Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres

Lumbricus terrestris is a deep-burrowing anecic earthworm that builds permanent, vertical burrows with linings (e.g., drilosphere) that are stable and long-lived microhabitats for bacteria, fungi, micro- and mesofauna. We conducted the first non-culture based field study to assess simultaneously the drilosphere (here sampled as 0–2 mm burrow lining) composition of microbial and micro/mesofaunal communities relative to bulk soil. Our study also included a treatment of surface-applied ¹³C- and ¹⁵N-labeled plant residue to trace the short-term (40 d) translocation of residue C and N into the drilosphere, and potentially the assimilation of residue C into drilosphere microbial phospholipid fatty acids (PLFAs). Total C concentration was 23%, microbial PLFA biomass was 58%, and PLFAs associated with protozoa, nematodes, Collembola and other fauna were 200-to-300% greater in the drilosphere than in nearby bulk soil. Principal components analysis of community PLFAs revealed that distributions of Gram-negative bacteria and actinomycetes and other Gram-positive bacteria were highly variable among drilosphere samples, and that drilosphere communities were distinct from bulk soil communities due to the atypical distribution of PLFA biomarkers for micro- and mesofauna. The degree of microbial PLFA ¹³C enrichment in drilosphere soils receiving ¹³C-labeled residue was highly variable, and only one PLFA, 18:1ω9c, was significantly enriched. In contrast, 11 PLFAs from diverse microbial groups where enriched in response to residue amendment in bulk soil 0–5 cm deep. Among control soils, however, a significant δ¹³C shift between drilosphere and bulk soil at the same depth (5–15 cm) revealed the importance of L. terrestris for translocating perennial ryegrass-derived C into the soil at depth, where we estimated the contribution of the recent grass C (8 years) to be at least 26% of the drilosphere soil C. We conclude that L. terrestris facilitates the translocation of plant C into soil at depth and promotes the maintenance of distinct soil microbial and faunal communities that are unlike those found in the bulk soil.     

Low importance for a fungal based food web in arable soils under mineral and organic fertilization indicated by Collembola grazers

We investigated the Collembola community at an arable field where mineral and organic fertilizers have been applied at low and high rates for 27 years. As food resources for Collembola, the soil microbial community was analyzed using phospholipid fatty acids (PLFAs). A special focus was put on AM fungi, which were estimated by the marker 16:1ω5 in PLFA (viable hyphae) and neutral lipid fatty acid (NLFA – storage fat in spores) fractions. Additionally, whole cellular lipids in crop plant tissues and manure were assessed. Greater Collembola species richness occurred in plots where mineral fertilizer was added. In contrast, soil microbial biomass including AM fungal hyphae increased with addition of organic fertilizer, while the amount of AM fungal spores and biomass of saprotrophic fungi were not affected by fertilizer type. The lipid pattern in wheat roots was altered by fertilizer type, application rate and their interaction, indicating different rhizosphere communities. In sum, the availability and composition of food resources for Collembola changed considerably due to farm management practice. The major diet of three dominant Collembola species, Isotoma viridis, Willemia anophthalma and Polyacanthella schäffer was determined by lipid profiling. Multivariate analysis demonstrated species specific lipid patterns, suggesting greater importance of species than management practice on the diet choice. Nevertheless, feeding strategy was affected by fertilizer type and availability of resources, as trophic biomarker fatty acids indicated feeding on wheat roots (and to some extent saprotrophic fungi) with mineral and a shift to soil organic matter (litter, detritus) with organic fertilization. Although AM fungi dominated the soil fungal community, the AMF marker 16:1ω5 was not detected in Collembola lipids, indicating that these were not consumed. The very low amount of saprotrophic fungi in the soil and the fact that Collembola as major fungal grazers did not feed on AM fungi indicates that the fungal energy channel in the investigated arable field is of little importance to the faunal food web.     

