Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

Abstract The effect of temperature on CH₄ production, turnover of dissolved H₂, and enrichment of H₂-utilizing anaerobic bacteria was studied in anoxic paddy soil and sediment of Lake Constance. When anoxic paddy soil was incubated under an atmosphere of H₂/CO₂, rates of CH₄ production increased 25°C, but decreased at temperatures lower than 20°C. Chloroform completely inhibited methano-genesis in anoxic paddy soil and lake sediment, but did not or only partially inhibit the turnover of dissolved H₂, especially at low incubation temperatures. Cultures with H₂ as energy source resulted in the enrichment of chemolithotrophic homoacetogenic bacteria whenever incubation temperatures were lower than 20°C. Hydrogenotrophic methanogens could only be enriched at 30°C from anoxic paddy soil. A homoacetogen     

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Abstract Interspecies H₂ transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of ¹⁴CO₂ to ¹⁴CH₄ in anoxic paddy soil. Only 3–5% of the ¹⁴CH₄ were produced from the turnover of dissolved H₂. The H₂-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H₂-dependent methanogenesis and the turnover of dissolved H₂ did not change significantly for up to 7 months of incubation. However, while the rates of H₂-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H₂-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H₂ by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH₄ production was almost completely restricted to H₂-syntrophic MBA. The activity of MBA, as measured by the conversion of ¹⁴CO₂ to ¹⁴CH₄, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H₂. Propionate and acetate had no effect.     

Soil type links microbial colonization of rice roots to methane emission

Most of the methane (CH₄) emission from rice fields is derived from plant photosynthates, which are converted to CH₄. Rice cluster I (RC-1) archaea colonizing the rhizosphere were found to be the methanogens responsible for this process. Hence, RC-1 methanogens seem to play a crucial role in emission of the greenhouse gas CH₄. We determined the community composition and activity of methanogens colonizing the roots of eight different rice cultivars after growth on both Italian rice soil and river bank soil, which contained different communities of methanogenic archaea. The community composition was analyzed by terminal restriction fragment length polymorphism and cloning/sequencing of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase. When grown on rice field soil, the methanogenic community of the different rice cultivars was always dominated by RC-1 methanogens. In contrast, roots were colonized by Methanomicrobiales when grown on river bank soil, in which RC-1 methanogens were initially not detectable. Roots colonized with Methanomicrobiales compared with RC-1 exhibited lower CH₄ production and CH₄ emission rates. The results show that the type of methanogens colonizing rice roots has a potentially important impact on the global CH₄ cycle.     

Sensitivity of methanogenic bacteria from paddy soil to oxygen and desiccation

Abstract The effects of combinations of desiccation and exposure to O₂ were studied in pure cultures of Methanosarcina barkeri strain Fusaro and in a new Methanosarcina strain and a new Methanobacterium strain which were both isolated from dry oxic paddy soil. Incubation of bacterial suspensions under air for 200 min resulted in a decreased potential to produce CH₄, but not in a decreased viability. The inhibitory effect of O₂ slightly increased with increased salt concentration. Desiccation of bacterial suspensions under N₂ resulted in reduction of viability to 10% and of potential CH₄ production to 0.6%. Desiccation of bacterial suspensions under air resulted in a larger decrease of both viability (0.5%) and potential CH₄ production (0.03%). This decrease was smaller at rapid compared to slow desiccation. Survival and potential CH₄ production were further inhibited when the suspension was dried in the presence of sand grains or glass beads coated with FeS or FeNH₄PO₄. However, survival and potential CH₄ production increased dramatically in the presence of pyrite (FeS₂) grains. Then, as much as 10% of the initial methanogenic population survived oxic desiccation. This relatively good resistance is in agreement with observations that methanogens in rice fields survive the periods when the paddy soil is dry and oxic.     

