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.     

The Use of Neutral Lipid Fatty Acids to Indicate the Physiological Conditions of Soil Fungi

The usefulness of measuring neutral lipid fatty acids (NLFAs) and phospholipid fatty acids (PLFAs) separately in order to interpret perturbation effects on soil and compost microorganisms has been studied. Initially the NLFA/PLFA ratios were studied in different soils. Low ratios were found for fatty acids common in bacteria, especially for cyclopropane fatty acids. Higher ratios were found for fatty acids common in eukaryotic organisms such as fungi (18:1omega9 and 18:2omega6,9) or in saturated fatty acids, common to many types of organisms. Adding glucose to a forest soil increased the amounts of the fungal NLFAs 18:1omega9 and 18:2 omega6,9 up to 60 and 10 times, respectively, after 10 days, followed by a gradual decrease. After 3 months incubation, higher levels of these NLFAs were still found compared with the control samples. Adding glucose together with nitrogen (N) and phosphorus (P) resulted in no increase in NLFAs but a 10-fold increase in the PLFAs 18:1omega9 and 18:2omega6,9. Thus, the NLFA/PLFA ratios for these fatty acids were lower than in the no-addition control when glucose was added together with N and P, but higher when glucose was added alone, even 3 months after the addition. Adding N+P without glucose did not affect the NLFA/PLFA ratio for any fatty acid. Increasing NLFA/PLFA ratios for the fungal fatty acids were also found with time after the thermophilic phase in a compost, indicating increased availability of easily available carbon.     

Cold acclimation and lipid composition in the earthworm Dendrobaena octaedra

We have investigated the lipid chemistry during cold acclimation in the freeze tolerant earthworm Dendrobaena octaedra. The dominant phospholipid fatty acids (PLFA) of D. octaedra were 20:4, 20:5 and 20:1 (50% of total PLFA) followed by 18:0, 18:1 and 18:2omega6,9 (25% of total PLFA). The ability to tolerate freezing in this species was acquired after acclimation at low temperature for 2-4 weeks. During this period one particular membrane PLFA, 18:2omega6,9, increased significantly and there was a good correlation between the proportion of this PLFA and the survival of freezing. The composition of neutral lipid fatty acids (NLFA), most likely representing storage lipids (triacylglycerides), also changed during cold acclimation so that the overall degree of unsaturation increased. Using a common-garden experiment approach, we compared lipid composition of three genetically different populations (Denmark, Finland and Greenland) that differed in their freeze tolerance. Inter-populational differences and differences due to cold acclimation in overall fatty acid composition were evident in both PLFAs and NLFAs. Specifically, the PLFAs, 20:4 and 20:5, were considerably more represented in worms from Greenland, and this contributed to a higher UI of PLFAs in this population.     

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.     

Phospholipid Fatty Acid Composition, Biomass, and Activity of Microbial Communities from Two Soil Types Experimentally Exposed to Different Heavy Metals

The phospholipid fatty acid (PLFA) pattern was analyzed in a forest humus and in an arable soil experimentally polluted with Cd, Cu, Ni, Pb, or Zn at different concentrations. In both soil types, there were gradual changes in the PLFA patterns for the different levels of metal contamination. The changes in the forest soil were similar irrespective of which metal was used, while in the arable soil the changes due to Cu contamination differed from those due to the other metals. Several PLFAs reacted similarly to the metal amendments in the two soil types, while others showed different responses. In both soils, the metal pollution resulted in a decrease in the iso-branched PLFAs i15:0 and i17:0 and in the monounsaturated 16:1ω5 and 16:1ω7c fatty acids, while increases were found for i16:0, the branched br17:0 and br18:0, and the cyclopropane cy17:0 fatty acids. In the forest soil, the methyl branched PLFAs 10Me16:0, 10Me17:0, and 10Me18:0 increased in metal-polluted soils, indicating an increase in actinomycetes, while in the arable soil a decrease was found for 10Me16:0 and 10Me18:0 in response to most metals. The bacterial PLFAs 15:0 and 17:0 increased in all metal-contaminated samples in the arable soil, while they were unaffected in the forest soil. Fatty acid 18:2ω6, which is considered to be predominantly of fungal origin, increased in the arable soil, except in the Cu-amended samples, in which it decreased instead. Effects on the PLFA patterns were found at levels of metal contamination similar to or lower than those at which effects on ATP content, soil respiration, or total amount of PLFAs had occurred.     

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 (13)C 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 16:1omega5 PLFA were more active in utilizing (13)C-labeled rhizodeposits in the second labeling period than in the first labeling period. In both labeling periods, however, the fungal PLFA 18:2omega6,9 was the most highly labeled. These results demonstrate the effectiveness of using (13)C labeling and PLFA analysis to examine the microbial dynamics associated with rhizosphere C cycling by focusing on the members actively involved.     

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.     

Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations

Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1omega7c, 16:1omega7t, 16:0, cy17:0, and 18:1omega7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (beta-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities.     

