Total Phospholipid Fatty Acid Content Research Articles

Multiple Climate Change Factors Interact to Alter Soil Microbial Community Structure in an Old‐Field Ecosystem

Climate change has the potential to alter both the composition and function of a soil’s microbial community, and interactions among climate change factors may alter soil communities in ways that are not possible to predict from experiments based on a single factor. This study evaluated the direct and interactive effects of three climate change factors—elevated CO 2 , altered amounts of precipitation, and elevated air temperature—on soil microbial communities from an old-field climate change experiment being conducted at Oak Ridge, TN. Soil microbial community composition and biomass were determined by phospholipid fatty acid (PLFA) and neutral lipid fatty acid composition. We found that the interactive effects of precipitation and temperature treatments, as well as the interactive effects of precipitation and CO 2 treatments, had significant impacts on microbial community composition. We found that total soil PLFA concentration, a measure of microbial biomass, was greater in the low-precipitation treatments, especially when low precipitation was combined with ambient CO 2 concentrations or ambient temperature. Ordination analysis indicated that temperature was the most significant predictor of shifts in the soil microbial community composition, explaining approximately 12% of the variance in relative abundance of PLFA biomarkers. The elevated-temperature treatment increased the abundance of Firmicutes (low-guanine–cytosine Gram positive) and decreased the abundance of Gram-negative bacteria. Elevated temperature also reduced the abundance of the arbuscular mycorrhizal fungi PLFA biomarker 16:1π5c and saprophytic fungal PLFA biomarker 18:2π6,9. Overall, our data indicate that the interactions among climate change factors alter the composition of soil microbial communities in old-field ecosystems, suggesting potential for changes in microbial community function under predicted future climate conditions.

Soil microbial community structure affected by 53 years of nitrogen fertilisation and different organic amendments

The Ultuna long-term soil organic matter experiment in Sweden (59′82° N, 17′65° E) was started in 1956 to study the effects of different N fertilisers and organic amendments on soil properties. In this study, samples were taken from 11 of the treatments, including unfertilised bare fallow and cropped fallow, straw with and without N addition, green manure, peat, farmyard manure, sawdust, sewage sludge, calcium nitrate and ammonium sulphate, with n = 4 for each treatment. Samples were taken from topsoil (0–20 cm) and subsoil (27–40 cm depth) and analysed for concentrations of phospholipid fatty acids (PLFAs), organic C, total N and pH. The results showed that the subsoil samples reflected the total PLFA content of the topsoil, but not the microbial community structure. Total PLFA content was well correlated with total organic C and total N in both topsoil and subsoil. Total PLFA content in topsoil samples was highest in the sewage sludge treatment (89 ± 22 nmol PLFA g dw−1). This contradicts earlier findings on microbial biomass in this sewage sludge-treated soil, which indicated inhibition of microorganisms, probably by heavy metals added with sludge. A switch towards microbial growth and faster decomposition of organic matter occurred around 2000, coinciding with lowered heavy metal content in the sludge. According to the PLFA data, the microbial community in the sewage sludge treatment is now dominated by Gram-positive bacteria. A lack of Gram-negative bacteria was also observed for the ammonium sulphate treatment, obviously caused by a drop in pH to 4.2.

Phospholipid fatty acids analysis of the vertical distribution of microbial communities in eutrophic lake sediments

Vertical distribution of microbial communities in a eutrophic lake sediments of Lake Xuanwu was quantified by phospholipid fatty acids analysis and multivariate statistical analysis was employed to interprete the data. Principle component analysis of sediment characteristics parameters, including total nitrogen, total phosphorus, organic matters and pH produced clustering of sampling sites for two distinct groups. These groups corresponded with the two sampling stations and the levels of nutrient enrichment. Total phospholipid fatty acids concentration, which is indicative of microbial biomass, reduced with depth, however, the relative percentage of anaerobic prokaryotes increased. To assess changes of microbial community along depth, phospholipid fatty acids compositions were analyzed by cluster analysis. Distinct clusters were observed in different sampling stations. Canonical correspondence analysis was carried out to infer the relationship between sediment characteristics and microbial communities. Phospholipid fatty acids samples collected at the same sampling site clustered together. Canonical correspondence analysis revealed that the environmental parameter with the greatest bearing on the phospholipid fatty acids profiles was pH. This study proved the successful application of phospholipid fatty acids and multivariate analysis to investigate the relationship between environment factors and microbial community composition.

