Microbial responses to warming enhance soil carbon loss following translocation across a tropical forest elevation gradient.

Tropical soils contain huge carbon stocks, which climate warming is projected to reduce by stimulating organic matter decomposition, creating a positive feedback that will promote further warming. Models predict that the loss of carbon from warming soils will be mediated by microbial physiology, but no empirical data are available on the response of soil carbon and microbial physiology to warming in tropical forests, which dominate the terrestrial carbon cycle. Here we show that warming caused a considerable loss of soil carbon that was enhanced by associated changes in microbial physiology. By translocating soils across a 3000 m elevation gradient in tropical forest, equivalent to a temperature change of ± 15 °C, we found that soil carbon declined over 5 years by 4% in response to each 1 °C increase in temperature. The total loss of carbon was related to its original quantity and lability, and was enhanced by changes in microbial physiology including increased microbial carbon-use-efficiency, shifts in community composition towards microbial taxa associated with warmer temperatures, and increased activity of hydrolytic enzymes. These findings suggest that microbial feedbacks will cause considerable loss of carbon from tropical forest soils in response to predicted climatic warming this century.

Isotopic evidence of biotrophy and unusual nitrogen nutrition in soil-dwelling Hygrophoraceae.

Several lines of evidence suggest that the agaricoid, non-ectomycorrhizal members of the family Hygrophoraceae (waxcaps) are biotrophic with unusual nitrogen nutrition. However, methods for the axenic culture and lab-based study of these organisms remain to be developed, so our current knowledge is limited to field-based investigations. Addition of nitrogen, lime or organophosphate pesticide at an experimental field site (Sourhope) suppressed fruiting of waxcap basidiocarps. Furthermore, stable isotope natural abundance in basidiocarps were unusually high in 15 N and low in 13 C, the latter consistent with mycorrhizal nutritional status. Similar patterns were found in waxcap basidiocarps from diverse habitats across four continents. Additional data from 14 C analysis of basidiocarps and 13 C pulse label experiments suggest that these fungi are not saprotrophs but rather biotrophic endophytes and possibly mycorrhizal. The consistently high but variable δ15 N values (10-20‰) of basidiocarps further indicate that N acquisition or processing differ from other fungi; we suggest that N may be derived from acquisition of N via soil fauna high in the food chain.

Microbes follow Humboldt: temperature drives plant and soil microbial diversity patterns from the Amazon to the Andes.

More than 200 years ago, Alexander von Humboldt reported that tropical plant species richness decreased with increasing elevation and decreasing temperature. Surprisingly, coordinated patterns in plant, bacterial, and fungal diversity on tropical mountains have not yet been observed, despite the central role of soil microorganisms in terrestrial biogeochemistry and ecology. We studied an Andean transect traversing 3.5 km in elevation to test whether the species diversity and composition of tropical forest plants, soil bacteria, and fungi follow similar biogeographical patterns with shared environmental drivers. We found coordinated changes with elevation in all three groups: species richness declined as elevation increased, and the compositional dissimilarity among communities increased with increased separation in elevation, although changes in plant diversity were larger than in bacteria and fungi. Temperature was the dominant driver of these diversity gradients, with weak influences of edaphic properties, including soil pH. The gradients in microbial diversity were strongly correlated with the activities of enzymes involved in organic matter cycling, and were accompanied by a transition in microbial traits towards slower-growing, oligotrophic taxa at higher elevations. We provide the first evidence of coordinated temperature-driven patterns in the diversity and distribution of three major biotic groups in tropical ecosystems: soil bacteria, fungi, and plants. These findings suggest that interrelated and fundamental patterns of plant and microbial communities with shared environmental drivers occur across landscape scales. These patterns are revealed where soil pH is relatively constant, and have implications for tropical forest communities under future climate change.

Temporal and spatial influences incur reconfiguration of Arctic heathland soil bacterial community structure.