Turnover of soil organic matter and of microbial biomass under C3-C4 vegetation change: Consideration of C fractionation and preferential substrate utilization

Two processes contribute to changes of the δ¹³C signature in soil pools: ¹³C fractionation per se and preferential microbial utilization of various substrates with different δ¹³C signature. These two processes were disentangled by simultaneously tracking δ¹³C in three pools - soil organic matter (SOM), microbial biomass, dissolved organic carbon (DOC) - and in CO₂ efflux during incubation of 1) soil after C₃-C₄ vegetation change, and 2) the reference C₃ soil.The study was done on the Ap horizon of a loamy Gleyic Cambisol developed under C₃ vegetation. Miscanthus giganteus - a perennial C₄ plant - was grown for 12 years, and the δ¹³C signature was used to distinguish between ‘old’ SOM (>12 years) and ‘recent’ Miscanthus-derived C (<12 years). The differences in δ¹³C signature of the three C pools and of CO₂ in the reference C₃ soil were less than 1‰, and only δ¹³C of microbial biomass was significantly different compared to other pools. Nontheless, the neglecting of isotopic fractionation can cause up to 10% of errors in calculations. In contrast to the reference soil, the δ¹³C of all pools in the soil after C₃-C₄ vegetation change was significantly different. Old C contributed only 20% to the microbial biomass but 60% to CO₂. This indicates that most of the old C was decomposed by microorganisms catabolically, without being utilized for growth. Based on δ¹³C changes in DOC, CO₂ and microbial biomass during 54 days of incubation in Miscanthus and reference soils, we concluded that the main process contributing to changes of the δ¹³C signature in soil pools was preferential utilization of recent versus old C (causing an up to 9.1‰ shift in δ¹³C values) and not ¹³C fractionation per se.Based on the δ¹³C changes in SOM, we showed that the estimated turnover time of old SOM increased by two years per year in 9 years after the vegetation change. The relative increase in the turnover rate of recent microbial C was 3 times faster than that of old C indicating preferential utilization of available recent C versus the old C.Combining long-term field observations with soil incubation reveals that the turnover time of C in microbial biomass was 200 times faster than in total SOM. Our study clearly showed that estimating the residence time of easily degradable microbial compounds and biomarkers should be done at time scales reflecting microbial turnover times (days) and not those of bulk SOM turnover (years and decades). This is necessary because the absence of C reutilization is a prerequisite for correct estimation of SOM turnover. We conclude that comparing the δ¹³C signature of linked pools helps calculate the relative turnover of old and recent pools.     

Linking microbial community structure and allocation of plant-derived carbon in an organic agricultural soil using 13CO2 pulse-chase labelling combined with 13C-PLFA profiling

We conducted a ¹³CO₂ pulse-chase labelling experiment in a drained boreal organic (peat) soil cultivated with perennial crop, reed canary grass (RCG; Phalaris arundinacea) to study the flow of carbon from plants to soil microbes. Both limed and unlimed soils were studied, since liming is a common agricultural practice for acidic organic soils. Soil samples taken within three months after the labelling and three times in the following year were used for the δ¹³C analysis of microbial phospholipid fatty acids (PLFAs), root sugars and root lipids. We estimated the contribution of carbon from root exudates to microbial PLFA synthesis. The flow of carbon from plants to microbes was fast as the label allocation in PLFAs had a peak 1–3 days after labelling. The results showed that fungi were important in the incorporation of fresh, plant-derived carbon, including root sugars. None of the main microbial PLFA biomarker groups (fungi, Gram-positive bacteria, Gram-negative bacteria, arbuscular mycorrhizal fungi) was completely lacking label over the measurement period. One year after the labelling, when the labelled carbon was widely distributed into plant biomass and soil, bacterial biomarkers increased their share of the label allocation. Liming had a minor effect on the label allocation rate into PLFAs. The mixing model approach used to calculate the root exudate contribution to microbial biomass resulted in a highly conservative estimate of utilization of this important C-source (0–6.5%, with highest incorporation into fungi). In summary, the results of this study provide new information about the role of various microbial groups in the turnover of plant-derived, fresh carbon in boreal organic soil.     