Changes in microbial‐community structure with depth and time in a chronosequence of restored grassland soils on the Loess Plateau in northwest China

AbstractMicrobial‐community structure is closely associated with plant‐community composition. The objective of this study was to evaluate the effects of depth and revegetation time on the microbial‐community structure of restored grassland soils of the Loess Plateau of China. Microbial‐community structure at 0–10 cm (depth 1) and 10–20 cm (depth 2) of eight sites of a grassland chronosequence with revegetation time ranging from 1 to 78 y was determined using phospholipid fatty acid (PLFA) analysis. Except for the youngest site microbial‐community structure at the two depths varied distinctly with actinomycetes and vesicular arbuscular mycorrhiza as the most important discriminators. Total PLFA content decreased with depth and increased with revegetation time. Protozoa were the only functional group whose proportion did not change with depth. Their relative abundance tended to decrease with increasing revegetation time. The microbial community at all sites was bacteria‐dominated with Gram‐negative bacteria representing the largest proportion. Principal‐component and cluster analysis revealed that microbial‐community structure in the surface soil (depth 1) of the older sites (23–78 y of revegetation) was rather similar which may be due to only small effects of the plant species present on the soil environment. Differences in microbial‐community composition at 0–20 cm depth of the eight sites were partly related to variations in the physico‐chemical characteristics. Relations to organic C, alkali‐extractable N, pH, and available P were found. Revegetation of former agricultural sites on the Loess Plateau improves the soil status and leads to shifts in microbial‐community structure more pronounced with depth than time.

Microbial biomass and community composition in boreal lake sediments

We used phospholipid fatty acids (PLFA) to determine microbial biomass and community structure in the sediments of eight boreal lakes with different loadings of allochthonous organic carbon and total phosphorus (TP) in the water during the course of a year. The total concentration of PLFA, an estimate of the microbial biomass, depended more on TP, a proxy for pelagic primary production, but not on dissolved organic carbon, a proxy for terrestrial organic carbon input. The composition of PLFAs varied considerably over time, demonstrating seasonal dynamics in microbial community composition. When PLFA profiles in all lakes and seasons are compared, community composition is more similar within season than within lakes.

Response of soil microbial communities to different management practices in surface soils of a soybean agroecosystem in Argentina

Argentina is the world’s third most important soybean producer; hence, there is an urgent need to preserve soil health by applying appropriate agricultural practices to maintain sustainable production in the upcoming years. Because productivity of agricultural systems largely depends on soil microbial processes, the influence of different management strategies on soil microbial community structure was analyzed in a long-term field trial started in 1992. The experimental design was a split-plot arrangement of treatments, consisting in two tillage treatments: zero tillage (ZT) and reduced tillage (RT), in combination with two crop rotation treatments: soybean monoculture (SS) and corn-soybean (CS). Phospholipid fatty acid (PLFA) profiles were used to assess total microbial community structure. Denaturing gradient gel electrophoresis (DGGE) profiles of 18S rRNA were generated to describe the influence of crop practices on fungal communities. Total PLFA content was lowest in soil under reduced tillage and soybean monoculture; therefore the use of reduced tillage-soybean monoculture in agroecosystems might produce important reductions in total microbial biomass. The structure of total microbial communities, as estimated by PLFA, was affected by crop rotation. Moreover, the fungal communities, as estimated by DGGE analysis, were influenced by combined effects of crop rotation and tillage system.