木焦油污染土壤中微生物特性的空间变异性研究

运用地统计学方法研究了木焦油污染土壤中微生物量、微生物群落结构、微生物活性等的空间变异特征.分别采用26种主要磷脂类脂肪酸(PLFA)的总含量(totPLFA)、PLFA的第一主成分和第二主成分(PLFA PC1和PC2)以及土壤培养过程中CO₂-C的累积释放量(Cre)来表征土壤中的微生物量、微生物群落结构以及微生物活性.结果表明,多数微生物特性指标均存在不同程度的空间自相关性,其半变异函数曲线可用带块金效应的球状模型进行拟合.变量的空间相关距离在2.50～16.60 m之间.PLFA PC1、totPLFA和Cre均具有较强的空间依赖性,其相对结构变差（由结构性因素引起的空间变异）分别为82.3%、79.6%和64.7%,而PLFA PC2 不存在明显的空间依赖性.克立格空间插值图表明,样地中存在几处微生物相对密集分布且代谢活性较高的区域,其中优势微生物菌群是由PLFAs 16:1ω7t,cy17:0,18:1ω7 和cy19:0所表征的革兰氏阴性细菌.土壤中主要污染物多环芳烃含量和空间分布是影响微生物特性空间分布格局的重要因素之一.     

氮肥添加对高寒藏嵩草(Kobresia tibetica)沼泽化草甸和土壤微生物群落的影响

以藏嵩草沼泽化草甸为研究对象,利用磷脂脂肪酸(PLFA)技术,研究连续6年N素添加对地上植被群落数量特征、土壤微生物群落结构的影响。结果表明:①藏嵩草沼泽化草甸群落生物量、枯枝落叶对施肥处理无明显响应,且莎草科植物对土壤氮素的吸收和利用率较低。②施肥增加了0-10 cm土壤微生物类群PLFAs丰富度尤其细菌和革兰氏阳性菌PLFAs,降低了10-20 cm PLFAs丰富度;③磷脂脂肪酸饱和脂肪酸/单烯不饱和脂肪酸、细菌PLFAs/真菌PLFAs的比值随土壤层次增加而增加;④0-10 cm土层革兰氏阳性菌、真菌PLFAs含量与pH、土壤速效磷、速效氮、土壤有机质显著正相关(P0.05或P0.01);10-20 cm土层,细菌、革兰氏阳性菌、真菌和总PLFAs含量与土壤有机质含量显著正相关(P0.05或P0.01)。表明藏嵩草沼泽化草甸微生物PLFAs含量和丰富度对施肥的响应存在明显的土层梯度效应,土壤微生物PLFAs含量和丰富度主要受表层土壤初始养分含量的影响。     

Application of thymol and iprodione to control garlic white rot (Sclerotium cepivorum) and its effect on soil microbial communities

The effect of different dosages of thymol alone, iprodione alone and combinations of thymol and iprodione on white rot disease of garlic and its impact on soil microbial community structure were investigated under greenhouse conditions. Thymol alone or in combination with the fungicide iprodione did not appear to reduce either white rot incidence or soil sclerotia density as compared to an infected control. However, iprodione alone or in combination with thymol reduced soil fungal biomass. In addition, iprodione alone decreased soil microbial activity as estimated by fluorescein diacetate (FDA). Soil bacterial community structure as estimated by phospholipid fatty acid (PLFA) profiles was also was affected by both thymol and iprodione applications. The correlation biplot of the individual PLFAs and biocide treatment indicated that the treatments with thymol alone increased cyclopropyl fatty acid (cy17:0 and cy19:0), while the treatments with iprodione alone increased some saturated and branched fatty acids (principally i16:0, a15:0 and 18:0). In addition, taking into account PLFA biomarkers, thymol applications reduced Gram-negative bacteria in soil. To our knowledge, this research is the first report about the effect of a monoterpene (thymol) on soil microflora.     

Variation in Microbial Community Structure in Two Boreal Peatlands as Determined by Analysis of Phospholipid Fatty Acid Profiles

Analyses of phospholipid fatty acids (PLFAs) were used to assess variation in community structure and total microbial biomass in two boreal peatlands in Sweden. The total PLFA concentration in peat ranged from 0.16 to 7.0 nmol g of wet peat(sup-1) (median, 0.70 nmol g of wet peat(sup-1)). Principal-component analysis of PLFA data revealed that the degree of depth-related variation in PLFA composition was high among peatland habitats, with general differences between wet sites, with water tables within a few centimeters of the moss surface, and dry sites, with water tables >10 cm below the moss surface. However, variation in PLFA composition over the growing season was negligible. In the principal-component analyses, most PLFAs were determined to be parts of clusters of covarying fatty acids, suggesting that they originated in the same functional groups of microorganisms. Major clusters were formed by monounsaturated (typical of gram-negative eubacteria), terminally branched (gram-positive or anaerobic gram-negative eubacteria), methyl-branched and branched unsaturated (sulfate-reducing bacteria and/or actinomycetes), (omega)8 monounsaturated (methane-oxidizing bacteria), and polyunsaturated (eucaryotes) PLFAs. Within the clusters, PLFAs had rather distinct concentration-depth distributions. For example, PLFAs from sulfate-reducing bacteria and/or actinomycetes and those from methane-oxidizing bacteria had maximum concentrations slightly below and at the average water table depth, respectively.     

