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.     

The active methanotrophic community in hydromorphic soils changes in response to changing methane concentration

Methanotrophic communities were studied in several periodically water-saturated gleyic soils. When sampled, each soil had an oxic upper layer and consumed methane from the atmosphere (at 1.75 ppmv). In most gleyic soils the K(m(app)) values for methane were between 70 and 800 ppmv. These are higher than most values observed in dry upland soils, but lower than those measured in wetlands. Based on cultivation-independent retrieval of the pmoA-gene and quantification of partial pmoA gene sequences, type II (Alphaproteobacteria) methanotrophs of the genus Methylocystis spp. were abundant (> 10(7) pmoA target molecules per gram of dry soil). Type I (Gammaproteobacteria) methanotrophs related to the genera Methylobacter and Methylocaldum/Methylococcus were detected in some soils. Six pmoA sequence types not closely related to sequences from cultivated methanotrophs were detected as well, indicating that diverse uncultivated methanotrophs were present. Three Gleysols were incubated under different mixing ratios of (13)C-labelled methane to examine (13)C incorporation into phospholipid fatty acids (PLFAs). Phospholipid fatty acids typical of type II methanotrophs, 16:0 and 18:1omega7c, were labelled with (13)C in all soils after incubation under an atmosphere containing 30 ppmv of methane. Incubation under 500 ppmv of methane resulted in labelling of additional PLFAs besides 16:0 and 18:1omega7c, suggesting that the composition of the active methanotrophic community changed in response to increased methane supply. In two soils, 16:1 PLFAs typical of type I methanotrophs were strongly labelled after incubation under the high methane mixing ratio only. Type II methanotrophs are most likely responsible for atmospheric methane uptake in these soils, while type I methanotrophs become active when methane is produced in the soil.     

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 δ¹³C 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 18:1ω7c, with 16:1ω5 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 × 10⁶ 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 (14)CH(4) followed by analysis of radiolabelled phospholipid ester-linked fatty acids ((14)C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The (14)C-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 17:1omega8c (up to 9.0% of the total (14)C-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., 18:1omega9, 18:1omega7, and 18:0). The (14)C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH(4). The (14)C-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 (14)C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.     

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.     

Soil microbial community structure and activity in a 100-year-old fertilization and crop rotation experiment

Aims Nitrogen (N) fertilization and lime addition may affect soil microbial and nematode communities and ecosystem functions through changing environmental conditions, such as soil pH and soil organic carbon. The objectives of this experiment were to examine the impact of N input and liming on soil microbial and nematode communities and to identify the key environmental determinant of community composition in a century-old fertilization and crop rotation experiment.Methods The field experiment consisting of a 3-year crop rotation regime was established in 1911 in southeastern USA. Four treatments, (i) no-input control, (ii) NPK with winter legume, (iii) PK with legume and lime and (iv) NPK with legume and lime, were included in this study. Soil samples collected at the 0–5cm depth were used to determine the bacterial growth rate by the 3 H-thymidine incorporation technique. Incorporation of 13 C into neutral lipids, glycolipids and phospholipid fatty acids (PLFAs) was measured after incubation of soil with 13 C-labeled acetate for 24h. Free-living nematodes in fresh soil were extracted using a density sucrose centrifugal flotation method and identified to trophic group level.Important findings Liming resulted in a 10-fold increase in bacterial growth rates compared with the no-input control, whereas N fertilization had no significant effect. Multivariate analysis of PLFA profiles showed that soil microbial community composition was different among the four treatments; the difference was primarily driven by soil pH. PLFAs indicative of Gram-negative bacteria covaried with soil pH, but not those of fungi and actinobacteria. Liming enhanced 13 C incorporation into neutral lipids, glycolipids and phospholipids by 2–15 times. In addition, 13 C incorporation into 16:0, 16:1ω9, 18:1ω9, 18:1ω7 and 18:2ω6 were greater than other PLFAs, suggesting that Gram-negative bacteria and fungi were more active and sensitive to simple C input. Bacterivorous nematodes were the dominant trophic group in the soil, but no significant differences in nematode communities were found among the treatments. Our results suggest that soil pH had a greater impact than N fertilization on soil microbial community composition and activity in a crop rotation system including legumes.     

南亚热带3种人工林土壤微生物生物量和微生物群落结构特征

以我国南亚热带格木、红椎和马尾松人工林为对象,采用氯仿熏蒸浸提法和磷脂脂肪酸法(PLFA)分析了林地土壤微生物生物量和微生物群落结构组成.结果表明: 林分和季节因素均显著影响土壤微生物生物量、总PLFAs量、细菌PLFAs量和真菌PLFAs量,且干季林分下的土壤微生物生物量、总PLFAs量、单个PLFA量均大于雨季.红椎人工林土壤微生物生物量碳(MBC)和总PLFAs量最高,而格木人工林土壤微生物生物量氮(MBN)最高.土壤pH值对土壤丛枝菌根真菌(16:1ω5c)的影响达到极显著正相关水平.土壤总PLFAs量、革兰氏阳性菌(G⁺)以及腐生真菌(18:2ω6,9c)、革兰氏阳性菌/革兰氏阴性菌(G⁺/G^-)与土壤有机碳、全氮和全磷显著相关,表明土壤有机碳、全氮、全磷含量是影响该地区土壤微生物数量和种类的重要因素.外生菌根真菌(18:1ω9c)和丛枝菌根真菌与土壤碳氮比值呈极显著相关.     

