, and from 1.64 μM (IQR 1.04–2.09) to 6.26 μM (IQR 3.83–8.16) after 5 days at 4559 m. Moreover, association analyses unmasked moderate correlations between measured EBC breath nitrite and ENO levels both at sea level,  and at altitude . However, this analysis was limited by low n values at sea level, particularly in the treatment group , nitrite (p < 0.001) and RXNO (p = 0.04) see . Median Initially, similar increases were also seen to median salivary RXNO concentration with the intervention, with levels going from 547.9 nM (IQR 340.6–3090.1) to 5696.4 nM (IQR 483.8–14897.4) at sea level after dietary nitrate supplementation. However the difference between the two randomisation groups was much smaller after 5 days at altitude. The median RXNO concentration in the placebo group increased from sea level to 1897.5 nM (IQR 462.2–4909.9) by day 5, whilst it fell in the supplemented group to 3258.4 nM (IQR 1084.3–7211.9).A secondary sensitivity analysis did not alter the significance of any of these results. However, despite significant increases in all of these biochemical markers of NO availability, there was no identifiable difference between the intervention and placebo group in any resting physiological variable or score of AMS severity see .4The novelty of the present study is that it was performed under field conditions  with mountaineer subjects residing at altitude over a prolonged period of time. This experimental design allowed us to take serial measurements in order to examine how dietary nitrate supplementation  affects physiological acclimatization to altitude.4.1NO), and nitrate and nitrite levels in saliva as well as nitrite concentrations in EBC throughout the duration of altitude exposure. In addition, nitrate supplementation also increased the steady-state concentrations of nitrosation products (S-nitrosothiols and N-nitrosamines) in saliva and compensated for the drop in total nitroso species content in EBC observed following ascent to high altitude. Unexpectedly, despite these significant biochemical changes there was no measureable difference in SpO2, RR, BP, HR or AMS severity associated with dietary nitrate supplementation, either at sea level or at high altitude.These results demonstrate that dietary nitrate supplementation with a beetroot/fruit juice mixture at altitude successfully increases nitrate availability; these increases are associated with markedly enhanced levels of exhaled NO  where only baseline values for FeNO were measured and subjects did not receive any dietary intervention NO values dropped significantly on acute exposure to high altitude found no association between ENO values and reported symptoms of AMS While it has been suggested that EThe two most striking differences between these studies and the results presented here are our dietary intervention to boost nitrate metabolism and the duration of hypoxic exposure. Our study measured changes over five days at altitude whereas the studies of MacInnis et al. and Brown et al. only looked at an acute exposure to hypoxia, measuring changes up to six hours and three hours, respectively. A recent study that explored a more prolonged exposure to hypobaric exposure where teenage subjects received dietary nitrate supplementation on a graded ascent over two weeks to 5300 m in Nepal also reported no change in AMS scores, lending further support to the unexpected findings of the current study NO to generate a measurable physiological change in either baseline observations (e.g. SpO2) or AMS score. We are not aware of any specific data that would have quantified the increase in ENO required to induce a change in either AMS, SpO2 or any other measure reported here in native lowlanders. However, our nitrate supplement successfully doubled median ENO values compared to control so that by day 5 the median ENO in the intervention arm was almost three times higher than levels measured earlier in healthy Tibetan highlanders NO concentrations; therefore, those supra-normal values in our cohort should have been sufficient to trigger a physiologically meaningful response Conceivably, our nitrate supplement might not have induced a large enough increase in E4.2EBC is being increasingly used to detect levels of airway inflammation, monitor disease progression and also assess disease severity in a number of respiratory conditions including adults and children with atopic asthma 2α) have also been shown to significantly increase in EBC measurements in small numbers of both exercising biathletes (n = 10) and sedentary volunteers (n = 5) over a six week period at 2800 m, further supporting our findings presented here l-arginine supplementation – an intervention that should increase endogenous NO production via the classical nitric oxide synthase pathway - no improvement in AMS scores were seen with supplementation. In fact, this study found that the treatment group actually developed significantly worse headaches There is some evidence to suggest EBC could be useful in predicting altitude-related illness. Results from a small study (5 subjects + 11 controls) suggested that EBC analysis before and after 90 min of normobaric hypoxic (12% oxygen) exposure might be able to distinguish between individuals with and without a history of HAPE Again, the biggest difference between all of these previous studies and the results we present here is the scale of the study. To the best of our knowledge, the Xtreme Alps expedition is currently the largest study and 4559 m is also the highest altitude at which EBC samples have yet been collected. While no association was found between NO metabolite related changes in EBC and AMS in the present study, future well-powered studies are needed to determine whether or not this technique could help predict or diagnose altitude illness using other biomarkers.NO, even in the low nitrate/placebo group and particularly at altitude. We believe that, for the first time, this provides evidence to suggest that nitrite in airway secretions might be acting as a possible source of ENO. Whether or not these observations are of biological significance warrants further investigation.A curious finding was that nitrite and nitrate concentrations in EBC measured in the present study were almost identical. This is unlike any other compartment where nitrate levels are typically exceeding nitrite levels by at least an order of magnitude. Moreover, we found that dietary nitrate resulted in significant rises to levels of EBC nitrite - with almost identical rises seen both at sea level and at altitude - but almost no difference to EBC nitrate. This strongly suggests that both EBC nitrate and nitrite are under active regulatory control, even when the exact mechanisms of sensing and regulation