C fractionation at the root-microorganisms-soil interface: A review and outlook for partitioning studies

Natural variations of the ¹³C/¹²C ratio have been frequently used over the last three decades to trace C sources and fluxes between plants, microorganisms, and soil. Many of these studies have used the natural-¹³C-labelling approach, i.e. natural δ¹³C variation after C₃-C₄ vegetation changes. In this review, we focus on ¹³C fractionation in main processes at the interface between roots, microorganisms, and soil: root respiration, microbial respiration, formation of dissolved organic carbon, as well as microbial uptake and utilization of soil organic matter (SOM). Based on literature data and our own studies, we estimated that, on average, the roots of C₃ and C₄ plants are ¹³C enriched compared to shoots by +1.2 ± 0.6‰ and +0.3 ± 0.4‰, respectively. The CO₂ released by root respiration was ¹³C depleted by about −2.1 ± 2.2‰ for C₃ plants and −1.3 ± 2.4‰ for C₄ plants compared to root tissue. However, only a very few studies investigated ¹³C fractionation by root respiration. This urgently calls for further research. In soils developed under C₃ vegetation, the microbial biomass was ¹³C enriched by +1.2 ± 2.6‰ and microbial CO₂ was also ¹³C enriched by +0.7 ± 2.8‰ compared to SOM. This discrimination pattern suggests preferential utilization of ¹³C-enriched substances by microorganisms, but a respiration of lighter compounds from this fraction. The δ¹³C signature of the microbial pool is composed of metabolically active and dormant microorganisms; the respired CO₂, however, derives mainly from active organisms. This discrepancy and the preferential substrate utilization explain the δ¹³C differences between microorganisms and CO₂ by an ‘apparent’ ¹³C discrimination. Preferential consumption of easily decomposable substrates and less negative δ¹³C values were common for substances with low C/N ratios. Preferential substrate utilization was more important for C₃ soils because, in C₄ soils, microbial respiration strictly followed kinetics, i.e. microorganisms incorporated heavier C (? = +1.1‰) and respired lighter C (? = −1.1‰) than SOM. Temperature and precipitation had no significant effect on the ¹³C fractionation in these processes in C₃ soils. Increasing temperature and decreasing precipitation led, however, to increasing δ¹³C of soil C pools.Based on these ¹³C fractionations we developed a number of consequences for C partitioning studies using ¹³C natural abundance. In the framework of standard isotope mixing models, we calculated CO₂ partitioning using the natural-¹³C-labelling approach at a vegetation change from C₃ to C₄ plants assuming a root-derived fraction between 0% and 100% to total soil CO₂. Disregarding any ¹³C fractionation processes, the calculated results deviated by up to 10% from the assumed fractions. Accounting for ¹³C fractionation in the standard deviations of the C₄ source and the mixing pool did not improve the exactness of the partitioning results; rather, it doubled the standard errors of the CO₂ pools. Including ¹³C fractionations directly into the mass balance equations reproduced the assumed CO₂ partitioning exactly. At the end, we therefore give recommendations on how to consider ¹³C fractionations in research on carbon flows between plants, microorganisms, and soil.     

Isotopic fractionation by saprotrophic microfungi: Effects of species, temperature and the age of colonies

Saprotrophic fungi represent an important resource for a number of fungivorous and omnivorous soil animals, but little is known about the patterns of isotopic fractionation by soil fungi. We grew five common species of saprotrophic microfungi in laboratory cultures on simple artificial substrate based on carbohydrates derived either from C3 or C4 plants. Fungal cultures were kept at 15, 20 or 25 °C. Isotopic composition of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) in bulk fungal tissue was determined after 11, 21 and 32 days. The fractionation of carbon and nitrogen stable isotopes was species-specific, but generally did not differ in C3- and C4-based growth media. The Zygomycete Mucor plumbeus did not differ in δ¹³C from the carbon source used, though Ascomycetes (Alternaria alternata, Cladosporium cladosporioides, Trichoderma harzianum and Ulocladium botrytis) were depleted in heavy carbon relative to the carbon source by 0.5-0.9‰. Three species were significantly depleted in ¹⁵N relative to the sodium nitrate that was used as a single source of nitrogen. In all species, δ¹⁵N but not δ¹³C tended to increase with the age of fungal colonies. The effect of temperature on δ¹⁵N was weak and inconsistent in different species. In contrast, all fungi except T. harzianum accumulated more ¹³С at 25 °C than at 15 °C. The overall variation in the isotopic signatures of saprotrophic fungi growing in identical conditions reached 8‰ for δ¹⁵N and 2.5‰ for δ¹³C due to species-specific differences in the isotopic fractionation and the age of individual fungal colonies. This variation should be incorporated into the interpretation of the isotopic composition of fungivorous soil animals.     

