Effect of immune responses on breath methane dynamics.

Methane (CH4) which can be detected in human breath has long been exclusively associated with anaerobic microbial activity (methanogenesis) in the gastrointestinal tract. However, recent studies challenge this understanding by revealing that CH4might also be produced endogenously in cells through oxidative-reductive stress reactions. Consequently, variations in breath CH4levels compared to an individual's baseline level might indicate enhanced oxidative stress levels, and, therefore, monitoring breath CH4levels might offer great potential for 'in vivo' diagnostics such as disease diagnosis, monitoring the efficacy of treatments, or during the application of personalized medicine. To evaluate the effects from immune responses triggered by infections, inflammations, and induced perturbation by vaccination on CH4dynamics in breath, two subjects were monitored over a period of almost 2 years. Breath CH4levels were measured by gas chromatography equipped with a flame-ionization detector. Both subjects exhibited significant deviations (positive and negative, respectively) from their normal CH4breath levels during periods of potential enhanced immune activity. Deviations from the 'healthy state' were indicated by the exceeding of individual CH4ranges. Moreover, for the first time we could clearly prove CH4degradation induced through vaccination by measuring stable carbon isotopes of CH4using gas chromatograph-combustion-isotope ratio mass spectrometry. Hence, breath CH4concentration and isotopic analyses may be used as a biomarker to evaluate specific immune responses and individual immune states.

Radical-Driven Methane Formation in Humans Evidenced by Exogenous Isotope-Labeled DMSO and Methionine.

Methane (CH4), which is produced endogenously in animals and plants, was recently suggested to play a role in cellular physiology, potentially influencing the signaling pathways and regulatory mechanisms involved in nitrosative and oxidative stress responses. In addition, it was proposed that the supplementation of CH4 to organisms may be beneficial for the treatment of several diseases, including ischemia, reperfusion injury, and inflammation. However, it is still unclear whether and how CH4 is produced in mammalian cells without the help of microorganisms, and how CH4 might be involved in physiological processes in humans. In this study, we produced the first evidence of the principle that CH4 is formed non-microbially in the human body by applying isotopically labeled methylated sulfur compounds, such as dimethyl sulfoxide (DMSO) and methionine, as carbon precursors to confirm cellular CH4 formation. A volunteer applied isotopically labeled (2H and 13C) DMSO on the skin, orally, and to blood samples. The monitoring of stable isotope values of CH4 convincingly showed the conversion of the methyl groups, as isotopically labeled CH4 was formed during all experiments. Based on these results, we considered several hypotheses about endogenously formed CH4 in humans, including physiological aspects and stress responses involving reactive oxygen species (ROS). While further and broader validation studies are needed, the results may unambiguously serve as a proof of concept for the endogenous formation of CH4 in humans via a radical-driven process. Furthermore, these results might encourage follow-up studies to decipher the potential physiological role of CH4 and its bioactivity in humans in more detail. Of particular importance is the potential to monitor CH4 as an oxidative stress biomarker if the observed large variability of CH4 in breath air is an indicator of physiological stress responses and immune reactions. Finally, the potential role of DMSO as a radical scavenger to counteract oxidative stress caused by ROS might be considered in the health sciences. DMSO has already been investigated for many years, but its potential positive role in medical use remains highly uncertain.

ROS-driven cellular methane formation: Potential implications for health sciences.

Recently it has been proposed that methane might be produced by all living organisms via a mechanism driven by reactive oxygen species that arise through the metabolic activity of cells. Here, we summarise details of this novel reaction pathway and discuss its potential significance for clinical and health sciences. In particular, we highlight the role of oxidative stress in cellular methane formation. As several recent studies also demonstrated the anti-inflammatory potential for exogenous methane-based approaches in mammalians, this article addresses the intriguing question if ROS-driven methane formation has a general physiological role and associated diagnostic potential.

Effects of CO2 enrichment on the anaerobic digestion of sewage sludge in continuously operated fermenters.

The effect of CO2 enrichment in sewage sludge anaerobic digestion (AD) as a potential strategy to improve the biogas yield was assessed at increasing organic loading rates (OLR). Effects on process performance and resilience were evaluated in long-term continuous AD experiments at lab-scale. The specific methane production (SMP) was sustainably enhanced in the test digester compared to a control at elevated OLRs, reaching an increase of 6 ± 12% on average at the highest OLR tested (4.0 kgVS/(m3·d)). The reduction of CO2 via homoacetogenesis, facilitating acetoclastic CH4 formation is proposed as the dominant conversion pathway. Results suggest that sufficient load of easily degradable substances is a prerequisite for intrinsic formation of the reduction equivalent H2 via acidogenesis. The enhanced resilience of the process under CO2-enriched conditions in response to acid accumulation further qualifies this approach as a viable option for improving AD performance by converting a waste stream into a valuable product.

