Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid 13C labeling

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The delta13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1omega7c, with 16:1omega5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 10(6) cells g(-1) of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.     

Radioactive Fingerprinting of Microorganisms That Oxidize Atmospheric Methane in Different Soils

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with ¹⁴CH₄ followed by analysis of radiolabelled phospholipid ester-linked fatty acids (¹⁴C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The ¹⁴C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1ω8c (up to 9.0% of the total ¹⁴C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1ω9, 18:1ω7, and 18:0). The ¹⁴C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH₄. The ¹⁴C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the ¹⁴C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.     

Partitioning of CH4 and CO2 Production Originating from Rice Straw,Soil and Root Organic Carbon in Rice Microcosms

Flooded rice fields are an important source of the greenhouse gas CH₄. Possible carbon sources for CH₄ and CO₂ production in rice fields are soil organic matter (SOM), root organic carbon (ROC) and rice straw (RS), but partitioning of the flux between the different carbon sources is difficult. We conducted greenhouse experiments using soil microcosms planted with rice. The soil was amended with and without ¹³C-labeled RS, using two ¹³C-labeled RS treatments with equal RS (5 g kg⁻¹ soil) but different δ¹³C of RS. This procedure allowed to determine the carbon flux from each of the three sources (SOM, ROC, RS) by determining the δ¹³C of CH₄ and CO₂ in the different incubations and from the δ¹³C of RS. Partitioning of carbon flux indicated that the contribution of ROC to CH₄ production was 41% at tillering stage, increased with rice growth and was about 60% from the booting stage onwards. The contribution of ROC to CO₂ was 43% at tillering stage, increased to around 70% at booting stage and stayed relatively constant afterwards. The contribution of RS was determined to be in a range of 12–24% for CH₄ production and 11–31% for CO₂ production; while the contribution of SOM was calculated to be 23–35% for CH₄ production and 13–26% for CO₂ production. The results indicate that ROC was the major source of CH₄ though RS application greatly enhanced production and emission of CH₄ in rice field soil. Our results also suggest that data of CH₄ dissolved in rice field could be used as a proxy for the produced CH₄ after tillering stage.     

Bacteria and Fungi Respond Differently to Multifactorial Climate Change in a Temperate Heathland,Traced with 13C-Glycine and FACE CO2

It is vital to understand responses of soil microorganisms to predicted climate changes, as these directly control soil carbon (C) dynamics. The rate of turnover of soil organic carbon is mediated by soil microorganisms whose activity may be affected by climate change. After one year of multifactorial climate change treatments, at an undisturbed temperate heathland, soil microbial community dynamics were investigated by injection of a very small concentration (5.12 µg C g⁻¹ soil) of ¹³C-labeled glycine (¹³C₂, 99 atom %) to soils in situ. Plots were treated with elevated temperature (+1°C, T), summer drought (D) and elevated atmospheric carbon dioxide (510 ppm [CO2]), as well as combined treatments (TD, TCO2, DCO2 and TDCO2). The ¹³C enrichment of respired CO₂ and of phospholipid fatty acids (PLFAs) was determined after 24 h. ¹³C-glycine incorporation into the biomarker PLFAs for specific microbial groups (Gram positive bacteria, Gram negative bacteria, actinobacteria and fungi) was quantified using gas chromatography-combustion-stable isotope ratio mass spectrometry (GC-C-IRMS).Gram positive bacteria opportunistically utilized the freshly added glycine substrate, i.e. incorporated ¹³C in all treatments, whereas fungi had minor or no glycine derived ¹³C-enrichment, hence slowly reacting to a new substrate. The effects of elevated CO₂ did suggest increased direct incorporation of glycine in microbial biomass, in particular in G⁺ bacteria, in an ecosystem subjected to elevated CO₂. Warming decreased the concentration of PLFAs in general. The FACE CO₂ was ¹³C-depleted (δ¹³C = 12.2‰) compared to ambient (δ¹³C = ∼−8‰), and this enabled observation of the integrated longer term responses of soil microorganisms to the FACE over one year. All together, the bacterial (and not fungal) utilization of glycine indicates substrate preference and resource partitioning in the microbial community, and therefore suggests a diversified response pattern to future changes in substrate availability and climatic factors.     