Translocation of surface litter carbon into soil by Collembola

Soil invertebrates are important in nutrient cycling in soils, but the degree to which mesofauna such as Collembola are responsible for the direct movement of carbon (C) from the litter layer into soil has not yet been ascertained. We used naturally occurring stable C isotopic differences between a C₄ soil and alder leaves (C₃) to examine the effect of the collembolan Folsomia candida on C translocation into soil in laboratory microcosms. Collembolan numbers greatly increased in the presence of alder, but despite large collembolan populations there were no changes in decomposition rate (measured as litter mass loss, cumulative respired CO₂ and alder C:N ratios). Small changes in the δ¹³C values of bulk soil organic matter were detected, but could not be assigned to collembolan activity. However, mean δ¹³C values of soil microbial phospholipid fatty acids (PLFAs) were significantly lower in the presence of alder and Collembola together, demonstrating that collembolan activities resulted in greater availability of litter-derived C to the soil microbial community. Additionally, the presence of Collembola resulted in the translocation of alder-derived compounds (chlorophyll and its breakdown product pheophytin) into soil, demonstrating that Collembola modify soil organic matter at the molecular level. These results are consistent with deposition of collembolan faeces in underlying soil and demonstrate that despite their small size, Collembola contribute directly to C transport in the litter-soil environment.     

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

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

Carbon isotopic ratios of wetland and terrace soil sequences in the Maya Lowlands of Belize and Guatemala

This article provides new data and synthesizes earlier findings on the carbon isotope ratios of the humin part of soil organic matter from a range of sites in the central Maya Lowlands. Changes down the soil profile in carbon isotope ratios can provide an important line of evidence for vegetation change and erosion over time, especially in well dated aggrading profiles. Research thus far has provided substantial evidence for significant inputs from C₄ vegetation in buried layers from the Ancient Maya periods in depositional soils but equivocal evidence from sloping soils. We present new findings from soil profiles through ancient Maya wetland fields, upland karst wetlands, ancient Maya aguadas (reservoirs), and ancient Maya terraces. Most of the profiles exhibited δ¹³C enrichment greater than the 2.5–3‰ typical from bacterial fractionation. Seven of nine ancient Maya wetland profiles showed δ¹³C enrichment ranging from 4.25 to 8.56‰ in ancient Maya-dated sediments that also contained phytolith and pollen evidence of grass (C₄ species) dominance. Upland karst sinks and ancient reservoirs produced more modest results for δ¹³C enrichment. These seasonal wetland profiles exhibited δ¹³C enrichment ranging from 1 to 7.3‰ from the surface to ancient Maya-period sediments. Agricultural terraces produced mixed results, with two terraces having substantial δ¹³C enrichment of 5.34 and 5.66‰ and two producing only equivocal results of 1.88 and 3.03‰ from modern topsoils to Maya Classic-period buried soils. Altogether, these findings indicate that C₄ plants made up c. 25% of the vegetation at our sites in the Maya Classic period and only a few percent today. These findings advance the small corpus of studies from ancient terraces, karst sinks, and ancient wetland fields by demonstrating substantial δ¹³C and thus C₄ plant enrichment in soil profile sections dated to ancient Maya times. These studies are also providing a new line of evidence about local and regional soil and ecological change in this region of widespread environmental change in the Late Holocene.     