Development of a gas diffusion probe for the determination of methane concentrations and diffusion characteristics in flooded paddy soil

Abstract A probe for the measurement of dissolved CH₄ in anoxic methanogenic environments was developed. The probe was based on the diffusion of dissolved CH₄ through a silicone membrane into a gas space at the end of the probe. This gas space was flushed with N₂ and analyzed gas-chromatographically for CH₄. The probe had a spatial resolution of < 1.3 mm, the detection limit was about 20 μM CH₄, the precision of the measurement was 9%, and consecutive measurements could be made every 4 min. Memory effects after analysis of high CH₄ concentrations could be avoided by flushing the probe with N₂ between each measurement. The probe was sensitive for water movement and, therefore, was calibrated in an artificial sediment of glass beads (100 μm diam.) immersed by aqueous solutions of known CH₄ concentrations. Sensitivity of the probe for changes in the sediment's porosity could not presently be excluded. The probe was used to measure vertical profiles of dissolved CH₄ in microcosms of anoxic paddy soil. The vertical CH₄ profiles measured with the probe compared fairly well with those measured after an extraction procedure. The profiles clearly showed that CH₄ was produced in deeper layers and diffused upwards to be consumed in the oxic top 2 mm soil layers. The probe was also used to determine the diffusion coefficient of CH₄ in an inactivated paddy soil microcosm using a set-up which allowed modelling of a measured CH₄ concentration profile using Fick's 2nd law.     

Effects of vegetation on the emission of methane from submerged paddy soil

Summary Methane emission rates from rice-vegetated paddy fields followed a seasonal pattern different to that of weed-covered or unvegetated fields. Presence of rice plants stimulated the emission of CH₄ both in the laboratory and in the field. In unvegetated paddy fields CH₄ was emitted almost exclusively by ebullition. By contrast, in rice-vegetated fields more than 90% of the CH₄ emission was due to plant-mediated transport. Rice plants stimulated methanogenesis in the submerged soil, but also enhanced the CH₄ oxidation rates within the rhizosphere so that only 23% of the produced CH₄ was emitted. Gas bubbles in vegetated paddy soils contained lower CH₄ mixing ratios than in unvegetated fiels. Weed plants were also efficient in mediating gas exchnage between submerged soil and atmosphere, but did not stimulate methanogenesis. Weed plants caused a relatively high redox potential in the submerged soil so that 95% of the produced CH₄ was oxidized and did not reach the atmosphere. The emission of CH₄ was stimulated, however, when the cultures were incubated under gas atmospheres containing acetylene or consisting of O₂-free nitrogen.     

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH₄ into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH₄ flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using ¹³C-labelled CH₄ directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to ¹³CH₄ for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.     

Latitudinal differentiated water table control of carbon dioxide, methane and nitrous oxide fluxes from hydromorphic soils: feedbacks to climate change

The possibility of carbon (C) being locked away from the atmosphere for millennia is given in hydromorphic soils. However, the water-table-dependent feedback from soil organic matter (SOM) decomposition to the climate system is less clear. At least three greenhouse gases are produced: carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O). These gases show emission peaks at different water table positions and have different global warming potentials (GWP), for example a factor of 23 for CH₄ and 296 for N₂O as compared with the equivalent mass of CO₂ on a 100-year time horizon. This review of available annual data on all three gases revealed that the radiative forcing effect of SOM decomposition is principally dictated by CO₂ despite its low GWP. Anaerobic SOM decomposition generally has a lower potential feedback to the climatic system than aerobic SOM decomposition. Concrete values are constrained by a lack of data from tropical and subarctic regions. Furthermore, data on N₂O and on plant effects are generally rare. However, there is a clear latitudinal differentiation for the GWP of soils under anaerobic conditions compared with aerobic conditions when looking at CO₂ and CH₄: in the tropical and temperate regions, the anaerobic GWP showed a range of 25–60% of the aerobic value, but values varied between 80% and 110% in the boreal zone. Hence, particularly in the vulnerable boreal zone, the feedback from ecosystems to climate change will highly depend on plant responses to changing water tables at elevated temperatures.     