Changing land use reduces soil CH4 uptake by altering biomass and activity but not composition of high-affinity methanotrophs

Forest ecosystems assimilate more CO₂ from the atmosphere and store more carbon in woody biomass than most nonforest ecosystems, indicating strong potential for afforestation to serve as a carbon management tool. However, converting grasslands to forests could affect ecosystem–atmosphere exchanges of other greenhouse gases, such as nitrous oxide and methane (CH₄), effects that are rarely considered. Here, we show that afforestation on a well-aerated grassland in Siberia reduces soil CH₄ uptake by a factor of 3 after 35 years of tree growth. The decline in CH₄ oxidation was observed both in the field and in laboratory incubation studies under controlled environmental conditions, suggesting that not only physical but also biological factors are responsible for the observed effect. Using incubation experiments with ¹³CH₄ and tracking ¹³C incorporation into bacterial phospholipid fatty acid (PLFA), we found that, at low CH₄ concentrations, most of the ¹³C was incorporated into only two PLFAs, 18 : 1ω7 and 16 : 0. High CH₄ concentration increased total ¹³C incorporation and the number of PLFA peaks that became labeled, suggesting that the microbial assemblage oxidizing CH₄ shifts with ambient CH₄ concentration. Forests and grasslands exhibited similar labeling profiles for the high-affinity methanotrophs, suggesting that largely the same general groups of methanotrophs were active in both ecosystems. Both PLFA concentration and labeling patterns indicate a threefold decline in the biomass of active methanotrophs due to afforestation, but little change in the methanotroph community. Because the grassland consumed CH₄ at a rate five times higher than forest soils under laboratory conditions, we concluded that not only biomass but also cell-specific activity was higher in grassland than in afforested plots. While the decline in biomass of active methanotrophs can be explained by site preparation (plowing), inorganic N (especially NH₄⁺) could be responsible for the change in cell-specific activity. Overall, the negative effect of afforestation of upland grassland on soil CH₄ uptake can be largely explained by the reduction in biomass and to a lesser extent by reduced cell-specific activity of CH₄-oxidizing bacteria.     

Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils

Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the “forest sequence cluster” (USC α), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel “upland soil cluster γ” (USC γ) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with ¹³CH₄ at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of ¹³C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC γ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC α sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.     

Microbial 13C utilization patterns via stable isotope probing of phospholipid biomarkers in Mojave Desert soils exposed to ambient and elevated atmospheric CO2

Changes in plant inputs under changing atmospheric CO₂ can be expected to alter the size and/or functional characteristics of soil microbial communities which can determine whether soils are a C sink or source. Stable isotope probing was used to trace autotrophically fixed ¹³C into phospholipid fatty acid (PLFA) biomarkers in Mojave Desert soils planted with the desert shrub, Larrea tridentata. Seedlings were pulse‐labeled with ¹³CO₂ under ambient and elevated CO₂ in controlled environmental growth chambers. The label was chased into the soil by extracting soil PLFAs after labeling at Days 0, 2, 10, 24, and 49. Eighteen of 29 PLFAs identified showed ¹³C enrichment relative to nonlabeled control soils. Patterns of PLFA enrichment varied temporally and were similar for various PLFAs found within a microbial functional group. Enrichment of PLFA ¹³C generally occurred within the first 2 days in general and fungal biomarkers, followed by increasingly greater enrichment in bacterial biomarkers as the study progressed (Gram‐negative, Gram‐positive, actinobacteria). While treatment CO₂ level did not affect total PLFA‐C concentrations, microbial functional group abundances and distribution responded to treatment CO₂ level and these shifts persisted throughout the study. Specifically, ratios of bacterial‐to‐total PLFA‐C decreased and fungal‐to‐bacterial PLFA‐C increased under elevated CO₂ compared with ambient conditions. Differences in the timing of ¹³C incorporation into lipid biomarkers coupled with changes in microbial functional groups indicate that microbial community characteristics in Mojave Desert soils have shifted in response to long‐term exposure to increased atmospheric CO₂.     