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

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

Origin of positive δC of emitted CO2 from soils after application of biogas residues

Bioenergy production from renewable organic material is known to be a clean energy source and therefore its use is currently much promoted in many countries. Biogas by-products also called biogas residues (BGR) are rich in partially stable organic carbon and can be used as an organic fertilizer for crop production. However so far, many environmental issues relevant when BGR are applied to agricultural land (soil C sequestration, increased denitrification and nutrient leaching) still have to be studied. Therefore a field experiment was set up to investigate the degradation of BGR and its impact on the decomposition of native soil organic matter based on a natural abundance stable isotope approach. Maize, a C₄ plant has been used as bioenergy crop, therefore the δ¹³C of total C in BGR was −16.0‰_PDB and soil organic matter was mostly derived from C₃ plant based detritus, SOM thus showed a δ¹³C of −28.4‰_PDB. Immediately after BGR application, soil-emitted CO₂ showed unexpectedly high δ¹³C of up to +23.6‰_PDB, which has never been reported earlier. A subsequent laboratory scale experiment confirmed the positive δ¹³C of soil-emitted CO₂ after BGR addition and showed that obviously, the added BGR led to a consumption of dissolved inorganic C in soils. Additionally, it was observed that the δ¹³C of CO₂ driven from inorganic C of BGR (BGR-IC) by acid treatment was +35.6‰_PDB. Therefore, we suggest that also under field conditions the transformation of BGR-IC into CO₂ contributed largely to CO₂ emissions in addition to the decomposition of organic matter, which affected both the amount and the carbon isotope signature of emitted CO₂ in the initial period after BGR application. Positive δ¹³C of inorganic C contained in BGR was attributed to processes with strong fractionation of C isotopes during anaerobic fermentation in the biogas formation process.     

Plant species influence microbial diversity and carbon allocation in the rhizosphere

Plant species effects on microbial communities are attributed to changes in microbial community composition and biomass, and may depend on plant species specific differences in the quality of resources (carbon) inputs. We examined the idea that plant-soil feedbacks can be explained by a chance effect, which is the probability of a highly productive or keystone plant species is present in the community and will influence the functions more than the number of species per se. A ¹³C pulse labelling technique was applied to three plant species and a species mixture in a greenhouse experiment to examine the carbon flow from plants to soil microbial communities. The ¹³C label was given as CO₂ to shoots of a legume (Lotus corniculatus), a forb (Plantago lanceolata), a grass (Holcus lanatus) and a mixture of the three species. Microbial phospholipid fatty acids (PLFA) was analysed in order to determine the biomass and composition of the soil microbial community. The incorporation of the stable isotope into soil microorganisms was determined through GC-IRMS analyses of the microbial PLFAs. Plant species identity did not influence the microbial biomass when determined as total carbon of microbial phospholipid fatty acids. However, the labelled carbon showed that the grass monoculture (H. lanatus) and the plant mixture allocated more ¹³C into bacteria and actinomycete biomass than the other plant species. H. lanatus monocultures had also the highest amounts of ¹³C allocated to AM-fungi and saprophytic fungi. The carbon allocation from plants to soil microorganisms in a plant species mixture can thus be explained by the presence of a highly productive species that influence soil functions.     

C signature of CO2 evolved from incubated maize residues and maize-derived sheep faeces

Analyses of the spatial and temporal variations in the natural abundance of ¹³C are frequently employed to study transformations of plant residues and soil organic matter turnover on sites where long continued vegetation with the C₃-type photosynthesis pathway has been replaced with a C₄-type vegetation (or vice versa). One controversial issue associated with such analyses is the significance of isotopic fractionation during the microbial turnovers of C in complex substrates. To evaluate this issue, C₃-soil and quartz sand were amended with maize residues and with faeces from sheep feed exclusively on maize silage. The samples were incubated at 15 °C for 117 days (maize residues) or 224 days (sheep faeces). CO₂ evolved during incubation was trapped in NaOH and analysed for C isotopic contents. At the end of incubation, 63 and 50% of the maize C was evolved as CO₂ in the soil and sand, respectively, while 32% of the faeces C incubated with soil and with sand was recovered as CO₂. Maize and faeces showed a similar decomposition pattern but maize decomposed twice as fast as faeces. The δ¹³C of faeces was 0.3‰ lower than that of the maize residue (δ¹³C −13.4‰), while the δ¹³C of the C₃-soil used for incubation was −31.6‰. The δ¹³C value of the CO₂ recovered from unamended C₃-soil was similar or slightly lower (up to −1.5‰) than that of the C₃-soil itself except for an initial flush of ¹³C enriched CO₂. The δ¹³C values of the CO₂ from sand-based incubations typically ranged −15‰ to −17‰, i.e. around −3‰ lower than the δ¹³C measured for maize and faeces. Our study clearly demonstrates that the decomposition of complex substrates is associated with isotopic fractionation, causing evolved CO₂ to be depleted in ¹³C relative to substrates. Consequently the microbial products retained in the soil must be enriched in ¹³C.     