Microbial responses to Arctic climate change could radically alter the stability of major stores of soil carbon. However, the sensitivity of plot-scale experiments simulating climate change effects on Arctic heathland soils to potential confounding effects of spatial and temporal changes in soil microbial communities is unknown. Here, the variation in heathland soil bacterial communities at two survey sites in Sweden between spring and summer 2013 and at scales between 0-1 m and, 1-100 m and between sites (> 100 m) were investigated in parallel using 16S rRNA gene T-RFLP and amplicon sequencing. T-RFLP did not reveal spatial structuring of communities at scales < 100 m in any site or season. However, temporal changes were striking. Amplicon sequencing corroborated shifts from r- to K-selected taxon-dominated communities, influencing in silico predictions of functional potential. Network analyses reveal temporal keystone taxa, with a spring betaproteobacterial sub-network centred upon a Burkholderia operational taxonomic unit (OTU) and a reconfiguration to a summer sub-network centred upon an alphaproteobacterial OTU. Although spatial structuring effects may not confound comparison between plot-scale treatments, temporal change is a significant influence. Moreover, the prominence of two temporally exclusive keystone taxa suggests that the stability of Arctic heathland soil bacterial communities could be disproportionally influenced by seasonal perturbations affecting individual taxa.

Effects of species evenness and dominant species identity on multiple ecosystem functions in model grassland communities.

Ecosystems provide multiple services upon which humans depend. Understanding the drivers of the ecosystem functions that support these services is therefore important. Much research has investigated how species richness influences functioning, but we lack knowledge of how other community attributes affect ecosystem functioning. Species evenness, species spatial arrangement, and the identity of dominant species are three attributes that could affect ecosystem functioning, by altering the relative abundance of functional traits and the probability of synergistic species interactions such as facilitation and complementary resource use. We tested the effect of these three community attributes and their interactions on ecosystem functions over a growing season, using model grassland communities consisting of three plant species from three functional groups: a grass (Anthoxanthum odoratum), a forb (Plantago lanceolata), and a N-fixing forb (Lotus corniculatus). We measured multiple ecosystem functions that support ecosystem services, including ecosystem gas exchange, water retention, C and N loss in leachates, and plant biomass production. Species evenness and dominant species identity strongly influenced the ecosystem functions measured, but spatial arrangement had few effects. By the end of the growing season, evenness consistently enhanced ecosystem functioning and this effect occurred regardless of dominant species identity. The identity of the dominant species under which the highest level of functioning was attained varied across the growing season. Spatial arrangement had the weakest effect on functioning, but interacted with dominant species identity to affect some functions. Our results highlight the importance of understanding the role of multiple community attributes in driving ecosystem functioning.

Vegetation type, not the legacy of warming, modifies the response of microbial functional genes and greenhouse gas fluxes to drought in Oro-Arctic and alpine regions.

Climate warming and summer droughts alter soil microbial activity, affecting greenhouse gas (GHG) emissions in Arctic and alpine regions. However, the long-term effects of warming, and implications for future microbial resilience, are poorly understood. Using one alpine and three Arctic soils subjected to in situ long-term experimental warming, we simulated drought in laboratory incubations to test how microbial functional-gene abundance affects fluxes in three GHGs: carbon dioxide, methane, and nitrous oxide. We found that responses of functional gene abundances to drought and warming are strongly associated with vegetation type and soil carbon. Our sites ranged from a wet, forb dominated, soil carbon-rich systems to a drier, soil carbon-poor alpine site. Resilience of functional gene abundances, and in turn methane and carbon dioxide fluxes, was lower in the wetter, carbon-rich systems. However, we did not detect an effect of drought or warming on nitrous oxide fluxes. All gene-GHG relationships were modified by vegetation type, with stronger effects being observed in wetter, forb-rich soils. These results suggest that impacts of warming and drought on GHG emissions are linked to a complex set of microbial gene abundances and may be habitat-specific.

Do soil depth and plant community composition interact to modify the resistance and resilience of grassland ecosystem functioning to drought?

