Methanotrophic communities in a landfill cover soil as revealed by [13C] PLFAs and respiratory quinones: Impact of high methane addition and landfill leachate irrigation

The soil microbial communities of a landfill cover substrate, which was treated with landfill gas (100 l CH₄ m^?2 d^?1) and landfill leachate for 1.5 years, were investigated by phospholipid fatty acid (PLFA), ergosterol and respiratory quinone analyses. The natural ¹³C depletion of methane was used to assess the activity of methanotrophs and carbon turnover in the soil system. Under methane addition, the soil microbial community was dominated by PLFAs (14:0 and 16:1 isomers) and quinones (ubiquinone-8 and 18-methylene-ubiquinone-8) related to type I methanotrophs, and 18:1 PLFAs contained in type II methanotrophs. While type I methanotrophic PLFAs were ¹³C depleted, i.e. type I methanotrophs were actively oxidising and assimilating methane, ¹³C depletion of 18:1 PLFAs was low and inconsistent with their abundance. This, possibly reflects isotopic discrimination, assimilation of carbon derived from type I methanotrophs and a high contribution of non-methanotrophic bacteria to the 18:1 isomers. Landfill leachate irrigation caused the methanotrophic community to shift closer to the soil surface. It also decreased 18:1 PLFAs, while type I methanotrophs were probably stimulated. Gram positive bacteria, but not fungi, were also ¹³C depleted and consequently involved in the secondary turnover of carbon originating from methanotrophic bacteria. Cy17:0 PLFA was ¹³C depleted in deep soil layers, indicating anaerobic methane oxidation.     

The differential effects of ammonium and nitrate on methanotrophs in rice field soil

It has been known that nitrogenous fertilizers can either stimulate or inhibit methane oxidation in soils. The mechanism, however, remains unclear. Here we conducted laboratory incubation experiments to evaluate the effects of ammonium versus nitrate amendment on CH₄ oxidation in a rice field soil. The results showed that both N forms stimulated CH₄ oxidation. But nitrate stimulated CH₄ oxidation to a greater extent than ammonium per unit N base. The 16S rRNA genes and the pmoA genes were analyzed to determine the dynamics of total bacterial and methanotrophic populations, respectively. The methanotrophic community consisted of type I and type II methanotrophs and was dominated by type I group after two weeks of incubation. Nitrate promoted both types of methanotrophs, but ammonium promoted only type I. DNA-based stable isotope probing confirmed that ammonium stimulated the incorporation of ¹³CH₄ into type I methanotrophs but not type II, while nitrate caused almost homogenous distribution of ¹³CH₄ in type I and type II methanotrophs. Our study suggests that nitrate can promote CH₄ oxidation more significantly than ammonium and is probably a better N source for both types of methanotrophs in rice field soil. More investigations, e.g. using ¹⁵N labeling, are necessary to elucidate this possibility.     

CH4 oxidation and emissions of CH4 and N2O from Lolium perenne swards under elevated atmospheric CO2

Emissions of N₂O and CH₄ and CH₄ oxidation rates were measured from Lolium perenne swards in a short-term study under ambient (36 Pa) and elevated (60 Pa) atmospheric CO₂ at the Free Air Carbon dioxide Enrichment experiment, Eschikon, Switzerland. Elevated pCO₂ increased (P<0.05) N₂O emissions from high N fertilised (11.2 g N m⁻²) swards by 69%, but had no significant effect on net emissions of CH₄. Application of ¹³C-CH₄ (11 μl l⁻¹; 11 at.% excess ¹³C) to closed chamber headspaces in microplots enabled determination of rates of ¹³C-CH₄ oxidation even when net CH₄ fluxes from main plots were positive. We found a significant interaction between fertiliser application rate and atmospheric pCO₂ on ¹³C-CH₄ oxidation rates that was attributed to differences in gross nitrification rates and C and N availability. CH₄ oxidation was slower and thought to be temporarily inhibited in the high N ambient pCO₂ sward. The most rapid CH₄ oxidation of 14.6 μg ¹³C-CH₄ m⁻² h⁻¹ was measured in the high fertilised elevated pCO₂ sward, and we concluded that either elevated pCO₂ had a stimulatory effect on CH₄ oxidation or inhibition of oxidation following fertiliser application was lowered under elevated pCO₂. Application of ¹⁴NH₄¹⁵NO₃ and ¹⁵NH₄¹⁵NO₃ (10 at.% excess ¹⁵N) to different replicates enabled determination of the respective contributions of nitrification and denitrification to N₂O emissions. Inhibition of CH₄ oxidation in the high fertilised ambient pCO₂ sward, due to competition between NH₃ and CH₄ for methane monooxygenase enzymes or toxic effects of NH₂OH or NO₂⁻ produced during nitrification, was hypothesised to increase gross nitrification (12.0 mg N kg dry soil⁻¹) and N₂O emissions during nitrification (327 mg ¹⁵N-N₂O m⁻² over 11 d). Our results indicate that increasing atmospheric concentrations of CO₂ may increase emissions of N₂O by denitrification, lower nitrification rates and either increase or decrease the ability of soil to act as a sink for atmospheric CH₄ depending on fertiliser management.     