Use of 13C and 15N natural abundance techniques to characterize carbon and nitrogen dynamics in composting and in compost-amended soils

Isotope fractionation during composting may produce organic materials with a more homogenous δ¹³C and δ¹⁵N signature allowing study of their fate in soil. To verify this, C, N, δ¹³C and δ¹⁵N content were monitored during nine months covered (thermophilic; >40 °C) composting of corn silage (CSC). The C concentration reduced from 10.34 to 1.73 g C (g ash)⁻¹, or 83.3%, during composting. Nitrogen losses comprised 28.4% of initial N content. Compost δ¹³C values became slightly depleted and increasingly uniform (from −12.8±0.6‰ to −14.1±0.0‰) with composting. Compost δ¹⁵N values (0.3±1.3 to 8.2±0.4‰) increased with a similar reduced isotope variability.The fate of C and N of diverse composts in soil was subsequently examined. C, N, δ¹³C, δ¹⁵N content of whole soil (0-5 cm), light (<1.7 g cm⁻³) and heavy (>1.7 g cm⁻³) fraction, and (250-2000 μm; 53-250 μm and <53 μm) size separates, were characterized. Measurements took place one and two years following surface application of CSC, dairy manure compost (DMC), sewage sludge compost (SSLC), and liquid dairy manure (DM) to a temperate (C₃) grassland soil. The δ¹³C values and total C applied (Mg C ha⁻¹) were DM (−27.3‰; 2.9); DMC (−26.6‰; 10.0); SSLC (−25.9‰; 10.9) and CSC (−14.0‰; 4.6 and 9.2). The δ¹³C of un-amended soil exhibited low spatial (−28.0‰±0.2; n=96) and temporal (±0.1‰) variability. All C₄ (CSC) and C₃ (DMC; SSLC) composts, except C₃ manure (DM), significantly modified bulk soil δ¹³C and δ¹⁵N. Estimates of retention of compost C in soil by carbon balance were less sensitive than those calculated by C isotope techniques. One and two years after application, 95 and 89% (CSC), 75 and 63% (SSLC) and 88 and 42% (DMC) of applied compost C remained in the soil, with the majority (80-90%) found in particulate (>53 μm) and light fractions. However, C₄ compost (CSC) was readily detectable (12% of compost C remaining) in mineral (<53 μm) fractions. The δ¹⁵N-enriched N of compost supported interpretation of δ¹³C data. We can conclude that composts are highly recalcitrant with prolonged C storage in non-mineral soil fractions. The sensitivity of the natural abundance tracer technique to characterize their fate in soil improves during composting, as a more homogeneous C isotope signature develops, in addition to the relatively large amounts of stable C applied in composts.     

Microbial phospholipid biomarkers and stable isotope methods help reveal soil functions

This review targets microbial phospholipid biomarkers, their isotope analysis and their ability to reveal soil functions. The amount and composition of phospholipid fatty acids (PLFAs) measured in environmental samples strongly depend on the methodology. To achieve comparable results the extraction, separation and methylation method must be kept constant. PLFAs patterns are sensitive to microbial community shifts even though the taxonomic resolution of PLFAs is low. The possibility to easily link lipid biomarkers with stable isotope techniques is identified as a major advantage when addressing soil functions. Measurement of PLFA isotopic ratios is sensitive and enables detecting isotopic fractionation. The difference between the carbon isotopic ratio of single PLFAs and their substrate (Δ¹³C) can vary between −6 and +11‰. This difference derives from the fractionation during biosynthesis and from substrate inhomogeneity. Consequently, natural abundance studies are restricted to quantifying substrate uptake of the total microbial biomass. In contrast, artificial labelling enables quantifying carbon uptake into single PLFAs, but labelling success depends on homogeneous and undisturbed label application. Current developments in microbial ecology (e.g. ¹³C and ¹⁵N proteomics) and isotope techniques (online monitoring of CO₂ isotope ratios) will likely improve soil functional interpretations in the future. ¹³C PLFA analysis will continue to contribute because it is affordable, sensitive and allows frequent sampling combined with the use of small amounts of ¹³C label.     