Soil microbial community dynamics and phenanthrene degradation as affected by rape oil application

The effects of rape oil application on soil microbial communities and phenanthrene degradation were characterized by examining phenanthrene concentrations, changes in microbial composition and incorporation of [ 13C] phenanthrene-derived carbon into phospholipid fatty acids (PLFAs). A Haplic Chernozem was incubated with and without rape oil in combination with and without phenanthrene over 60 days. High-performance liquid chromatography (HPLC) analysis showed a net reduction in extractable phenanthrene in the soils treated with rape oil but no net reduction in the soils without rape oil. Rape oil application increased the total PLFA content and changed microbial community composition predominantly due to growth of fungal groups and Gram-positive bacterial groups. Under rape oil and phenanthrene amendment all detected microbial groups grew until day 24 of incubation. The 13C PLFA profiles showed 13C enrichment for the PLFAs i14:0, 15:0, 18:0, 18:1ω5 and the fungal biomarker 18:2ω6,9 under rape oil application. Fungal PLFA growth was highest among detected all PLFAs, but its 13C incorporation was lower compared to the Gram-positive and Gram-negative bacteria PLFAs. Our results demonstrate the effect of rape oil application on the abundance of microbial groups in soil treated with phenanthrene and its impact on phenanthrene degradation.

Impact of imazethapyr on the microbial community structure in agricultural soils

Large amounts of imazethapyr were applied for weed control in cultivation fields in China, but their effects on the soil microbial community remains unclear. In this study, two agricultural soils, a silty loam (HS) and a loamy soil (QL), were spiked with imazethapyr (CK, 0.1, 1 and 10 mg kg −1) and incubated for 1, 15, 30, 60, 90 and 120 d. In addition, untreated controls received only water. The soil microbial community structures were characterized by investigating the phospholipid fatty acids (PLFA) and microbial biomass C. Soil microbial biomass C and total concentration of PLFA were variable with incubation time, which were also reduced by the addition of imazethapyr. Imazethapyr addition also decreased the ratios of GN/GP and fungi/bacteria. A larger stress level, measured as the ratio of PLFA (cyc17:0 + cyc19:0)/(16:1 ω7c + 18:1 ω7c), was found in the high concentration (1 and 10 mg kg −1) herbicide treatment groups. The effects of imazethapyr at the field application on soil microbial biomass and microbial community were minor. Principal component analysis (PCA) of the PLFA clearly separated the treatments and incubation times. Both soils showed different total PLFA concentrations and ratios of GN/GP and fungi/bacteria, but similar changes in the PLFA pattern upon soil treatment. The soil microbial community structure was shifted by the addition of imazethapyr, which recovered after 60 d. In addition, the dissipation of imazethapyr was slow in both soils. Our results demonstrated that the addition of imazethapyr shifted the microbial community structure, but that it recovered after a period of incubation.

Allantoin-induced changes of microbial diversity and community in rice soil

Rice (Oryza sativa L.) may release allantoin into paddy soil. Currently, little attention has been paid to the effects of allantoin on soil microbial diversity and community. In this study, the diversity indices for the microbial community in rice soil and the allantoin-induced shifts in the microbial community composition across moisture regimes versus incubation times were examined by looking at phospholipid fatty acid (PLFA) profiles and PCR-denaturing gradient gel electrophoresis (DGGE) patterns. The total PLFA concentration increased with the amount of allantoin applied during incubation. A similar trend occurred for microbial biomass carbon (MBC) and pH. Furthermore, applying allantoin resulted in an increased ratio of anaerobe: aerobe and bacteria: fungi but decreased the ratio of Gram-negative bacteria: Gram-positive bacteria (GN: GP) and fungal marker 18:2ω6,9c under flooded rice soil. A stepwise discriminant analysis of the PLFA pattern clearly separated allantoin-treated samples from non-treated samples. There was also a clear split between different incubation times. Moisture treatments differed in the ratios of anaerobe: aerobe and GN: GP but showed similar trends in total PLFA concentrations and the bacteria: fungi ratio during incubation. These results suggest that application of allantoin stimulates shifts in microbial community composition and increases microbial diversity as well as living microbial biomass in rice soil.