Effects of raised CO2 on potential CH4 production and oxidation in, and CH4 emission from, a boreal mire

1 In a glasshouse experiment we studied the effect of raised CO₂ concentration (720 p.p.m.) on CH₄ emission at natural boreal peat temperatures using intact cores of boreal peat with living vascular plants and Sphagnum mosses. After the end of the growing season half of the cores were kept unnaturally warm (17–20 °C). The potential for CH₄ production and oxidation was measured at the end of the emission experiment.
2 The vascular cores ('Sedge') consisted of a moss layer with sedges, and the moss cores (' Sphagnum ') of Sphagnum mosses (some sedge seedlings were removed by cutting). Methane efflux was 6–12 times higher from the Sedge cores than from the Sphagnum cores. The release of CH ₄ from Sedge cores increased with increasing temperature of the peat and decreased with decreasing temperature. Methane efflux from Sphagnum cores was quite stable independent of the peat temperatures.
3 In both Sedge and Sphagnum samples, CO₂ treatment doubled the potential CH₄ production but had no effect on the potential CH₄ oxidation. A raised concentration of CO₂ increased CH₄ efflux weakly and only at the highest peat temperatures (17–20 °C).
4 The results suggest that in cool regions, such as boreal wetlands, temperature would restrict decomposition of the extra substrates probably derived from enhanced primary production of mire vegetation under raised CO₂ concentrations, and would thus retard any consequent increase in CH₄ emission.     

Methane emission from Texas rice paddy soils. 1. Quantitative multi-year dependence of CH4 emission on soil, cultivar and grain yield

Methane emissions at different rice productivity levels were observed from Texas rice paddy soils during the years 1991–95. Analysis of field measurements showed that seasonal methane emission (E) was strongly dependent on soil, cultivar, and rice grain yield. The relationship can be quantitatively described by E (g m^–2) = 0.048 × SI × VI × GY. SI is a soil index to characterize the relative effect of soil texture on emission and is linked with soil sand percentage. VI is a variety index to identify the intervarietal difference in methane emission and is related to the amount of methane emission per unit grain yield. GY is grain yield (g m^–2). Constant 0.048 was derived from the measurements of 10 cultivars planted in 1993. Computed emission applying the relationship is well matched with measured data. The comparison of computed with measured seasonal methane emission over an 80-day period using a total of 32 data sets yields a correlation coefficient r ² of 0.800. In addition, the ratio of seasonal methane emission to net primary productivity was calculated on a carbon to carbon basis, which produces an average value of 2.8%, ranging from 1.2% to 5.4%. A further investigation indicated that the ratio is soil and variety dependent and can be quantitatively explained by C[CH₄]/C[NPP] (%) = 3.21 × SI × VI + 0.12 ( r ² = 0.738, n = 32). Under the condition of 30% soil sand, this ratio is ≈ 3% for the majority of cultivars.     

Snow depth, soil freezing, and fluxes of carbon dioxide, nitrous oxide and methane in a northern hardwood forest

Soil–atmosphere fluxes of trace gases (especially nitrous oxide (N₂O)) can be significant during winter and at snowmelt. We investigated the effects of decreases in snow cover on soil freezing and trace gas fluxes at the Hubbard Brook Experimental Forest, a northern hardwood forest in New Hampshire, USA. We manipulated snow depth by shoveling to induce soil freezing, and measured fluxes of N₂O, methane (CH₄) and carbon dioxide (CO₂) in field chambers monthly (bi-weekly at snowmelt) in stands dominated by sugar maple or yellow birch. The snow manipulation and measurements were carried out in two winters (1997/1998 and 1998/1999) and measurements continued through 2000. Fluxes of CO₂ and CH₄ showed a strong seasonal pattern, with low rates in winter, but N₂O fluxes did not show strong seasonal variation. The snow manipulation induced soil freezing, increased N₂O flux and decreased CH₄ uptake in both treatment years, especially during winter. Annual N₂O fluxes in sugar maple treatment plots were 207 and 99 mg N m⁻² yr⁻¹ in 1998 and 1999 vs. 105 and 42 in reference plots. Tree species had no effect on N₂O or CO₂ fluxes, but CH₄ uptake was higher in plots dominated by yellow birch than in plots dominated by sugar maple. Our results suggest that winter fluxes of N₂O are important and that winter climate change that decreases snow cover will increase soil:atmosphere N₂O fluxes from northern hardwood forests.     