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

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

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

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

Incorporation of 13C-labelled rice rhizodeposition carbon into soil microbial communities under different water status

The overall processes by which carbon is fixed by plants in photosynthesis then released into the soil by rhizodeposition and subsequently utilized by soil micro-organisms, links the atmospheric and soil carbon pools. The objective of this study was to determine the plant derived ¹³C incorporated into the phospholipid fatty acid (PLFA) pattern in paddy soil, to test whether utilization of rice rhizodeposition carbon by soil micro-organisms is affected by soil water status. This is essential to understand the importance of flooded conditions in regulating soil microbial community structure and activity in wetland rice systems. Rice plants were grown in soil derived from a paddy system under controlled irrigation (CI), or with continuous waterlogging (CW). Most of the ¹³C-labelled rice rhizodeposition carbon was distributed into the PLFAs 16:0, 18:1ω7 and 18:1ω9 in both the CW and CI treatments. The bacterial PLFAs i15:0 and a15:0, both indicative of gram positive bacteria, were relatively more abundant in the treatments without rice plants. When rice plants were present rates of ¹³C-incorporation into i15:0 and a15:0 was slow; the microbes containing these PLFAs may derive most of their carbon from more recalcitrant C (soil organic matter). PLFAs, 18:1ω7 and 16:1ω7c, indicative of gram negative bacteria showed a greater amount incorporation of labelled plant derived carbon in the CW treatment. In contrast, 18:2ω6,9 indicative of fungi and 18:1ω9 indicative of aerobes but also potentially fungi and plant roots had greater incorporation in the CI treatment. The greater root mass concomitant with lower incorporation of ¹³C into the total PLFA pool in the CW treatment suggests that the microbial communities in wetland rice soil are limited by factors other than substrate availability in flooded conditions. In this study differing soil microbial communities were established through manipulating the water status of paddy soils. Steady state ¹³C labelling enabled us to determine that the microbial community utilizing plant derived carbon was also affected by water status.     

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.     

Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a C labelled-leaf litter experiment

Partitioning of the quantities of C lost by leaf litter through decomposition into (i) CO₂ efflux to the atmosphere and (ii) C input to soil organic matter (SOM) is essential in order to develop a deeper understanding of the litter-soil biogeochemical continuum. However, this is a challenging task due to the occurrence of many different processes contributing to litter biomass loss. With the aim of quantifying different fluxes of C lost by leaf litter decomposition, a field experiment was performed at a short rotation coppice poplar plantation in central Italy. Populus nigra leaf litter, enriched in ¹³C (δ¹³C ∼ +160‰) was placed within collars to decompose in direct contact with the soil (δ¹³C ∼ −26‰) for 11 months. CO₂ efflux from within the collars and its isotopic composition were determined at monthly intervals. After 11 months, remaining litter and soil profiles (0-20 cm) were sampled and analysed for their total C and ¹³C content. Gas chromatography (GC), GC-mass spectrometry (MS) and GC-combustion-isotope ratio (GC/C/IRMS) were used to analyse phospholipid fatty acids (PLFA) extracted from soil samples to identify the groups of soil micro-organisms that had incorporated litter-derived C and to determine the quantity of C incorporated by the soil microbial biomass (SMB). By the end of the experiment, the litter had lost about 80% of its original weight. The fraction of litter C lost as an input into the soil (67 ± 12% of the total C loss) was found to be twice as much as the fraction released as CO₂ to the atmosphere (30 ± 3%), thus demonstrating the importance of quantifying litter-derived C input to soils, in litter decomposition studies. The mean δ¹³C values of PLFAs in soil (δ¹³C = −12.5‰) showed sustained incorporation of litter-derived C after one year (7.8 ± 1.6% of total PLFA-C). Thus, through the application of stable ¹³C isotope analyses, we have quantified two major C fluxes contributing to litter decomposition, at macroscopic and microscopic levels.