Varying atmospheric methane concentrations affect soil methane oxidation rates and methanotroph populations in pasture,an adjacent pine forest,and a landfill

We describe experiments to better understand how CH₄ oxidation rates by different methanotroph communities respond to changing CH₄ concentrations. We used a novel system of automatically monitored chambers to investigate the response of CH₄ oxidation rates in a New Zealand pasture and adjacent pine forest soil exposed to varying atmospheric CH₄ concentrations.Type II methanotrophs that dominate CH₄ oxidation in the forest soil became progressively saturated as CH₄ concentrations rose from ambient (1.8 ppmv) to 570 ppmv, as shown by a decrease in uptake efficiency from 20% to 2% removal. By contrast, CH₄ oxidation in the pasture soil where Type I methanotrophs dominate increased in proportion to the increase in CH₄ inlet concentration, oxidising about 2% of the inlet CH₄ flux throughout. Modelling based on Michaelis-Menten kinetics revealed that low-affinity (Type I) methanotrophs were solely responsible for CH₄ oxidation in pasture soils, whereas high affinity (Type II) methanotrophs only contributed about 10% of the CH₄ oxidation in the forest soil. Increased aeration status using a soil–perlite (1:1) mixture doubled CH₄ oxidation rates at both ambient (1.8 ppmv) and 40 ppmv atmospheric CH₄. A similar volcanic soil previously exposed for 8 y to high CH₄ fluxes from a landfill had removal efficiencies consistently above 95% for atmospheric CH₄ concentrations up to 7500 ppmv when the CH₄ oxidation rate was7000 μg CH₄ kg⁻¹soil h⁻¹.     

Soil methane oxidation and methanotroph responses to afforestation of pastures with Pinus radiata stands

Afforestation of pastures in New Zealand reduces methane (CH₄) production from soil, while also stimulating oxidation of atmospheric CH₄ by soil methanotrophs. However, neither the mechanisms by which soil CH₄ oxidation is enhanced by afforestation, nor how long after forest planting tree-dependent responses in CH₄ oxidation become detectable are fully known. Here, we investigated the effects of different-aged stands (5-20 y) of the exotic pine (Pinus radiata (D. Don)) on CH₄ oxidation and methanotrophic community structure in soils, compared with adjacent, long-established pastures. Two of the pastures were on volcanic soils and two were on non-volcanic soils. Although the CH₄ fluxes in soils from these young stands were not significantly different from those in the associated pastures, the rate of oxidation of added ¹³CH₄ was higher in the pine soils. Both fluxes and ¹³CH₄ oxidation rates were higher in the volcanic than the non-volcanic soils. Combined phospholipid fatty acid (PLFA) and stable isotope probe (SIP) analyses suggested that type II methanotrophs (PLFA C18:1ω7) were most active in all soils followed by uncultivable bacteria (C17:0ai). Molecular analysis of the methanotrophic community structure using pmoA (particulate methane monooxygenase) genes suggested that a particular type II methanotroph (TRF 35) was dominant in all soils, but more so in the pine than in pasture soils. A type I methanotroph (TRF 245) was more prevalent in the pasture than in associated pine soils, whereas TRF 128 (a type II methanotroph) was slightly more dominant in soils under pine. Cloning and sequencing data suggest TRFs 35 and 128, which differ from one another, belong to distant relatives of Methylocapsa sp; TRF 245 is related to Methylococcus capsulatus. Land-use change resulted in changes in soil bulk density, porosity, moisture contents and in methanotrophic community structure. Methane oxidation rates were most closely related to soil moisture, as well as to the methanotrophic community structure, and nitrate-N, extractable C and total C concentrations. Stepwise multiple regression also suggested a weak effect (P = 0.06) of stand age on CH₄ oxidation rate. By contrast, the responses of the methanotrophic community structure to this land-use change were more readily detected by the specific molecular analyses, and indicated a predominance of type II methanotrophs in pine soils.     