Dynamics of a Pasture Soil Microbial Community after Deposition of Cattle Urine Amended with [13C]Urea

Within grazed pastures, urine patches are hot spots of nitrogen turnover, since dietary N surpluses are excreted mainly as urea in the urine. This short-term experiment investigated ¹³C uptake in microbial lipids after simulated deposition of cattle urine at 10.0 and 17.1 g of urea C m⁻². Confined field plots without or with cattle urine amendment were sampled after 4 and 14 days, and soil from 0- to 5-cm and 10- to 20-cm depths was analyzed for content and composition of phospholipid fatty acids (PLFAs) and for the distribution of urea-derived ¹³C among individual PLFAs. Carbon dioxide emissions were quantified, and the contributions derived from urea were assessed. Initial changes in PLFA composition were greater at the lower level of urea, as revealed by a principal-component analysis. At the higher urea level, osmotic stress was indicated by the dynamics of cyclopropane fatty acids and branched-chain fatty acids. Incorporation of ¹³C from [¹³C]urea was low but significant, and the largest amounts of urea-derived C were found in common fatty acids (i.e., 16:0, 16:1ω7c, and 18:1ω7) that would be consistent with growth of typical NH₄⁺-oxidizing (Nitrosomonas) and NO₂⁻-oxidizing (Nitrobacter) bacteria. Surprisingly, a 20‰ depletion of ¹³C in the cyclopropane fatty acid cy17:0 was observed after 4 days, which was replaced by a 10 to 20‰ depletion of that in cy19:0 after 14 days. Possible reasons for this pattern are discussed. Autotrophic nitrifiers could not be implicated in urea hydrolysis to any large extent, but PLFA dynamics and the incorporation of urea-derived ¹³C in PLFAs indicated a response of nitrifiers which differed between the two urea concentrations.     

Microbial Community Dynamics Associated with Rhizosphere Carbon Flow

Root-deposited photosynthate (rhizodeposition) is an important source of readily available carbon (C) for microbes in the vicinity of growing roots. Plant nutrient availability is controlled, to a large extent, by the cycling of this and other organic materials through the soil microbial community. Currently, our understanding of microbial community dynamics associated with rhizodeposition is limited. We used a ¹³C pulse-chase labeling procedure to examine the incorporation of rhizodeposition into individual phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of greenhouse-grown annual ryegrass (Lolium multiflorum Lam. var. Gulf). Labeling took place during a growth stage in transition between active root growth and rapid shoot growth on one set of plants (labeling period 1) and 9 days later during the rapid shoot growth stage on another set of plants (labeling period 2). Temporal differences in microbial community composition were more apparent than spatial differences, with a greater relative abundance of PLFAs from gram-positive organisms (i15:0 and a15:0) in the second labeling period. Although more abundant, gram-positive organisms appeared to be less actively utilizing rhizodeposited C in labeling period 2 than in labeling period 1. Gram-negative bacteria associated with the 16:1ω5 PLFA were more active in utilizing ¹³C-labeled rhizodeposits in the second labeling period than in the first labeling period. In both labeling periods, however, the fungal PLFA 18:2ω6,9 was the most highly labeled. These results demonstrate the effectiveness of using ¹³C labeling and PLFA analysis to examine the microbial dynamics associated with rhizosphere C cycling by focusing on the members actively involved.     