The microbial community structure of drinking water biofilms can be affected by phosphorus availability.

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 microg of phosphorus liter(-1) and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1 omega 7c and 18:1 omega 7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.     

Bacterial populations and processes involved in acetate and propionate consumption in anoxic brackish sediment

Bacterial populations and pathways involved in acetate and propionate consumption were studied in anoxic brackish sediment from the Grosser Jasmunder Bodden, German Baltic Sea. Uptake of acetate and propionate from the porewater was studied using stable carbon isotope-labeled compounds. Labeled acetate was not produced as an intermediate during propionate uptake experiments, and propionate consumption was not affected by the addition of acetate. In parallel, incorporation of labeled acetate and propionate into phospholipid-derived fatty acids (PLFA) was studied to indicate bacterial populations involved in the consumption of these substrates. The (13)C-acetate label was mainly recovered in even-numbered PLFA (16:1omega7c, 16:0 and 18:1omega7c). In contrast, primarily odd-numbered PLFA (a15:0, 15:0, 17:1omega6 and 17:0) and the even-numbered i16:0 were labeled after incubation with (13)C-propionate. Although single PLFA labeled with propionate are commonly found in sulfate reducers, the complete PLFA-labeling pattern does not resemble any of the know strains. However, the acetate-labeling pattern is similar to Desulfotomaculum acetoxidans and Desulfofrigus spp., two acetate-consuming, sulfate reducers. In conclusion, our data suggest that acetate and propionate were predominantly consumed by different, specialized groups of sulfate-reducing bacteria.     

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.     

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。     

Ethanol, BTEX and microbial community interactions in E-blend contaminated soil slurry

Degradation of benzene, toluene, ethylbenzene, m-, p- and o-xylenes (BTEX) and microbial community shifts in soil slurries contaminated with ethanol–gasoline blends (E-blends), containing 10, 50 or 90% (v/v) ethanol (E10, E50 and E90) were studied in soil slurries previously uncontaminated, contaminated by E-blends or ethanol. BTEX originating from E50 degraded fastest whereas from E10 slowest. Among the individual compounds, ethylbenzene degraded fastest (max 30% d⁻¹), and o-xylene slowest (min 1% d⁻¹) during aerobic conditions in previously not contaminated soils. Previous contamination by E-blends increased BTEX degradation significantly (3–19 times) compared with previously uncontaminated soils, whereas previous contamination with ethanol did not show significant difference in BTEX degradation. At least one type of the E-blends during aerobic conditions had a positive effect on total PLFAs (phospholipid fatty acids) and specific PLFAs, i.e. 10Me18:0, 16:1ω6 and cy17:0, but had a negative effect on cy19:0 and 18:2ω6,9c. The effects on total PLFAs, as well as the individual PLFAs, were particularly strong after repeated contamination. The single most affected PLFA was 16:1ω6, which increased 23 times during E10 treatment in soil slurries previously contaminated by E-blends. Altogether, the various E-blends had significantly different effects on BTEX degradation and also on individual PLFAs under aerobic conditions.     

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.     

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities

Phospholipid fatty acid (PLFA) as biomarkers, is widely used to profile microbial communities in environmental samples. However, PLFA extraction and derivatization protocols are not standardized and have widely varied among published studies. Specifically investigators have used either HCl/MeOH or KOH/MeOH or both for the methylation step of PLFA analysis, without justification or research to support either one. It seems likely that each method could have very different outcomes and conclusions for PLFA based studies. Therefore, the objective of this study was to determine the effect of catalyst type for methylation on detecting PLFAs and implications for interpreting microbial profiling in soil. Fatty acid samples extracted from soils obtained from a wetland, an intermittently flooded site, and an adjacent upland site were subjected to HCl/MeOH or KOH/MeOH catalyzed methylation procedures during PLFA analyses. The methylation method using HCl/MeOH resulted in significantly higher concentrations of most PLFAs than the KOH/MeOH method. Another important outcome was that fatty acids with a methyl group (18:1ω,7c 11Me, TBSA 10Me 18:0, 10Me 18:0, 17:0 10Me and 16:0 10Me being an actinomycetes biomarker) could not be detected by HCl/MeOH catalyzed methylation but were found in appreciable concentrations with KOH/MeOH method. From our results, because the HCl/MeOH method did not detect the fatty acids containing methyl groups that could strongly influence the microbial community profile, we recommend that the KOH/MeOH catalyzed transesterification method should become the standard procedure for PLFA profiling of soil microbial communities.     

Linking toluene degradation with specific microbial populations in soil

Phospholipid fatty acid (PLFA) analysis of a soil microbial community was coupled with (13)C isotope tracer analysis to measure the community's response to addition of 35 microg of [(13)C]toluene ml of soil solution(-1). After 119 h of incubation with toluene, 96% of the incorporated (13)C was detected in only 16 of the total 59 PLFAs (27%) extracted from the soil. Of the total (13)C-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 [(13)C]glucose. Our study showed that coupling (13)C 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.