Daniela Polag

Identification of methanogenic pathways in anaerobic digesters using stable carbon isotopes

In a series of anaerobic batch experiments, the stable carbon isotopes, δ13CCH?4? and δ13CCO?2?, were measured in biogas produced from various sources (maize, cellulose, inoculum) to identify the degradation kinetics and specific methanogenic pathways. Isotopic analysis was performed using a new absorption laser spectrometer in addition to conventional MS. A comparison of the isotopic evolution shows large isotope dynamics for maize and cellulose, indicating a temporal change in degradation pathways and/or a change in the relative contribution from different carbon fractions within the substrate. Further batch experiments with isotopically labelled acetate (either 13CH3CO2Na or CH  313 CO2Na) were carried out to study the degradation of acetate in inoculum systematically. The results suggest that the acetate is completely oxidized into CO2 which in turn is partly reduced to CH4. Furthermore, the distinct isotopic signature CH4 and CO2 (for acetate‐methyl labelling as well as for acetate‐carboxy labelling) indicate that only a minor part of the produced methane derives from acetate. A substantial fraction of methane may have been produced at an earlier stage of the reaction chain or by other potential methane precursors such as formate or methanol.

Online monitoring of stable carbon isotopes of methane in anaerobic digestion as a new tool for early warning of process instability

Effective control of anaerobic digestion in biogas plants requires the monitoring of process sensitive and rapid response parameters in order to ensure efficient biogas production and to prevent potential process failure. In this study, stable carbon isotopes of methane (δ(13)CCH4) produced in a full-scale continuous stirred-tank reactor were investigated as a potential new monitoring tool for this purpose. Over a six-month period with variable organic loading rates, δ(13)CCH4-values were measured online by a portable high-precision laser absorption spectrometer. During a stress period of consecutive high organic loading, δ(13)CCH4-values early indicated process changes in contrast to traditionally monitored parameters where a change was observed some five to ten days later. Comparison of the stable isotope values with data from microbial analyses showed a distinct relationship between the quantity of potentially acetoclastic methanogens and δ(13)CCH4-values. This finding indicates an association between dominant methanogenic pathways and carbon isotope values.

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 of13C 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% 13CH3COONa and 99% 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.

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.

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 carbon isotopes of methane for real‐time process monitoring in anaerobic digesters

Efficient operation and stability of biogas plants requires continuous monitoring of the digester content. Traditional laboratory analysis of digester sludge is often complex and time‐consuming and shows a delayed response to disruptions within the fermentation process. As a new approach, we applied an online measurement technique (laser absorption spectroscopy) for real‐time monitoring of stable carbon isotopes of methane ( δ13C CH 4 ) in a pilot‐scale biogas digester (3500 L) regularly fed with maize silage. Generally, isotopic composition of methane gives information about specific substrate degradation, that is, methanogenic pathways that reflect the actual digester state. First results of a 2‐wk monitoring experiment show that stable carbon isotopes of methane respond promptly and highly dynamic to changes in the process state of the digester. In combination with other monitoring parameters (methane production rate, concentration of volatile fatty acids, and pH) the fluctuations in δ13C CH 4 can be interpreted as a change in methanogenic pathways due to a high organic loading rate. In this context, δ13C CH 4 might be used as a new parameter tool for monitoring and characterization of the process state of the digester.

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 worlds 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.

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.