In Vitro Study of Lipid Biosynthesis in an Anaerobically Methane-Oxidizing Microbial Mat

The anaerobic oxidation of methane (AOM) is a key process in the global methane cycle, and the majority of methane formed in marine sediments is oxidized in this way. Here we present results of an in vitro ¹³CH₄ labeling study (δ¹³CH₄, ~5,400‰) in which microorganisms that perform AOM in a microbial mat from the Black Sea were used. During 316 days of incubation, the ¹³C uptake into the mat biomass increased steadily, and there were remarkable differences for individual bacterial and archaeal lipid compounds. The greatest shifts were observed for bacterial fatty acids (e.g., hexadec-11-enoic acid [16:1Δ11]; difference between the δ¹³C at the start and the end of the experiment [Δδ¹³C_start-end], ~160‰). In contrast, bacterial glycerol diethers exhibited only slight changes in δ¹³C (Δδ¹³C_start-end, ~10‰). Differences were also found for individual archaeal lipids. Relatively high uptake of methane-derived carbon was observed for archaeol (Δδ¹³C_start-end, ~25‰), a monounsaturated archaeol, and biphytanes, whereas for sn-2-hydroxyarchaeol there was considerably less change in the δ¹³C (Δδ¹³C_start-end, ~2‰). Moreover, an increase in the uptake of ¹³C for compounds with a higher number of double bonds within a suite of polyunsaturated 2,6,10,15,19-pentamethyleicosenes indicated that in methanotrophic archaea there is a biosynthetic pathway similar to that proposed for methanogenic archaea. The presence of group-specific biomarkers (for ANME-1 and ANME-2 associations) and the observation that there were differences in ¹³C uptake into specific lipid compounds confirmed that multiple phylogenetically distinct microorganisms participate to various extents in biomass formation linked to AOM. However, the greater ¹³C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.     

Linking Toluene Degradation with Specific Microbial Populations in Soil

Phospholipid fatty acid (PLFA) analysis of a soil microbial community was coupled with ¹³C isotope tracer analysis to measure the community’s response to addition of 35 μg of [¹³C]toluene ml of soil solution⁻¹. After 119 h of incubation with toluene, 96% of the incorporated ¹³C was detected in only 16 of the total 59 PLFAs (27%) extracted from the soil. Of the total ¹³C-enriched PLFAs, 85% were identical to the PLFAs contained in a toluene-metabolizing bacterium isolated from the same soil. In contrast, the majority of the soil PLFAs (91%) became labeled when the same soil was incubated with [¹³C]glucose. Our study showed that coupling ¹³C tracer analysis with PLFA analysis is an effective technique for distinguishing a specific microbial population involved in metabolism of a labeled substrate in complex environments such as soil.     

Linking activity,composition and seasonal dynamics of atmospheric methane oxidizers in a meadow soil

Microbial oxidation is the only biological sink for atmospheric methane. We assessed seasonal changes in atmospheric methane oxidation and the underlying methanotrophic communities in grassland near Giessen (Germany), along a soil moisture gradient. Soil samples were taken from the surface layer (0–10 cm) of three sites in August 2007, November 2007, February 2008 and May 2008. The sites showed seasonal differences in hydrological parameters. Net uptake rates varied seasonally between 0 and 70 μg CH₄ m⁻² h⁻¹. Greatest uptake rates coincided with lowest soil moisture in spring and summer. Over all sites and seasons, the methanotrophic communities were dominated by uncultivated methanotrophs. These formed a monophyletic cluster defined by the RA14, MHP and JR1 clades, referred to as upland soil cluster alphaproteobacteria (USCα)-like group. The copy numbers of pmoA genes ranged between 3.8 × 10⁵–1.9 × 10⁶ copies g⁻¹ of soil. Temperature was positively correlated with CH₄ uptake rates (P<0.001), but had no effect on methanotrophic population dynamics. The soil moisture was negatively correlated with CH₄ uptake rates (P<0.001), but showed a positive correlation with changes in USCα-like diversity (P<0.001) and pmoA gene abundance (P<0.05). These were greatest at low net CH₄ uptake rates during winter times and coincided with an overall increase in bacterial 16S rRNA gene abundances (P<0.05). Taken together, soil moisture had a significant but opposed effect on CH₄ uptake rates and methanotrophic population dynamics, the latter being increasingly stimulated by soil moisture contents >50 vol% and primarily related to members of the MHP clade.     