Effects of temperature, ultraviolet radiation and pectin methyl esterase on aerobic methane release from plant material

This study examines the effects of different irradiance types on aerobic methane (CH₄) efflux rates from terrestrial plant material. Furthermore, the role of the enzyme pectin methyl esterase (PME) on CH₄ efflux potential was also examined. Different types of plant tissue and purified pectin were incubated in glass vials with different combinations of irradiation and/or temperature. Purified dry pectin was incubated in solution, and with or without PME. Before and after incubation, the concentration of CH₄ was measured with a gas chromatograph. Rates of CH₄ emission were found to depend exponentially on temperature and linearly on UV-B irradiance. UV-B had a greater stimulating effect than UV-A, while visible light had no effect on emission rates. PME was found to substantially reduce the potential for aerobic CH₄ emissions upon demethylation of pectin.     

Missing methane emissions from leaves of terrestrial plants

The controversial claim that attached leaves of terrestrial plants emit CH₄ aerobically remains to be corroborated. Here, we report CH₄ fluxes and CO₂ exchange rates for leaves of the C₄ species Zea mays using a high-accuracy traceable online analytical system. In contrast to earlier results for Z. mays , our measurements provide no evidence for substantial aerobic CH₄ emissions from photosynthesizing leaves illuminated with photosynthetically active radiation ( λ =400–700 nm), or from dark-respiring leaves. Preliminary measurements with the same system indicated a similar lack of aerobic CH₄ emissions in the light or dark from leaves of the C₃ species Nicotiana tabacum . These findings are supported by independent high-precision ¹³C-labeling studies that also failed to confirm substantial aerobic CH₄ emissions from plants. Nevertheless, we are not able to exclude the possibility that CH₄ emissions from plants may be linked to nonenzymatic processes with an action spectrum lying outside the wavelength range for photosynthesis.     

A review of nitrogen enrichment effects on three biogenic GHGs: the CO2 sink may be largely offset by stimulated N2O and CH4 emission

Anthropogenic nitrogen (N) enrichment of ecosystems, mainly from fuel combustion and fertilizer application, alters biogeochemical cycling of ecosystems in a way that leads to altered flux of biogenic greenhouse gases (GHGs). Our meta-analysis of 313 observations across 109 studies evaluated the effect of N addition on the flux of three major GHGs: CO₂, CH₄ and N₂O. The objective was to quantitatively synthesize data from agricultural and non-agricultural terrestrial ecosystems across the globe and examine whether factors, such as ecosystem type, N addition level and chemical form of N addition influence the direction and magnitude of GHG fluxes. Results indicate that N addition increased ecosystem carbon content of forests by 6%, marginally increased soil organic carbon of agricultural systems by 2%, but had no significant effect on net ecosystem CO₂ exchange for non-forest natural ecosystems. Across all ecosystems, N addition increased CH₄ emission by 97%, reduced CH₄ uptake by 38% and increased N₂O emission by 216%. The net effect of N on the global GHG budget is calculated and this topic is reviewed. Most often N addition is considered to increase forest C sequestration without consideration of N stimulation of GHG production in other ecosystems. However, our study indicated that although N addition increased the global terrestrial C sink, the CO₂ reduction could be largely offset (53–76%) by N stimulation of global CH₄ and N₂O emission from multiple ecosystems.     

Potential of double-cropped rice ecology to conserve organic carbon under subtropical climate

Understanding the processes of soil organic carbon (SOC) accumulation or depletion under different management strategies is vital for maintaining soil health and curbing global warming. Using a 36-year-old fertility experiment under subtropical climate, we investigated the impact of long-term intensive rice–rice cropping system with different managements on the SOC stock. The mechanistic pathway of stabilization of the SOC into different pools, with a tentative C budgeting was also established. Biochemical composition of the organic residues involved, SOC pools of different oxidizability and methane (CH₄) emission were estimated for the experiment conducted using organic and inorganic sources of nutrients. Cultivation over the years caused a net decrease in SOC stocks but with balanced fertilization it increased. With increasing depth, the stock decreased on average, to the extent of 50%, 26% and 24% of the total at 0–0.2, 0.2–0.4 and 0.4–0.6 m, respectively. About 4.0% of the crop residues C incorporated into the soil were stabilized into SOC. This was further enhanced (1.6 times) by the application of compost. Carbon loss through CH₄ emission was very low (2.6% of the total). 'Summer fallow' had a positive significant influence on C loss from the system. As much as 29% of the compost C added to the soil was stabilized into SOC mostly in the less-labile or nonlabile recalcitrant pools preferentially in the surface layer of the soil. Large polyphenol and lignin contents of crop residues including compost, and the long period of soil submergence under rice cultivation might have conferred recalcitrant character to the SOC leading to its stabilization in nonlabile pools. This would result into an enrichment of the SOC stock and restriction to the gaseous C loading into the atmosphere.     