Methane turnover and temperature response of methane-oxidizing bacteria in permafrost-affected soils of northeast Siberia

The abundance, activity, and temperature response of aerobic methane-oxidizing bacteria were studied in permafrost-affected tundra soils of northeast Siberia. The soils were characterized by both a high accumulation of organic matter at the surface and high methane concentrations in the water-saturated soils. The methane oxidation rates of up to 835 nmol CH₄ h⁻¹ g⁻¹ in the surface soils were similar to the highest values reported so far for natural wetland soils worldwide. The temperature response of methane oxidation was measured during short incubations and revealed maximum rates between 22 °C and 28 °C. The active methanotrophic community was characterized by its phospholipid fatty acid (PLFA) concentrations and with stable isotope probing (SIP). Concentrations of 16:1ω8 and 18:1ω8 PLFAs, specific to methanotrophic bacteria, correlated significantly with the potential methane oxidation rates. In all soils, distinct 16:1 PLFAs were dominant, indicating a predominance of type I methanotrophs. However, long-term incubation of soil samples at 0 °C and 22 °C demonstrated a shift in the composition of the active community with rising temperatures. At 0 °C, only the concentrations of 16:1 PLFAs increased and those of 18:1 PLFAs decreased, whereas the opposite was true at 22 °C. Similarly, SIP with ¹³CH₄ showed a temperature-dependent pattern. When the soils were incubated at 0 °C, most of the incorporated label (83%) was found in 16:1 PLFAs and only 2% in 18:1 PLFAs. In soils incubated at 22 °C, almost equal amounts of ¹³C label were incorporated into 16:1 PLFAs and 18:1 PLFAs (33% and 36%, respectively). We concluded that the highly active methane-oxidizing community in cold permafrost-affected soils was dominated by type I methanotrophs under in situ conditions. However, rising temperatures, as predicted for the future, seem to increase the importance of type II methanotrophs, which may affect methane cycling in northern wetlands.     

CH4 oxidation and N2O emissions at varied soil water-filled pore spaces and headspace CH4 concentrations

Emission of N₂O and CH₄ oxidation rates were measured from soils of contrasting (30-75%) water-filled pore space (WFPS). Oxidation rates of ¹³C-CH₄ were determined after application of 10 μl ¹³C-CH₄ l⁻¹ (10 at. % excess ¹³C) to soil headspace and comparisons made with estimates from changes in net CH₄ emission in these treatments and under ambient CH₄ where no ¹³C-CH₄ had been applied. We found a significant effect of soil WFPS on ¹³C-CH₄ oxidation rates and evidence for oxidation of 2.2 μg ¹³C-CH₄ d⁻¹ occurring in the 75% WFPS soil, which may have been either aerobic oxidation occurring in aerobic microsites in this soil or anaerobic CH₄ oxidation. The lowest ¹³C-CH₄ oxidation rate was measured in the 30% WFPS soil and was attributed to inhibition of methanotroph activity in this dry soil. However, oxidation was lowest in the wetter soils when estimated from changes in concentration of ¹²⁺¹³C-CH₄. Thus, both methanogenesis and CH₄ oxidation may have been occurring simultaneously in these wet soils, indicating the advantage of using a stable isotope approach to determine oxidation rates. Application of ¹³C-CH₄ at 10 μl ¹³C-CH₄ l⁻¹ resulted in more rapid oxidation than under ambient CH₄ conditions, suggesting CH₄ oxidation in this soil was substrate limited, particularly in the wetter soils. Application of and (80 mg N kg soil⁻¹; 9.9 at.% excess ¹⁵N) to different replicates enabled determination of the respective contributions of nitrification and denitrification to N₂O emissions. The highest N₂O emission (119 μg ¹⁴⁺¹⁵N-N₂O kg soil⁻¹ over 72 h) was measured from the 75% WFPS soil and was mostly produced during denitrification (18.1 μg ¹⁵N-N₂O kg soil⁻¹; 90% of ¹⁵N-N₂O from this treatment). Strong negative correlations between ¹⁴⁺¹⁵N-N₂O emissions, denitrified ¹⁵N-N₂O emissions and ¹³C-CH₄ concentrations (r=−0.93 to −0.95, N₂O; r=−0.87 to −0.95, denitrified ¹⁵N-N₂O; P<0.05) suggest a close relationship between CH₄ oxidation and denitrification in our soil, the nature of which requires further investigation.     