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH₄ and CO₂ assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO₂ reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ¹³C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ¹⁴C from radiolabeled CH₄ or CO₂ revealed one hot spot for methanotrophy and CO₂ fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ¹⁴CH₄ or ¹⁴CO₂ revealed that there was interconversion of CH₄ and CO_2. The level of CO₂ reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Methane-Derived Carbon in the Benthic Food Web in Stream Impoundments

Methane gas (CH₄) has been identified as an important alternative source of carbon and energy in some freshwater food webs. CH₄ is oxidized by methane oxidizing bacteria (MOB), and subsequently utilized by chironomid larvae, which may exhibit low δ¹³C values. This has been shown for chironomid larvae collected from lakes, streams and backwater pools. However, the relationship between CH₄ concentrations and δ¹³C values of chironomid larvae for in-stream impoundments is unknown. CH₄ concentrations were measured in eleven in-stream impoundments located in the Queich River catchment area, South-western Germany. Furthermore, the δ¹³C values of two subfamilies of chironomid larvae (i.e. Chironomini and Tanypodinae) were determined and correlated with CH₄ concentrations. Chironomini larvae had lower mean δ¹³C values (−29.2 to −25.5 ‰), than Tanypodinae larvae (−26.9 to −25.3 ‰). No significant relationships were established between CH₄ concentrations and δ¹³C values of chironomids (p>0.05). Mean δ¹³C values of chironomid larvae (mean: −26.8‰, range: −29.2‰ to −25.3‰) were similar to those of sedimentary organic matter (SOM) (mean: −28.4‰, range: −29.3‰ to −27.1‰) and tree leaf litter (mean: −29.8 ‰, range: −30.5‰ to −29.1‰). We suggest that CH₄ concentration has limited influence on the benthic food web in stream impoundments.     

Use of Phospholipid Fatty Acids To Detect Previous Self-Heating Events in Stored Peat

The use of the phospholipid fatty acid (PLFA) composition of microorganisms to detect previous self-heating events was studied in naturally self-heated peat and in peat incubated under temperature-controlled conditions. An increased content of total PLFAs was found in self-heated peat compared to that in unheated peat. Two PLFAs, denoted T1 and T2, were detected only in the self-heated peat. Incubation of peat samples at 25 to 55°C for 4 days indicated that T1 and T2 were produced from microorganisms with different optimum temperatures. This was confirmed by isolation of bacteria at 55°C, which produced T2 but not T1. These bacteria produced another PLFA (denoted T3) which coeluted with 18:1ω7. T2 and T3 were identified as ω-cyclohexyltridecanoic acid and ω-cyclohexylundecanoic acid, respectively, indicating that the bacteria belonged to the genus Alicyclobacillus. T1 was tentatively identified as ω-cycloheptylundecanoic acid. T2 was detected 8 h after the peat incubation temperature was increased to 55°C, and maximum levels were found within 5 days of incubation. The PLFA 18:1ω7-T3 increased in proportion to T2. T1 was detected after 96 h at 55°C, and its level increased throughout the incubation period, so that it eventually became one of the dominant PLFAs after 80 days. In peat samples incubated at 55°C and then at 25°C, T1 and T2 disappeared slowly. After 3 months, detectable levels were still found. Incubation at 25°C after heating for 3 days at 55°C decreased the amounts of T2 and 18:1ω7-T3 faster than did incubation at 5°C. Thus, not only the duration and temperature during the heating event but also the storage temperature following heating are important for the detection of PLFAs indicating previous self-heating.     

A continuous labelling approach to recover photosynthetically fixed carbon in plant tissue and rhizosphere organisms of young beech trees (Fagus sylvatica L.) using 13C depleted CO2

A continuous labelling experiment using ¹³C-CO₂ was set up in open-top chambers in order to follow fluxes of assimilates from the plant into the rhizosphere. Labelling was performed for one growing season by adding low amounts of CO₂ depleted in ¹³C to the atmosphere of the open-top chambers, resulting in a difference of ? ¹³C 5‰ V-PDB compared to ambient conditions. The label was recovered in both plant parts and soil microbial communities, analysed via phospholipid fatty acid (PLFA) side chains. PLFA 18:2ω6,9 showed a significant incorporation of the ¹³C label in October, indicating that fungi utilized plant derived carbon. In bacterial PLFA no label incorporation was detected, probably due to a lower use of rhizodeposits or a preference to older carbon compounds as energy sources. This experimental setup represents a low-cost continuous labelling method for field experiments with only minor increase of CO₂ concentrations.     