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

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

Long-term monitoring of breath methane.

In recent years, methane as a component of exhaled human breath has been considered as a potential bioindicator providing information on microbial activity in the intestinal tract. Several studies indicated a relationship between breath methane status and specific gastrointestinal disease. So far, almost no attention has been given to the temporal variability of breath methane production by individual persons. Thus here, for the first time, long-term monitoring was carried out measuring breath methane of three volunteers over periods between 196 and 1002days. Results were evaluated taking into consideration the health status and specific medical intervention events for each individual during the monitoring period, and included a gastroscopy procedure, a vaccination, a dietary change, and chelate therapy. As a major outcome, breath methane mixing ratios show considerable variability within a person-specific range of values. Interestingly, decreased breath methane production often coincided with gastrointestinal complaints whereas influenza infections were mostly accompanied by increased breath methane production. A gastroscopic examination as well as a change to a low-fructose diet led to a dramatic shift of methane mixing ratios from high to low methane production. In contrast, a typhus vaccination as well as single chelate injections resulted in significant short-term methane peaks. Thus, this study clearly shows that humans can change from high to low methane emitters and vice versa within relatively short time periods. In the case of low to medium methane emitters the increase observed in methane mixing ratios, likely resulting from immune reactions and inflammatory processes, might indicate non-microbial methane formation under aerobic conditions. Although detailed reaction pathways are not yet known, aerobic methane formation might be related to cellular oxidative-reductive stress reactions. However, a detailed understanding of the pathways involved in human methane formation is necessary to enable comprehensive interpretation of methane breath levels.

Chloromethane emissions in human breath.

Chloromethane (CH3Cl), currently the most abundant chlorinated organic compound in the atmosphere at around ~550 parts per trillion by volume (pptv), is considered responsible for approximately 16% of halogen-catalyzed stratospheric ozone destruction. Although emissions of CH3Cl are known to occur from animals such as cattle, formation and release of CH3Cl from humans has not yet been reported. In this study a pre-concentration unit coupled with a gas chromatograph directly linked to a mass spectrometer was used to precisely measure concentrations of CH3Cl at the pptv level in exhaled breath from 31 human subjects with ages ranging from 3 to 87years. We provide analytical evidence that all subjects exhaled CH3Cl in the range of 2.5 to 33 parts per billion by volume, levels which significantly exceed those of inhaled air by a factor of up to 60. If the mean of these emissions was typical for the world's population, then the global source of atmospheric CH3Cl from humans would be around 0.66Ggyr-1 (0.33 to 1.48Ggyr-1), which is less than 0.03% of the total annual global atmospheric source strength. The observed endogenous formation of a chlorinated methyl group in humans might be of interest to biochemists and medical scientists as CH3Cl is also known to be a potent methylating agent and thus, could be an important target compound in future medical research diagnostic programs.

Exogenous addition of H2 for an in situ biogas upgrading through biological reduction of carbon dioxide into methane.

Biological reduction of CO2 into CH4 by exogenous addition of H2 is a promising technology for upgrading biogas into higher CH4 content. The aim of this work was to study the feasibility of exogenous H2 addition for an in situ biogas upgrading through biological conversion of the biogas CO2 into CH4. Moreover, this study employed systematic study with isotope analysis for providing comprehensive evidence on the underlying pathways of CH4 production and upstream processes. Batch reactors were inoculated with digestate originating from a full-scale biogas plant and fed once with maize leaf substrate. Periodic addition of H2 into the headspace resulted in a completely consumption of CO2 and a concomitant increase in CH4 content up to 89%. The microbial community and isotope analysis shows an enrichment of hydrogenotrophic Methanobacterium and the key role of hydrogenotrophic methanogenesis for biogas upgrading to higher CH4 content. Excess H2 was also supplied to evaluate its effect on overall process performance. The results show that excess H2 addition resulted in accumulation of H2, depletion of CO2 and inhibition of the degradation of acetate and other volatile fatty acids (VFA). A systematic isotope analysis revealed that excess H2 supply led to an increase in dissolved H2 to the level that thermodynamically inhibit the degradation of VFA and stimulate homo-acetogens for production of acetate from CO2 and H2. The inhibition was a temporary effect and acetate degradation resumed when the excess H2 was removed as well as in the presence of stoichiometric amount of H2 and CO2. This inhibition mechanism underlines the importance of carefully regulating the H2 addition rate and gas retention time to the CO2 production rate, H2-uptake rate and growth of hydrogenotrophic methanogens in order to achieve higher CH4 content without the accumulation of acetate and other VFA.