PLFA profiles of drinking water biofilters with different acetate and glucose loadings

The biofilters fed with acetate or glucose and their phospholipid fatty acid (PLFA) profiles were investigated to observe the impact of organic matter concentrations on the microbial community structure in the drinking water production system. PLFA markers for bacteria were predominant in all of the biofilters and made up over half of the total PLFA content. PLFA diversity was compared and the biofilters fed with glucose had higher diversity. The Shannon-Wiener (or sometimes known as just Shannon diversity index) indices in the biofilters fed with acetate were from 0.68 to 0.97, while the indices in the biofilters fed with glucose were from 0.95 to 1.25. Principle components analysis showed that carbon sources and media depth were responsible for 68 and 17% of the total PLFA variance, respectively. The results indicated that PLFA analysis could be useful in illustrating microbial community structure in drinking water bioreactors, and microbial community structure was impacted by carbon substrates.

Substrate inputs and pH as factors controlling microbial biomass, activity and community structure in an arable soil

Our aim was to determine whether the smaller biomasses generally found in low pH compared to high pH arable soils under similar management are due principally to the decreased inputs of substrate or whether some factor(s) associated with pH are also important. This was tested in a soil incubation experiment using wheat straw as substrate and soils of different pHs (8.09, 6.61, 4.65 and 4.17). Microbial biomass ninhydrin-N, and microbial community structure evaluated by phospholipid fatty acids (PLFAs), were measured at 0 (control soil only), 5, 25 and 50 days and CO 2 evolution up to 100 days. Straw addition increased biomass ninhydrin-N, CO 2 evolution and total PLFA concentrations at all soil pH values. The positive effect of straw addition on biomass ninhydrin-N was less in soils of pH 4.17 and 4.65. Similarly total PLFA concentrations were smallest at the lowest pH. This indicated that there is a direct pH effect as well as effects related to different substrate availabilities on microbial biomass and community structure. In the control soils, the fatty acids 16:1ω5, 16:1ω7c, 18:1ω7c&9t and i17:0 had significant and positive linear relationships with soil pH. In contrast, the fatty acids i15:0, a15:0, i16:0 and br17:0, 16:02OH, 18:2ω6,9, 17:0, 19:0, 17:0c9,10 and 19:0c9,10 were greatest in control soils at the lowest pHs. In soils given straw, the fatty acids 16:1ω5, 16:1ω7c, 15:0 and 18:0 had significant and positive linear relationships with pH, but the concentration of the monounsaturated 18:1ω9 PLFA decreased at the highest pHs. The PLFA profiles indicative of Gram-positive bacteria were more abundant than Gram-negative ones at the lowest pH in control soils, but in soils given straw these trends were reversed. In contrast, straw addition changed the microbial community structures least at pH 6.61. The ratio: [fungal PLFA 18:2w6,9]/[total PLFAs indicative of bacteria] indicated that fungal PLFAs were more dominant in the microbial communities of the lowest pH soil. In summary, this work shows that soil pH has marked effects on microbial biomass, community structure, and response to substrate addition.

Effects of different soil weights, storage times and extraction methods on soil phospholipid fatty acid analyses

Phospholipid fatty acid (PLFA) analysis has become a popular and powerful method for characterizing soil microbial communities. However, little work has been done to compare different extraction procedures. In this study, the effects of single or consecutive extractions, varying soil weights, alternative solid phase extraction (SPE) tubes and a comparison of extractions from fresh soil and that from soil previously stored freeze-dried at − 70 °C for 1 year were determined. About 90–95% of PLFAs were extracted by the method used in this paper. More individual PLFAs and changes in PLFA peak distributions (profiles) were found with increasing soil sample weights, which suggests that for each set of experiments, the soil weight should be constant. Home-made SPE tubes used in this study can give almost as good results as the commercial standard ones, which significantly decreased costs. The total PLFA concentrations decreased by more than 28% following storage of the freeze-dried soils for 1 year at − 70 °C. The PLFA profiles also changed during storage over this period, suggesting that PLFA analyses are best done as soon as possible after sampling.