Quantification of methane oxidation in the rhizosphere of emergent aquatic macrophytes: defining upper limits

Rates of rhizospheric methane oxidation were evaluated by aerobic incubations of subcores collected in flooded anoxic soils populated by emergent macrophytes, by greenhouse whole plant incubations, and by CH₄ stable isotopic analysis. Subcore incubations defined upper limits for rhizospheric methane oxidation on an areal basis which were equal to or greater than emission rates. These rates are considered upper limits because O₂ did not limit CH₄ uptake as is likely to occur in situ. The ratio of maximum potential methane oxidation (MO) to methane emission (ME) ranged from 0.7 to 1.9 in Louisiana rice (Oryza sativa), from 1.0 to 4.0 in a N. Florida Sagittaria lancifolia marsh, and from 5.6 to 51 in Everglades Typha domingensis and Cladium jamaicense areas. Methane oxidation/methane emission ratios determined in whole plant incubations of Sagittaria lancifolia under oxic and anoxic conditions ranged from 0.5 to 1.6. Methane oxidation activity associated with emergent aquatic macrophytes was found primarily in fine root material. A weak correlation was observed between live root biomass and CH₄ uptake in Typha. Rhizomes showed small or zero rates of methane uptake and no uptake was associated with plant stems. Methane stable isotope data from a S. lancifolia marsh were as follows: CH₄ emitted from plants: −61.6 ± 0.3%; CH₄ within stems: −42.0 ± 0.2%; CH₄ within sedimentary bubbles: −51.7 ± 0.3%). The ¹³C enrichment observed relative to emitted CH₄ could be due to preferential mobilization of CH₄ containing the lighter isotope and/or the action of methanotrophic bacteria.     

New Insights into the Ligninolytic Capability of a Wood Decay Ascomycete

Wood-grown cultures of Daldinia concentrica oxidized a permethylated β-¹⁴C-labeled synthetic lignin to ¹⁴CO₂ and also cleaved a permethylated α-¹³C-labeled synthetic lignin to give C_α-C_β cleavage products that were detected by ¹³C nuclear magnetic resonance spectrometry. Therefore, this ascomycete resembles white-rot basidiomycetes in attacking the recalcitrant nonphenolic structures that predominate in lignin.     

Microbial assimilation of new photosynthate is altered by plant species richness and nitrogen deposition

To determine how plant species richness impacts microbial assimilation of new photosynthate, and how this may be modified by atmospheric N deposition, we analyzed the microbial assimilation of recent photosynthate in a 6-year-long field experiment in which plant species richness, atmospheric N deposition, and atmospheric CO₂ concentration were manipulated in concert. The depleted δ¹³C of fumigation CO₂ enabled us to investigate the effect of plant species richness and atmospheric N deposition on the metabolism of soil microbial communities in the elevated CO₂ treatment. To accomplish this, we determined the δ¹³C of bacterial, actinobacterial, and fungal phospholipid fatty acids (PLFAs). In the elevated CO₂ conditions of this study, the δ¹³C of bacterial PLFAs (i15:0, i16:0, 16:1ω7c, 16:1ω9c, 10Me16:0, and 10Me18:0) and the fungal PLFA 18:1ω9c was significantly lower in species-rich plant communities than in species-poor plant communities, indicating that microbial incorporation of new C increased with plant species richness. Despite an increase in plant production, total PLFA decreased under N deposition. Moreover, N deposition also decreased fungal relative abundance in species-rich plant communities. In our study, plant species richness directly increased microbial incorporation of new photosynthate, providing a mechanistic link between greater plant detritus production in species-rich plant communities and larger and more active soil microbial community.