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.     

Soil Moisture Alters the Response of Soil Organic Carbon Mineralization to Litter Addition

Increasing rainfall and longer drought conditions lead to frequent changes in soil moisture that affect soil organic carbon (SOC) mineralization. However, how soil moisture affects response of SOC mineralization to litter addition in forest ecosystems remains unexplored. We added ¹³C-labeled litter to subtropical forest soils with three mass water contents (L, 21%; M, 33%; H, 45%). Carbon dioxide production was monitored, and the composition of soil microbial communities was determined by phospholipid fatty acid (PLFA). When no litter was added, SOC mineralization was greater in the M-treated soil. Litter addition promoted SOC mineralization, but this promotion was altered by soil moisture and litter type. Priming effects induced by P. massoniana leaf litter in the M-moistened soil were significantly (P < 0.05) higher than those in other treatments. Litter-derived C was approximately 55% incorporated into 18:1ω9c and 16:0 PLFAs, and this proportion was not significantly affected by soil moisture. Soil moisture affected the distribution of litter-¹³C in i15:0, i17:0, and cy19:0 individual PLFAs. The primed C evolution was significantly related to the ratio of Gram-positive to Gram-negative bacteria. These results suggest that changes in soil moisture could affect SOC mineralization in forest ecosystems.     

A Microbial Link between Elevated CO2 and Methane Emissions that is Plant Species-Specific

Rising atmospheric CO₂ levels alter the physiology of many plant species, but little is known of changes to root dynamics that may impact soil microbial mediation of greenhouse gas emissions from wetlands. We grew co-occurring wetland plant species that included an invasive reed canary grass (Phalaris arundinacea L.) and a native woolgrass (Scirpus cyperinus L.) in a controlled greenhouse facility under ambient (380 ppm) and elevated atmospheric CO₂ (700 ppm). We hypothesized that elevated atmospheric CO₂ would increase the abundance of both archaeal methanogen and bacterial methanotroph populations through stimulation of plant root and shoot biomass. We found that methane levels emitted from S. cyperinus shoots increased 1.5-fold under elevated CO₂, while no changes in methane levels were detected from P. arundincea. The increase in methane emissions was not explained by enhanced root or shoot growth of S. cyperinus. Principal components analysis of the total phospholipid fatty acid (PLFA) recovered from microbial cell membranes revealed that elevated CO₂ levels shifted the composition of the microbial community under S. cyperinus, while no changes were detected under P. arundinacea. More detailed analysis of microbial abundance showed no impact of elevated CO₂ on a fatty acid indicative of methanotrophic bacteria (18:2ω6c), and no changes were detected in the terminal restriction fragment length polymorphism (T-RFLP) relative abundance profiles of acetate-utilizing archaeal methanogens. Plant carbon depleted in ¹³C was traced into the PLFAs of soil microorganisms as a measure of the plant contribution to microbial PLFA. The relative contribution of plant-derived carbon to PLFA carbon was larger in S. cyperinus compared with P. arundinacea in four PLFAs (i14:0, i15:0, a15:0, and 18:1ω9t). The δ¹³C isotopic values indicate that the contribution of plant-derived carbon to microbial lipids could differ in rhizospheres of CO₂-responsive plant species, such as S. cyperinus in this study. The results from this study show that the CO₂–methane link found in S. cyperinus can occur without a corresponding change in methanogen and methanotroph relative abundances, but PLFA analysis indicated shifts in the community profile of bacteria and fungi that were unique to rhizospheres under elevated CO₂.     

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.     

Methane carbon supports aquatic food webs to the fish level

Large amounts of the greenhouse gas methane (CH(4)) are produced by anaerobic mineralization of organic matter in lakes. In spite of extensive freshwater CH(4) emissions, most of the CH(4) is typically oxidized by methane oxidizing bacteria (MOB) before it can reach the lake surface and be emitted to the atmosphere. In turn, it has been shown that the CH(4)-derived biomass of MOB can provide the energy and carbon for zooplankton and macroinvertebrates. In this study, we demonstrate the presence of specific fatty acids synthesized by MOB in fish tissues having low carbon stable isotope ratios. Fish species, zooplankton, macroinvertebrates and the water hyacinth Eichhornia crassipes were collected from a shallow lake in Brazil and analyzed for fatty acids (FA) and carbon stable isotope ratios (δ(13)C). The fatty acids 16∶1ω8c, 16∶1ω8t, 16∶1ω6c, 16∶1ω5t, 18∶1ω8c and 18∶1ω8t were used as signature for MOB. The δ(13)C ratios varied from -27.7‰ to -42.0‰ and the contribution of MOB FA ranged from 0.05% to 0.84% of total FA. Organisms with higher total content of MOB FAs presented lower δ(13)C values (i.e. they were more depleted in (13)C), while organisms with lower content of MOB signature FAs showed higher δ(13)C values. An UPGMA cluster analysis was carried out to distinguish grouping of organisms in relation to their MOB FA contents. This combination of stable isotope and fatty acid tracers provides new evidence that assimilation of methane-derived carbon can be an important carbon source for the whole aquatic food web, up to the fish level.     

Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from [13C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that (13)C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [13C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [13C]phenol, and (iii) 12 daily doses of [13C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of alpha-, beta-, and gamma-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.     

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 alpha), 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 gamma" (USC gamma) 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 (13)CH(4) 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 (13)C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC gamma 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 alpha sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.     

毛竹高速生长期土壤碳氮动态及其微生物特性

研究了典型毛竹林毛竹高速生长期间土壤碳氮动态及其微生物生态特性。结果表明:毛竹高速生长期间,3个试验地土壤全氮、碱解氮、铵态氮、硝态氮及总有机碳和水溶性有机碳(DOC)的含量均有不同幅度的下降,其中25℃蒸馏水提取DOC(25℃DOC)降幅分别达到51%、22%和223%,且25℃DOC下降幅度明显大于80℃DOC的下降幅度。随毛竹生长,土壤全氮和有机碳含量变化较为明显,相关分析表明两者呈极显著的正相关(R2=0.89**)。同时,土壤微生物量碳含量大幅度降低,由原来的800 mg/kg降到了525 mg/kg。采用PLFA法对土壤微生物群落结构进行了分析,代表细菌的饱和脂肪酸(14:0,16:0,18:0,20:0,i15:0,i16:0,i17:0,i18:0,a15:0,a17:0)基本上都分布在载荷图的右侧;代表真菌的不饱和脂肪酸(18:2w6,9c/18:0ANTE)分布在主成分载荷图的左侧,表明随着毛竹生长,土壤中细菌含量减少,真菌含量增加。说明毛竹的高速生长消耗了土壤中的碳氮,同时对土壤微生物群落结构产生了明显的影响。     

The biogeochemical controls of N2O production and emission in landfill cover soils: the role of methanotrophs in the nitrogen cycle

Emissions of N2O from cover soils of both abandoned (> 30 years) and active landfills greatly exceed the maximum fluxes previously reported for tropical soils, suggesting high microbial activities for N2O production. Low soil matrix potentials (<-0.7 MPa) indicate that nitrification was the most likely mechanism of N2O formation during most of the time of sampling. Soil moisture had a strong influence on N2O emissions. The production of N2O was stimulated by as much as 20 times during laboratory incubations, when moisture was increased from -2.0 MPa to -0.6 MPa. Additional evidence from incubation experiments and delta13C analyses of fatty acids (18:1) diagnostic of methanotrophs suggests that N2O is formed in these soils by nitrification via methanotrophic bacteria. In a NH3(g)-amended landfill soil, the rate of N2O production was significantly increased when incubated with 100 ppmv methane compared with 1.8 ppmv (atmospheric) methane. Preincubation of a landfill soil with 1% CH4 for 2 weeks resulted in higher rates of N2O production when subsequently amended with NH3(g) relative to a control soil preincubated without CH4. At one location, at the soil depth (9-16 cm) of maximum methane consumption and N2O production, we observe elevated concentrations of organic carbon and nitrogen and distinct minima in delta15N (+1.0%) and delta13C (-33.8%) values for organic nitrogen and organic carbon respectively. A delta13C value of -39.3% was measured for 18:1 carbon fatty acids in this soil, diagnostic of type II methanotrophs. The low delta15N value for organic nitrogen is consistent with N2 fixation by type II methanotrophs. These observations all point to a methanotrophic origin for the organic matter at this depth. The results of this study corroborate previous reports of methanotrophic nitrification and N2O formation in aqueous and soil environments and suggest a predominance of type II rather than type I or type X methanotrophs in this landfill soil.     

Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing

Methane is used as an alternative carbon source in the denitrification of wastewater lacking organic carbon sources because it is nontoxic and may be efficiently produced by anaerobic biological processes. Methane-dependent denitrification (MDD) in the presence of oxygen requires the co-occurrence of methanotrophy and denitrification. Activated sludge was incubated with 13C-labeled methane in either a nitrate-containing medium or a nitrate-free medium. Then, bacterial and methanotrophic populations were analyzed by cloning analysis and terminal restriction fragment length polymorphism analysis targeting 16S rRNA gene and cloning analysis targeting pmoA genes. DNA-based stable-isotope probing (DNA-SIP) analysis of the 16S rRNA gene revealed an association of the Methylococcaceae and the Hyphomicrobiaceae in a MDD ecosystem. Furthermore, supplementation of nitrate stimulated methane consumption and the activity of methanotrophic populations (i.e. the stimulation of uncultivated relatives of distinct groups of the Methylococcaceae). In particular, uncultured type-X methanotrophs of Gammaproteobacteria were dominant when nitrate was added, i.e. in the MDD incubations. On the other hand, most methanotrophs (types I, II, and X methanotrophs) were found to have been labeled with 13C under nitrate-free conditions. This DNA-SIP study identifies key bacterial populations involved in a MDD ecosystem.