Effects of different chloroform stabilizers on the extraction efficiencies of phospholipid fatty acids from soils

Chloroform is commonly used as an organic solvent to extract phospholipid fatty acids from soil samples. However, the extraction efficiency of the fatty acids depends on the particular chloroform stabilizers used. The effect of chloroform stabilizers 2-methyl-2-butene and ethanol was investigated at different steps of the extraction procedure. Only the ethanol stabilized chloroform prevented the loss of certain phospholipid fatty acids. In particular, the unsaturated fatty acids 16:1ω7c, 16:1ω6c, 16:1ω5, 17:1ω8, 18:1ω7c, 18:1ω5, the fungal biomarker 18:2ω6,9 and the saturated fatty acid 17:0 were absent when chloroform stabilized with 2-methyl-2-butene was used. In addition, the total phospholipid fatty acid concentrations were also significantly reduced when chloroform stabilized with 2-methyl-2-butene was used. Thus, the proper choice of chloroform stabilizer for the analysis of phospholipid fatty acids is very important.

Structural diversity and enzyme activity of volcanic soils at different stages of development and response to experimental disturbance

We investigated the phospholipid fatty acid (PLFA) diversity and enzyme activities in soils from the volcano, Mt. Etna (Sicily). The soils were at sites which have been developing for different periods of time and have formed in volcanic lava of differing ages that have been supplemented with volcanic ejecta from subsequent eruptions. However, the plant communities indicated a marked successional difference between the sites and we have used this as a proxy for developmental stage. We have compared the structural and functional properties of the microbial communities in soils from the two sites and tested experimentally the hypothesis that the more diverse community was more resistant and resilient to disturbance. The experimental disturbance imposed was heating (60 °C for 48 h) and the recovery of enzyme activities (β-glucosidase, acid phosphatase and arylsulfatase) and structural properties (PLFA profiles) were then followed over six months. The microbial community in the soil from the older site was more structurally diverse and had a larger total PLFA concentration before disturbance than that of the soil from the younger site. The older soil community was not more resistant and resilient following an environmental disturbance as the younger soil community was equally or more resistant and resilient for all parameters. Changes in enzyme activities following disturbance were almost entirely attributable to changes in biomass (total PLFA).

Impact of the antibiotic sulfadiazine and pig manure on the microbial community structure in agricultural soils

Large amounts of veterinary antibiotics enter soil via manure of treated animals. The effects on soil microbial community structure are not well investigated. In particular, the impact of antibiotics in the presence of manure is poorly understood. In this study, two agricultural soils, a sandy Cambisol (KS) and a loamy Luvisol (ML), were spiked with manure and sulfadiazine (SDZ; 0, 10 and 100 μg g −1) and incubated for 1, 4, 32 and 61 days. Untreated controls received only water. The microbial community structure was characterised by investigating phospholipid fatty acids (PLFA) and using PCR–denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. The total concentration of PLFA increased with addition of manure and was reduced by both SDZ concentrations at incubation times >4 days. The SDZ addition decreased the bacteria:fungi ratio. The largest stress level, measured as ratio of PLFA (cyc17:0 + cyc19:0)/(16:1ω7c + 18:1ω7c), was found for the controls, followed by the manure treatments and the SDZ treatments. A discriminant analysis of the PLFA clearly separated treatments and incubation times. Both soils differed in total PLFA concentrations and Gram −:Gram + ratios, but showed similar changes in PLFA pattern upon soil treatment. Effects of manure and SDZ on the bacterial community structure were also revealed by DGGE analysis. Effects on pseudomonads and β-proteobacteria were less pronounced. While community structure remained altered even after two months, the extractable concentrations of SDZ decreased exponentially and the remaining solution concentrations after 32 days were ≤27% of the spiking concentration. Our results demonstrate that a single addition of SDZ has prolonged effects on the microbial community structure in soils.