remain unclear. We also noted moderate associations between levels of EBC nitrite and Fe4.3l-arginine and oxygen to NO and citrulline by NOS enzymes was established two decades earlier The physiological importance of the enterosalivary nitrate circulation has only been realised recently whilst the conversion of Dietary nitrate supplementation initially increased the levels of salivary nitrate, nitrite and nitrosation products as expected – extra oral nitrate availability enables more bacterial nitrate to nitrite conversion, leading to enhanced systemic nitrite availability and formation of downstream nitrosation products 4.4NO after just a single dose of sodium nitrate. However, despite ENO increasing by 24% at a simulated altitude of 4300 m, there was no demonstrable benefit in isometric exercise performance, forearm blood flow or tissue oxygenation Our findings suggest that even though dietary nitrate supplementation at altitude results in increased biochemical nitrate availability, this does not improve acclimatization and/or reduce the incidence of AMS. Another hypobaric chamber study recently showed similar increases in E4.5The Xtreme Alps study is one of the earliest double-blinded randomised placebo controlled trials of dietary nitrate supplementation at altitude that the authors are aware of, and also one of the larger studies to investigate the effects of nitrate metabolism at altitude. Nevertheless, absolute numbers remain rather moderate in each arm of the trial; we also had a number of missing data points due to subjects not being able to participate on some test days, usually due to altitude related illness. Consequently, confidence intervals remained wide in all reported measures so we cannot completely exclude a type-2 error, although a sensitivity analysis to correct for these missing values did not change these outcomes.2 after nitrate supplementation Since the Xtreme Alps study was conducted, other studies have emerged that also investigated how nitrate supplementation may affect performance at altitude, however most have used a simulated (chamber) altitude environment. In accordance with our findings, AMS incidence was shown to be similar to controls in cyclists taking dietary nitrate supplements exposed to short sessions of 11% oxygen in a normobaric chamber Another study strength was the fact that the supplement we used to deliver nitrate  was almost identical in taste, appearance and composition to the placebo (the same mixture except that the beetroot/fruit juice was selectively reduced in nitrate). Ours is the only study we are aware of that also monitored nitrite and nitrate intake with the food.As alluded to before, ascent occurred in two successive phases but inclement weather conditions meant that these ascent profiles could not be identical. However, statistical modeling has allowed us to control for these differences in ascent profile as much as possible, and trends between both ascent groups were similar. With each subject starting their beetroot juice supplement (containing either high nitrate 'treatment' or very low nitrate 'placebo') three days prior to each testing period, this meant we were unable to record baseline values at either sea level or at altitude. We were also unable to take a complete third 'treatment free' control group to altitude without reducing the power of the study due to the space available at 4559 m. However, the few measurements that were taken on the small number of treatment free laboratory team members all closely matched those of the placebo group suggesting that the low nitrate placebo supplement taken by this group did indeed behave as a placebo agent.The majority of our subjects were young, non-smoking males with previous altitude experience, which is unsurprising given we primarily recruited from a mailing list of medical scientists and healthcare workers with an interest in high-altitude travel and hypoxia physiology. This sample remains representative of lowlanders who visit high altitude regions, as >83% of patients seen at the Himalayan Rescue Association's Everest Base Camp clinic over a ten-year period were male with an average age less than 40 5NO as well as nitrate and nitrite concentrations in saliva, and nitrite concentrations in EBC. The association between EBC nitrite and FeNO levels suggests that exhaled NO may originate, at least in part, from nitrite in alveolar lining fluid. However, these increases in NO and related metabolites were not associated with any measureable changes in AMS, peripheral oxygen saturation or vital physiological parameters such as heart rate and blood pressure after five days of hypobaric hypoxic exposure. Future efforts should be directed at investigating whether dietary nitrate supplementation at altitude is associated with other physiological changes.This study suggests that dietary nitrate supplementation is tolerable and safe at high altitude and robustly increases levels of ETogether MF and HP designed, and HP produced and provided both the intervention and placebo supplement juice drinks for this study. MPWG serves on the medical advisory board of Sphere Medical Ltd and is a director of Oxygen Control Systems Ltd. He has received honoraria for speaking for and/or travel expenses from BOC Medical (Linde Group), Edwards Lifesciences and Cortex GmBH. MPWG leads the Xtreme- Everest Oxygen Research Consortium and the Fit-4-Surgery research collaboration. Some of this work was undertaken at University Southampton NHS Foundation Trust - University of Southampton NIHR Biomedical Research Centre. MPWG serves as the UK NIHR CRN national specialty group lead for Anaesthesia Perioperative Medicine and Pain and is an elected council member of the Royal College of Anaesthetists, an elected board member of the Faculty of Intensive Care Medicine, and President of the Critical Care Medicine Section of the Royal Society of Medicine. All other authors have nothing to declare.University College Hospital NHS Foundation Trust as well as unrestricted research funding from Smiths Medical Ltd. and Deltex Medical Ltd. MP and MF acknowledge support from the Faculty of Medicine, University of Southampton. Analysis of the EBC and saliva samples was in partial fulfillment of the requirements for a MSc degree. Part of the work carried out at the University of Warwick was supported by funds from the Medical Research Council (G0701115) . None of the funding bodies or the institutions the authors are affiliated with had any role in the study design; collection, analysis and interpretation of data; manuscript preparation, or decision to publish. AC is a National Institute for Health Research Academic Clinical Fellow.Xtreme Alps received charitable support from the Friends of