Ecology of aerobic methanotrophs in controlling methane fluxes from wetlands

Methane (CH₄) is a critical greenhouse gas, with wetlands and flooded rice soils contributing >38% of the global emissions of CH₄. A key process that offsets CH₄ production is biological oxidation by methanotrophs that can consume as much as 90% of the CH₄ produced in aerobic soils. The objective was to review the literature on the biochemical pathways, ecology of methanotrophs, research methods, and environmental and management controls on CH₄ oxidation in wetlands. Methanotrophs have been extensively studied using phospholipid fatty acids (PLFA) but much less so using nucleic acid analysis or with the powerful stable-isotope probing technique of ¹³C-PLFA analysis. Of the environmental factors that affect CH₄ oxidation, temperature, water content and redox potential are the most important whereas a wide range of soil pH enables oxidation. Methane oxidation varies with season as functions of temperature and plant community shifts (changes with growth stage of C input chemistry and oxygenation from aquatic rhizospheres). Indirect evidence suggests there is greater CH₄ oxidation in pulsing than permanently flooded soils based on observations that static wetlands have greater emissions than pulsing wetlands. Basic research is needed on: the role of inorganic N species in controlling CH₄ oxidation pathways; phylogenetic analysis of methanotrophs; and environmental and edaphic effects on methanotroph growth and activity. Research is needed to develop wetland systems that optimize CH₄ oxidation relative to wetland type and environments and hydrology while maintaining and/or promoting other ecosystem services (e.g. C sequestration, pollution remediation, wildlife habitat, flood control).     

Immediate effects of nitrogen, phosphorus, and potassium amendments on the methanotrophic activity and abundance in a Chinese paddy soil under short-term incubation experiment

Purpose

Methane-oxidizing bacteria (methanotrophs) biologically consume and consequently affect the concentration of atmospheric methane (CH₄), the second most prominent greenhouse gas, and therefore play critical roles in the mitigation of global warming effect. Long-term fertilization often affects the methanotrophic community and CH₄ oxidation in various soils. Here, the immediate effects of nitrogen (N), phosphorus (P), and potassium (K) amendments on the CH₄ oxidation activity and methanotrophic community structure were evaluated.

Materials and methods

Paddy soil samples were collected from the Taoyuan Experimental Station of the Chinese Academy of Sciences in central Hunan Province of China. A laboratory-based incubation experiment was conducted to investigate the immediate effects of N, P, and K amendments on the methanotrophs in soil. The CH₄ oxidation rates and methanotrophic activities were determined by measuring the dynamic changes of CH₄ concentration in the incubation system. The methanotrophic abundance and community changes in all of the seven treatments with and without nutrients addition were studied using real-time PCR and denaturing gradient gel electrophoresis, respectively.

Results and discussion

All of the N, P, and K treatments significantly decreased the CH₄ oxidation activities. Compared with the control, the P and K amendments significantly increased the methanotrophic population size, but the N treatments have no effect on the methanotrophic abundance. A negative correlation was found between methanotrophic activity and methanotrophic abundance. We suggested that methanotrophic activity may not be inferred through the pmoA gene copies, especially in the short-term simulation experiments. Investigation of the methanotrophic population size and diversity is not enough to evaluate the soil CH₄ sink accurately.

Conclusions

We concluded that the additions of N, P, and K reduce the activity but enhance the abundance of methanotrophs in a Chinese paddy soil through a short-term incubation experiment. Additionally, we found that the CH₄ oxidation activity could be completely inhibited by Cl^? toxicity. Our results implied that caution should be exercised in the types and amounts of fertilizers, especially KCl in agricultural systems to control the instantaneous increase in CH₄ emission from the field.     

Atmospheric nitrate deposition and the microbial degradation of cellobiose and vanillin in a northern hardwood forest