Changes in soil microbial biomass and community composition along vegetation zonation in a coastal sand dune

We used phospholipid fatty acid (PLFA) analysis to examine the relation of microbial biomass and community composition to vegetation zonation on a coastal sand dune. Soil samples were collected along 3 line transects established from the shoreline to the inland bush. Total PLFA content and PLFA composition of soils were used as indices of total microbial biomass and community composition, respectively. The microbial biomass was much higher in the inland Vitex rotundifolia zone than in the seaside plots. The microbial community composition also differed among the vegetation zones, with a higher contribution of fungal biomarkers in the inland plots. The microbial biomass increased significantly with increasing soil organic matter (SOM) content, but was not correlated with soil salinity. These results suggest that microbial biomass in the coastal sand dune was controlled primarily by the accumulation of SOM. The microbial community composition also changed with SOM content in the seaside plots, but SOM had little effect in the inland plots. These results suggest that the factors limiting the microbial community composition differed with location on the dune.

Unraveling the effects of management regime and plant species on soil organic carbon and microbial phospholipid fatty acid profiles in grassland soils

In a field experiment we have examined the effect of long-term grassland management regimes ( viz., intensive versus extensive) and dominant plant species ( viz., Arrhenatherum elatius, Holcus lanatus and Dactylis glomerata) on soil organic carbon (SOC) build up, soil microbial communities using biomarker phospholipid fatty acids (PLFA), and the relationship between SOC and PLFAs of major groups of microorganisms ( viz., bacteria, fungi, and actinomycetes). The results have revealed that changes in SOC were not significantly affected by the intensity of management or by the plant species composition or by their interaction. The amount of PLFA of each microbial group was affected weakly by management regime and plant species, but the canonical variance analysis (CVA), based on individual PLFA values, demonstrated significant ( P < 0.05) effects of management regime and plant species on the composition of microbial community. Positive and significant ( P < 0.01) relationships were observed between PLFA of bacteria ( R 2 = 0.47), fungi ( R 2 = 0.33), actinomycetes ( R 2 = 0.71) and total microbial PLFA ( R 2 = 0.53) and SOC content.

Carbon and nitrogen limitation of soil microbial respiration in a High Arctic successional glacier foreland near Ny-Ålesund, Svalbard

The hypotheses that carbon and nitrogen availability limit microbial activity, and that the key factors limiting microbes vary along the successional gradient were tested in a High Arctic glacier foreland. We examined the responses of the respiration rate and the phospholipid fatty acid content to the addition of carbon and/or nitrogen. Soil samples were collected from the early stage and late stage of primary succession in the foreland of a glacier near Ny-Ålesund, Svalbard. The addition of both carbon (glucose) and nitrogen (ammonium nitrate) engendered an increase in the microbial respiration rate in the early stage of succession. In contrast, the addition of either carbon or nitrogen did not increase the microbial respiration rate. In the late stage of succession the addition of carbon alone, as well as the addition of both carbon and nitrogen, increased the microbial respiration rate. However, neither the addition of carbon nor the addition of nitrogen affected the total phospholipid fatty acid content (an index of microbial biomass) for any soil within 15 days of incubation at 10 ° C. An increase in the respiration rate was therefore attributed to changes in the physiological activities of the microbial community, such as enzymatic activity. Our study suggests that microbial respiration was limited by the low availability of both carbon and nitrogen in the early stage of succession. Thereafter, nitrogen limitation is mitigated.