Emission of hydrogen from deep and shallow freshwater environments

In-situ partial pressures of hydrogen in anoxic profundal lake sediments reached values of up to 5 Pa which were more than 5 orders of magnitude lower than the partial pressures of methane. Analysis of gas bubbles collected from anoxic submerged paddy soil showed H₂ partial pressures in the range of 1.8 ± 1.3 Pa being ca. 4 orders of magnitude lower than the CH₄ partial pressures. H₂ emission rates, on the other hand, were less than 3 orders of magnitude lower than the CH₄ emission rates indicating that H₂ and CH₄ were oxidized to a different extent in the rhizosphere of the soil before they reached the atmosphere, or that H₂ was produced by the plants. More than 70% of the emitted H₂ reached the atmosphere via plant-mediated flux. The rest was emitted via ebullition from the anoxic soil and, in addition, was produced in the paddy water. A significant amount of H₂ was indeed found to be produced in the water under conditions where thallic algae and submerged parts of the rice plants produced oxygen by photosynthesis. Very little H₂ was emitted via molecular diffusion through the paddy water; in addition, this amount was less than expected from the degree of supersaturation and the diffusional emission rate of CH₄ indicating a relatively high rate of H₂ consumption in the surface film of the paddy water. The total H₂ source strength of rice paddies and other freshwater environments was estimated to be less than 1 Tg yr^-1, being negligible in the atmospheric budget of H₂.     

Links between methanotroph community composition and CH4 oxidation in a pine forest soil

The main gap in our knowledge about what determines the rate of CH₄ oxidation in forest soils is the biology of the microorganisms involved, the identity of which remains unclear. In this study, we used stable-isotope probing (SIP) following ¹³CH₄ incorporation into phospholipid fatty acids (PLFAs) and DNA/RNA, and sequencing of methane mono-oxygenase ( pmoA ) genes, to identify the influence of variation in community composition on CH₄ oxidation rates. The rates of ¹³C incorporation into PLFAs differed between horizons, with low ¹³C incorporation in the organic soil and relatively high ¹³C incorporation into the two mineral horizons. The microbial community composition of the methanotrophs incorporating the ¹³C label also differed between horizons, and statistical analyses suggested that the methanotroph community composition was a major cause of variation in CH₄ oxidation rates. Both PLFA and pmoA -based data indicated that CH₄ oxidizers in this soil belong to the uncultivated 'upland soil cluster α'. CH₄ oxidation potential exhibited the opposite pattern to ¹³C incorporation, suggesting that CH₄ oxidation potential assays may correlate poorly with in situ oxidation rates. The DNA/RNA-SIP assay was not successful, most likely due to insufficient ¹³C-incorporation into DNA/RNA. The limitations of the technique are briefly discussed.     

Consumption of NO by methanotrophic bacteria in pure culture and in soil

Abstract The methanotrophs Methylomonas angile (type I) and Methylosinus trichosporium (type II) produced nitrite, nitrate and N₂O during growth on methane, apparently by heterotrophic nitrification of ammonium. The methanotrophs were also able to consume NO but did not produce it. After incubation of soil from a drained paddy field in the presence of CH₄ the numbers of methanotrophs increased from 10⁵ to 10⁷ per gram dry weigth. The thus enriched soil showed increased rates of NO consumption while rates of NO production did not change.     

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H₂ turnover per gram soil. Dilution did not affect the fraction of H₂/CO₂-dependent methanogenesis which made up 22% of total CH₄ production. However, dilution resulted in a ten fold decrease of the H₂ steady state partial pressure from approximately 4 to 0.4 Pa indicating that H₂/CO₂-dependent methanogenesis was more or less independent of the H₂ pool. The rates of H₂ production calculated from the H₂ turnover rate constants and the H₂ steady state partial pressures accounted for only < 5% of H₂/CO₂-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H₂/CO₂-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H₂ transfer within syntrophic bacterial associations and was not significantly affected by the outside H₂ pool.     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.