Human activity has increased the amount of N entering terrestrial ecosystems from atmospheric NO₃⁻ deposition. High levels of inorganic N are known to suppress the expression of phenol oxidase, an important lignin-degrading enzyme produced by white-rot fungi. We hypothesized that chronic NO₃⁻ additions would decrease the flow of C through the heterotrophic soil food web by inhibiting phenol oxidase and the depolymerization of lignocellulose. This would likely reduce the availability of C from lignocellulose for metabolism by the microbial community. We tested this hypothesis in a mature northern hardwood forest in northern Michigan, which has received experimental atmospheric N deposition (30 kg NO₃⁻-N ha⁻¹ y⁻¹) for nine years. In a laboratory study, we amended soils with ¹³C-labeled vanillin, a monophenolic product of lignin depolymerization, and ¹³C-labeled cellobiose, a disaccharide product of cellulose degradation. We then traced the flow of ¹³C through the microbial community and into soil organic carbon (SOC), dissolved organic carbon (DOC), and microbial respiration. We simultaneously measured the activity of enzymes responsible for lignin (phenol oxidase and peroxidase) and cellobiose (β-glucosidase) degradation. Nitrogen deposition reduced phenol oxidase activity by 83% and peroxidase activity by 74% when compared to control soils. In addition, soil C increased by 76%, whereas microbial biomass decreased by 68% in NO₃⁻ amended soils. ¹³C cellobiose in bacterial or fungal PLFAs was unaffected by NO₃⁻ deposition; however, the incorporation of ¹³C vanillin in fungal PLFAs extracted from NO₃⁻ amended soil was 82% higher than in the control treatment. The recovery of ¹³C vanillin and ¹³C cellobiose in SOC, DOC, microbial biomass, and respiration was not different between control and NO₃⁻ amended treatments. Chronic NO₃⁻ deposition has stemmed the flow of C through the heterotrophic soil food web by inhibiting the activity of ligninolytic enzymes, but it increased the assimilation of vanillin into fungal PLFAs.     

Agricultural factors affecting methane oxidation in arable soil

CH₄ oxidation activity in a sandy soil (Ardoyen) and agricultural practices affecting this oxidation were studied under laboratory conditions. CH₄ oxidation in the soil proved to be a biological process. The instantaneous rate of CH₄ consumption was in the order of 800 mol CH₄ kg^–1 day^–1 (13 mg CH₄ kg^–1 day^–1) provided the soil was treated with ca. 4.0 mmol CH₄ kg^–1 soil. Upon repeated supplies of a higher dose of CH₄, the oxidation was accelerated to a rate of at least 198 mg CH₄ kg^–1 day^–1. Addition of the plant-growth promoting rhizopseudomonad strains Pseudomonas aeruginosa 7NSK2 and Pseudomonas fluorescens ANP15 significantly decreased the CH₄ oxidation by 20 to 30% during a 5-day incubation. However, with further incubation this suppression was no longer detectable. Growing maize plants prevented the suppression of CH₄ oxidation. The numbers of methanotrophic bacteria and fungi increased significantly after the addition of CH₄, but there were no significant shifts in the population of total bacteria and fluorescent pseudomonads. Drying and rewetting of soil for at least 1 day significantly reduced the activity of the indigenous methanotrophs. Upon rewetting, their activity was regained after a lag phase of about 3 days. The herbicide dichlorophenoxy acetic acid (2,4-D) had a strong negative effect on CH₄ oxidation. The application of 5 ppm increased the time for CH₄ removal; at concentrations above 25 ppm 2,4-D CH₄–oxidizing activity was completely hampered. After 3 days of delay, only the treatments with below 25 ppm 2,4-D showed recovery of CH₄–oxidizing activity. This finding suggests that it can be important to include a CH₄–removal bioassay in ecotoxicology studies of the side effects of pesticides. Changes in the native soil pH also affected the CH₄–oxidizing capacity. Permanent inhibition occurred when the soil pH was altered by 2 pH units, and partial inhibition by 1 pH unit change. A rather narrow pH range (5.9–7.7) appeared to allow CH₄ oxidation. Soils pre-incubated with NH ₄ ⁺ had a lower CH₄–removal capacity. Moreover, the nitrification inhibitor 2-chloro-6-trichloromethyl pyridine (nitrapyrin) strongly inhibited CH₄ oxidation. Probably methanotrophs rather than nitrifying microorganisms are mainly responsible for CH₄ removal in the soil studied. It appears that the causal methanotrophs are remarkably sensitive to soil environmental disturbances.     