Mean annual temperatures of mid-latitude regions derived from δ2H values of wood lignin methoxyl groups and its implications for paleoclimate studies.

Tree-rings are widely used climate archives providing annual resolutions on centennial to millennial timescales. Stable isotope ratios of tree-rings have been applied to assist with the delineation of climate parameters such as temperature and precipitation. Here, we investigated stable hydrogen isotope ratios (expressed as δ2H values) of lignin methoxyl groups of wood from various tree species collected along a ~3500km north-south transect across Europe with mean annual temperatures (MAT) ranging from -4 to +17°C. We found a strong linear relationship between MATs and δ2H values of wood lignin methoxyl groups. We used this relationship to predict MATs from randomly collected wood samples and found general agreement between predicted and observed MATs for the mid-latitudes on a global scale. Our results are discussed in context of their paleoclimate relevance and suggest that δ2H values of lignin methoxyl groups might have the potential to reconstruct MATs when applied on mid-latitudinal tree-ring chronologies of the Late Holocene.

Stable isotope and high precision concentration measurements confirm that all humans produce and exhale methane.

Mammalian formation of methane (methanogenesis) is widely considered to occur exclusively by anaerobic microbial activity in the gastrointestinal tract. Approximately one third of humans, depending on colonization of the gut by methanogenic archaea, are considered methane producers based on the classification terminology of high and low emitters. In this study laser absorption spectroscopy was used to precisely measure concentrations and stable carbon isotope signatures of exhaled methane in breath samples from 112 volunteers with an age range from 1 to 80 years. Here we provide analytical evidence that volunteers exhaled methane levels were significantly above background (inhaled) air. Furthermore, stable carbon isotope values of the exhaled methane unambiguously confirmed that this gas was produced by all of the human subjects studied. Based on the emission and stable carbon isotope patterns of various age groups we hypothesize that next to microbial sources in the gastrointestinal tracts there might be other, as yet unidentified, processes involved in methane formation supporting the idea that humans might also produce methane endogenously in cells. Finally we suggest that stable isotope measurements of volatile organic compounds such as methane might become a useful tool in future medical research diagnostic programs.

Age dependent breath methane in the German population.

Methane which can sometimes be found in exhaled breath of humans is known to reflect in situ intestinal methanogenic activity. In recent years, several factors have been studied in order to understand their relevance to methane production in the intestinal tract. However, the relationship between age and methane producing status has hitherto not been sufficiently investigated. In the present study we evaluated the relationship between age and percentage of breath methane producers in the German population in 428 subjects with ages ranging from 4 to 95 years. When subjects were divided into age groups of 15 years, an increase in the percentage of breath methane producers with age was observed. The near linear increase (R(2)=0.977) from 5% for children (1-15 years) to 57% for the elderly (>75 years) may indicate a continuous development in the human gut methanogenic flora throughout lifetime. However, when subjects were compared on 5 year age intervals, an interruption in the percentage of methane producers in the sixth and seventh decade was noted. We further revealed an age dependence on the ratio of female to male producers. This is shown by a dominance in female breath methane producers during the first half of life which afterwards is replaced by a dominance in male breath methane producers with an approximately linear decrease in the ratio between 20 and 65 years (R(2)=0.926). These observations might suggest a relationship between methanogenic activity and hormonal factors. Using our data, we predict that the percentage of breath CH4 producers within the German population will increase from its current value of 30% (2013) to 35% by 2050.

Evidence of anaerobic syntrophic acetate oxidation in biogas batch reactors by analysis of 13C carbon isotopes.

Between 2008 and 2010 various batch experiments were carried out to study the stable carbon isotopic composition of biogas (CH4 and CO2) produced from (i) pure sludge and (ii) sludge including maize. From the evolution of the natural isotopic signature, a temporal change of methanogenic pathways could be detected for the treatment with maize showing that a dominance in acetotrophic methanogenesis was replaced by a mixture of hydrogenotrophic and acetotrophic methanogenesis. For pure sludge, hydrogenotrophic methanogenesis was the dominant or probably exclusive pathway. Experiments with isotopically labelled acetate (99% (13)CH3COONa and 99% CH3(13)COONa) indicated a significant contribution of syntrophic acetate oxidation (SAO) for all the investigated treatments. In the case of pure sludge, experiments from 2008 showed that acetate was almost entirely oxidised to CO2, i.e. acetotrophic methanogenesis was negligible. However, in 2010, the sludge showed a clear dominance in acetotrophic methanogenesis with a minor contribution by SAO indicating a significant change in the metabolic character. Our results indicate that SAO during anaerobic degradation of maize might be a significant process that needs to be considered in biogas research.