Application of near infrared reflectance (NIR) and fluorescence spectroscopy to analysis of microbiological and chemical properties of arctic soil

Applicability of near infrared reflectance (NIR) and fluorescence spectroscopic techniques was tested on highly organic arctic soil. Soil samples were obtained at a long-term climate change manipulation experiment at a subarctic fell heath in Abisko, northern Sweden. The ecosystem had been exposed to treatments simulating increasing temperature (open-top greenhouses), higher nutrient availability (NPK fertilization) and increasing cloudiness (shading cloths) for 15 years prior to the sampling. For each of the 72 samples from the 0 to 5 cm soil depth and 36 samples from the 5 to 10 cm depth, the wavelength range of 400–2500 nm (visible and near infrared spectrum) was scanned with a NIR spectrophotometer and fluorescence excitation-emission matrices (EEMs) were recorded with a spectrofluorometer. Principal component analyses of the visible, NIR and fluorescence spectra clearly separated the treatments, which indicates that the chemical composition of the soil and its spectral properties had changed during the climate change simulation. Similarly to the results from the conventional analyses of soil chemical and microbiological properties, fertilization treatment posed strongest effects on the spectra. Partial least-squares (PLS) regression methods with cross-validation were used to analyse relationships between the spectroscopic data and the chemical and microbiological data derived from the conventional analyses. The fluorescence EEMs of the dried solid soil samples were moderately related to soil ergosterol content (correlation coefficient r=0.84), bacterial activity analysed by leucine incorporation technique ( r=0.78) and total phospholipid fatty acid (PLFA) content ( r=0.74), but in general fluorescence provided inferior predictions of the chemical and microbiological variables to NIR. NIR was highly related to soil organic matter content ( r>0.9) and showed promising predictions of soil ergosterol content ( r>0.9), microbial biomass C, microbial biomass P, and total PLFA contents ( r=0.78–0.79). These results suggest that especially NIR could be used to predict soil organic matter and fungal biomass. Since it is rapid and inexpensive, and requires little sample mass, it could be used as a ‘quick and dirty’ technique to estimate progression of the treatment responses in long-term ecosystem experiments, where extensive soil sampling is to be avoided.

Nine years of enriched CO2 changes the function and structural diversity of soil microorganisms in a grassland

SummaryTo gain insight into microbial function following increased atmospheric CO2 concentration, we investigated the influence of 9 years of enriched CO2 (600 μl litre−1) on the function and structural diversity of soil microorganisms in a grassland ecosystem under free air carbon dioxide enrichment (FACE), as affected by plant species (Trifolium repens L. and Lolium perenne L. in monocultures and mixed culture) and nitrogen (N) supply. We measured biomass and activities of enzymes covering cycles of the most important elements (C, N and P). The microbial community was profiled by molecular techniques of phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analysis. The enrichment in CO2 increased soil microbial biomass (+48.1%) as well as activities of invertase (+36.2%), xylanase (+22.9%), urease (+23.8%), protease (+40.2%) and alkaline phosphomonoesterase (+54.1%) in spring 2002. In autumn, the stimulation of microbial biomass was 25% less and that of enzymes 3–12% less than in spring. Strong correlations between activities of invertase, protease, urease and alkaline phosphomonoesterase and microbial biomass were found. The stimulation of microbial activity in the enriched atmosphere was probably caused by changes in the quantity and kind of root litter and rhizodeposition. The response of soil microorganisms to enriched CO2 was most pronounced under Trifolium monoculture and under greater N supply. The PLFA analysis revealed that total PLFA contents were greater by 24.7% on average, whereby the proportion of bioindicators representative of Gram‐negative bacteria increased significantly in the enriched CO2 under less N‐fertilized Lolium culture. Discriminant analysis showed marked differences between the PLFA profiles of the three plant communities. Shannon diversity indices calculated from DGGE patterns were greater (+12.5%) in the enriched CO2, indicating increased soil bacterial diversity. We conclude that greater microbial biomass and enzyme activity buffer the potential increase in C sequestration occurring from greater C addition in enriched CO2 due to greater mineralization of soil organic matter.