Methane concentrations and methanotrophic community structure influence the response of soil methane oxidation to nitrogen content in a temperate forest

Methane oxidation in forest soils removes atmospheric CH₄. Many studies have determined methane uptake rates and their controlling variables, yet the microorganisms involved have rarely been assessed simultaneously over the longer term. We measured methane uptake rates and the community structure of methanotrophic bacteria in temperate forest soil (sandy clay loam) on a monthly basis for two years in South Korea. Methane uptake rates at the field site did not show any seasonal patterns, and net uptake occurred throughout both years. In situ uptake rates and uptake potentials determined in the laboratory were 2.92 ± 4.07 mg CH₄ m⁻² day⁻¹ and 51.6 ± 45.8 ng CH₄ g⁻¹ soil day⁻¹, respectively. Contrary to results from other studies, in situ oxidation rates were positively correlated with soil nitrate concentrations. Short-term experimental nitrate addition (0.20-1.95 μg N g⁻¹ soil) significantly stimulated oxidation rates under low methane concentrations (1.7-2.0 ppmv CH₄), but significantly inhibited oxidation under high methane concentrations (300 ppmv CH₄). We analyzed the community structures of methanotrophic bacteria using a DNA-based fingerprinting method (T-RFLP). Type II methanotrophs dominated under low methane concentrations while Type I methanotrophs dominated under high methane concentrations. Nitrogen addition selectively inhibited Type I methanotrophic bacteria. Overall, the results of this study indicate that the effects of inorganic N on methane uptake depend on methane concentrations and that such a response is related to the dissimilar activation or inhibition of different types of methanotrophic bacteria.     

Response of CH<Subscript>4</Subscript> oxidation and methanotrophic diversity to NH<Subscript>4</Subscript><Superscript>+</Superscript> and CH<Subscript>4</Subscript> mixing ratios

Methane oxidising activity and community structure of 11, specifically targeted, methanotrophic species have been examined in an arable soil. Soils were sampled from three different field plots, receiving no fertilisation (C), compost (G) and mineral fertiliser (M), respectively. Incubation experiments were carried out with and without pre-incubation at elevated CH₄ mixing ratios (100 ml CH₄ l⁻¹) and with and without ammonium (100 mg N kg⁻¹) pre-incubation. Four months after fertilisation, plots C, G and M did not show significant differences in physicochemical properties and CH₄ oxidising activity. The total number of methanotrophs (determined as the sum the 11 specifically targeted methanotrophs) in the fresh soils was 17.0×10⁶, 13.7×10⁶ and 15.5×10⁶ cells g⁻¹ for treatment C, G and M, respectively. This corresponded to 0.11 to 0.32% of the total bacterial number. The CH₄ oxidising activity increased 10⁵-fold (20–26 mg CH₄ g⁻¹ h⁻¹), the total number of methanotrophs doubled (28–76×10⁶ cells g⁻¹) and the methanotrophic diversity markedly increased in treatments with a pre-incubation at elevated CH₄ concentrations. In all soils and treatments, type II methanotrophs (62–91%) outnumbered type I methanotrophs (9–38%). Methylocystis and Methylosinus species were always most abundant. After pre-incubation with ammonium, CH₄ oxidation was completely inhibited; however, no change in the methanotrophic community structure could be detected.     

Effect of summer throughfall exclusion, summer drought, and winter snow cover on methane fluxes in a temperate forest soil

Soil moisture strongly controls the uptake of atmospheric methane by limiting the diffusion of methane into the soil, resulting in a negative correlation between soil moisture and methane uptake rates under most non-drought conditions. However, little is known about the effect of water stress on methane uptake in temperate forests during severe droughts. We simulated extreme summer droughts by exclusion of 168 mm (2001) and 344 mm (2002) throughfall using three translucent roofs in a mixed deciduous forest at the Harvard Forest, Massachusetts, USA. The treatment significantly increased CH₄ uptake during the first weeks of throughfall exclusion in 2001 and during most of the 2002 treatment period. Low summertime CH₄ uptake rates were found only briefly in both control and exclusion plots during a natural late summer drought, when water contents below 0.15 g cm⁻³ may have caused water stress of methanotrophs in the A horizon. Because these soils are well drained, the exclusion treatment had little effect on A horizon water content between wetting events, and the effect of water stress was smaller and more brief than was the overall treatment effect on methane diffusion. Methane consumption rates were highest in the A horizon and showed a parabolic relationship between gravimetric water content and CH₄ consumption, with maximum rate at 0.23 g H₂O g⁻¹ soil. On average, about 74% of atmospheric CH₄ was consumed in the top 4-5 cm of the mineral soil. By contrast, little or no CH₄ consumption occurred in the O horizon. Snow cover significantly reduced the uptake rate from December to March. Removal of snow enhanced CH₄ uptake by about 700-1000%, resulting in uptake rates similar to those measured during the growing season. Soil temperatures had little effect on CH₄ uptake as long as the mineral soil was not frozen, indicating strong substrate limitation of methanotrophs throughout the year. Our results suggest that the extension of snow periods may affect the annual rate of CH₄ oxidation and that summer droughts may increase the soil CH₄ sink of temperate forest soils.     

Identification of Metabolically Active Rhizosphere Microorganisms by Stable Isotopic Probing of PLFA in Switchgrass

Root-derived rhizodeposits of recent photosynthetic carbon (C) are the foremost source of energy for microbial growth and development in rhizosphere soil. A substantial amount of photosynthesized C by the plants is translocated to belowground and is released as root exudates that influence the structure and function of soil microbial communities with potential inference in nutrient and C cycling in the ecosystem. We applied the ¹³C pulse chase labeling technique to evaluate the incorporation of rhizodeposit-C into the phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of switchgrass (Panicum virgatum L.). Soil samples of bulk and rhizosphere were taken at 1, 5, 10 and 20 days after labeling and analyzed for ¹³C enrichment in the microbial PLFAs. Temporal differences of ¹³C enrichment in PLFAs were more prominent than spatial differences. Among the microbial PLFA biomarkers, fungi and Gram-negative (GM-ve) bacterial PLFAs showed rapid enrichment with ¹³C compared to Gram-positive (GM+ve) and actinomycetes in rhizosphere soil. The ¹³C enrichment of actinomycetes biomarker PLFA significantly increased along with sampling time in both soils. PLFAs indicative to fungi, GM-ve and GM+ve showed a significant decrease in ¹³C enrichment over sampling time in the rhizosphere, but a decrease was also observed in GM-ve (16:1ω5c) and fungal biomarker PLFAs in the bulk soil. The relative ¹³C concentration in fungal PLFA decreased on day 10, whereas those of GM-ve increased on day 5 and GM+ve remained constant in the rhizosphere soil. However, the relative ¹³C concentrations of GM-ve and GM+ve increased on days 5 and 10, respectively, and those of fungal remain constant in the bulk soil. The present study demonstrates the usefulness of ¹³C pulse chase labeling together with PLFA analysis to evaluate the active involvement of microbial community groups for utilizing rhizodeposit-C.     

Structure and activity of soil-inhabiting methanotrophic communities in northern forest of Japan

In a Japanese forest, CH₄ uptake rate and methanotrophic community structure in the soil were investigated at four sites of different vegetation. At two of these sites, undergrowth was dominated by Sasa senanensis, and that of another was dominated by Sasa kurilensis. At the rest site, undergrowth had been removed artificially. The tree-layer composition differed between the two sites with S. senanensis, but tree layer of the other two sites were dominated by the same species. At the site lacking undergrowth, observed CH₄ uptake rate was twice as high as that at the other sites. Under laboratory conditions, soil sample from the site lacking undergrowth exhibited CH₄ consumption rate higher than that of the adjacent site with the same dominating tree species. The community structures of methanotrophs were investigated with denaturing gradient gel electrophoresis (DGGE) of the gene encoding particulate methane monooxygenase (pmoA). The banding patterns observed were different depending on the type of undergrowth vegetation. The sequences of the DGGE bands were closely related to each other and belonged to the “upland soil cluster alpha” (USCα). These results imply possible close relationship between the undergrowth vegetation and methanotrophic communities in forest soils.     

Methane production and consumption in a cultivated humisol

Summary Laboratory studies were conducted on a cultivated humisol containing populations of both methanotrophs and methanogens. The molar ratio CO₂ produced : O₂ consumed :CH₄ consumed was 0.27:1.0:1.0. Methane oxidation showed typical Michaelis-Menten kinetics with apparent K _m values for CH4 and O₂ of 66.2 M and 37.0 M, respectively. The low CO₂ yields and the effects of low dissolved oxygen indicated the presence of aerobic obligate methanotrophs. It is suggested that the methanotrophs in this soil are not entirely dependent on atmospheric CH₄ for growth and survival in situ.     

Microbial community composition and rhizodeposit-carbon assimilation in differently managed temperate grassland soils

Rhizodeposit-carbon provides a major energy source for microbial growth in the rhizosphere of grassland soils. However, little is known about the microbial communities that mediate the rhizosphere carbon dynamics, especially how their activity is influenced by changes in soil management. We combined a ¹³CO₂ pulse-labeling experiment with phospholipid fatty acid (PLFA) analysis in differently managed Belgian grasslands to identify the active rhizodeposit-C assimilating microbial communities in these grasslands and to evaluate their response to management practices. Experimental treatments consisted of three mineral N fertilization levels (0, 225 and 450 kg N ha⁻¹ y⁻¹) and two mowing frequencies (3 and 5 times y⁻¹). Phospholipid fatty acids were extracted from surface (0-5 cm) bulk (BU) and root-adhering (RA) soil samples prior to and 24 h after pulse-labeling and were analyzed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-c-IRMS). Soil habitats significantly differed in microbial community structure (as revealed by multivariate analysis of mol% biomarker PLFAs) as well as in gram-positive bacterial rhizodeposit-C uptake (as revealed by greater ¹³C-PLFA enrichment following pulse-labeling in RA compared to BU soil in the 450N/5M treatment). Mowing frequency did not significantly alter the relative abundance (mol%) or activity (¹³C enrichment) of microbial communities. In the non-fertilized treatment, the greatest ¹³C enrichment was seen in all fungal biomarker PLFAs (C16:1ω5, C18:1ω9, C18:2ω6,9 and C18:3ω3,6,9), which demonstrates a prominent contribution of fungi in the processing of new photosynthate-C in non-fertilized grassland soils. In all treatments, the lowest ¹³C enrichment was found in gram-positive bacterial and actinomycetes biomarker PLFAs. Fungal biomarker PLFAs had significantly lower ¹³C enrichment in the fertilized compared to non-fertilized treatments in BU soil (C16:1ω5, C18:1ω9) as well as RA soil (all fungal biomarkers). While these observations clearly indicated a negative effect of N fertilization on fungal assimilation of plant-derived C, the effect of N fertilization on fungal abundance could only be detected for the arbuscular mycorrhizal fungal (AMF) PLFA (C16:1ω5). On the other hand, increases in the relative abundance of gram-positive bacterial PLFAs with N fertilization were found without concomitant increases in ¹³C enrichment following pulse-labeling. We conclude that in situ¹³C pulse-labeling of PLFAs is an effective tool to detect functional changes of those microbial communities that are dominantly involved in the immediate processing of new rhizosphere-C.     

Interactive effects of tree species and soil moisture on methane consumption

Methane consumption by temperate forest soils is a major sink for this important greenhouse gas, but little is known about how tree species influence CH₄ uptake by soils. Here, we show that six common tree species in Siberian boreal and temperate forests significantly affect potential CH₄ consumption in laboratory microcosms. Overall, soils under hardwood species (aspen and birch) consumed CH₄ at higher rates than soils under coniferous species and grassland. While NH₄⁺ addition often reduces CH₄ uptake, we found no effect of NH₄⁺ addition, possibly because of the relatively high ratio of CH₄-to-NH₄⁺ in our incubations. The effects of soil moisture strongly depended on plant species. An increase in soil moisture enhanced CH₄ consumption in soils under spruce but had the opposite effect under Scots pine and larch. Under other species, soil moisture did not affect CH₄ consumption. These results could be explained by specific responses of different groups of CH₄-oxidizing bacteria to elevated moisture.     

Linking dynamics of soil microbial phospholipid fatty acids to carbon mineralization in a C natural abundance experiment: Impact of heavy metals and acid rain

A ¹³C natural abundance experiment including GC-c-IRMS analysis of phospholipid fatty acids (PLFAs) was conducted to assess the temporal dynamics of the soil microbial community and carbon incorporation during the mineralization of plant residues under the impact of heavy metals and acid rain. Maize straw was incorporated into (i) control soil, (ii) soil irrigated with acid rain, (iii) soil amended with heavy metal-polluted filter dust and (iv) soil with both, heavy metal and acid rain treatment, over a period of 74 weeks. The mineralization of maize straw carbon was significantly reduced by heavy metal impact. Reduced mineralization rate of the added carbon likely resulted from a reduction of the microbial biomass due to heavy metal stress, while the efficiency of ¹³C incorporation into microbial PLFAs was hardly affected. Since acid rain did not significantly change soil pH, little impact on soil microorganisms and mineralization rate was found. Temporal dynamics of labelling of microbial PLFAs were different between bacterial and fungal PLFA biomarkers. Utilization of maize straw by bacterial PLFAs peaked immediately after the application (2 weeks), while labelling of the fungal biomarker 18:2ω6,9 was most pronounced 5 weeks after the application. In general, ¹³C labelling of microbial PLFAs was closely linked to the amounts of maize carbon present in the soil. The distinct higher labelling of microbial PLFAs in the heavy metal-polluted soils 74 weeks after application indicated a large fraction of available maize straw carbon still present in the soil.