While the effect of drought on plant communities and their associated ecosystem functions is well studied, little research has considered how responses are modified by soil depth and depth heterogeneity. We conducted a mesocosm study comprising shallow and deep soils, and variable and uniform soil depths, and two levels of plant community composition, and exposed them to a simulated drought to test for interactive effects of these treatments on the resilience of carbon dioxide fluxes, plant functional traits, and soil chemical properties. We tested the hypotheses that: (a) shallow and variable depth soils lead to increased resistance and resilience of ecosystem functions to drought due to more exploitative plant trait strategies; (b) plant communities associated with intensively managed high fertility soils, will have more exploitative root traits than extensively managed, lower fertility plant communities. These traits will be associated with higher resistance and resilience to drought and may interact with soil depth and depth heterogeneity to amplify the effects on ecosystem functions. Our results showed that while there were strong soil depth/heterogeneity effects on plant-driven carbon fluxes, it did not affect resistance or resilience to drought, and there were no treatment effects on plant-available carbon or nitrogen. We did observe a significant increase in exploitative root traits in shallow and variable soils relative to deep and uniform, which may have resulted in a compensation effect which led to the similar drought responses. Plant community compositions representative of intensive management were more drought resilient than more diverse "extensive" communities irrespective of soil depth or soil depth heterogeneity. In intensively managed plant communities, root traits were more representative of exploitative strategies. Taken together, our results suggest that reorganization of root traits in response to soil depth could buffer drought effects on ecosystem functions.

Vegetation affects the relative abundances of dominant soil bacterial taxa and soil respiration rates in an upland grassland soil.

Plant-derived organic matter inputs are thought to be a key driver of soil bacterial community composition and associated soil processes. We sought to investigate the role of acid grassland vegetation on soil bacterial community structure by assessing bacterial diversity in combination with other soil variables in temporally and spatially distinct samples taken from a field-based plant removal experiment. Removal of aboveground vegetation resulted in reproducible differences in soil properties, soil respiration and bacterial diversity. Vegetated soils had significantly increased carbon and nitrogen concentrations and exhibited higher rates of respiration. Molecular analyses revealed that the soils were broadly dominated by Alphaproteobacterial and Acidobacterial lineages, with increased abundances of Alphaproteobacteria in vegetated soils and more Acidobacteria in bare soils. This field-based study contributes to a growing body of evidence documenting the effect of soil nutrient status on the relative abundances of dominant soil bacterial taxa, with Proteobacterial taxa dominating over Acidobacteria in soils exhibiting higher rates of C turnover. Furthermore, we highlight the role of aboveground vegetation in mediating this effect by demonstrating that plant removal can alter the relative abundances of dominant soil taxa with concomitant changes in soil CO(2)-C efflux.

Litter evenness influences short-term peatland decomposition processes.

There is concern that changes in climate and land use could increase rates of decomposition in peatlands, leading to release of stored C to the atmosphere. Rates of decomposition are driven by abiotic factors such as temperature and moisture, but also by biotic factors such as changes in litter quality resulting from vegetation change. While effects of litter species identity and diversity on decomposition processes are well studied, the impact of changes in relative abundance (evenness) of species has received less attention. In this study we investigated effects of changes in short-term peatland plant species evenness on decomposition in mixed litter assemblages, measured as litter weight loss, respired CO(2) and leachate C and N. We found that over the 307-day incubation period, higher levels of species evenness increased rates of decomposition in mixed litters, measured as weight loss and leachate dissolved organic N. We also found that the identity of the dominant species influenced rates of decomposition, measured as weight loss, CO(2) flux and leachate N. Greatest rates of decomposition were when the dwarf shrub Calluna vulgaris dominated litter mixtures, and lowest rates when the bryophyte Pleurozium schreberi dominated. Interactions between evenness and dominant species identity were also detected for litter weight loss and leachate N. In addition, positive non-additive effects of mixing litter were observed for litter weight loss. Our findings highlight the importance of changes in the evenness of plant community composition for short-term decomposition processes in UK peatlands.

Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands.

Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.

Soil bacterial networks are less stable under drought than fungal networks.

Soil microbial communities play a crucial role in ecosystem functioning, but it is unknown how co-occurrence networks within these communities respond to disturbances such as climate extremes. This represents an important knowledge gap because changes in microbial networks could have implications for their functioning and vulnerability to future disturbances. Here, we show in grassland mesocosms that drought promotes destabilising properties in soil bacterial, but not fungal, co-occurrence networks, and that changes in bacterial communities link more strongly to soil functioning during recovery than do changes in fungal communities. Moreover, we reveal that drought has a prolonged effect on bacterial communities and their co-occurrence networks via changes in vegetation composition and resultant reductions in soil moisture. Our results provide new insight in the mechanisms through which drought alters soil microbial communities with potential long-term consequences, including future plant community composition and the ability of aboveground and belowground communities to withstand future disturbances.

Land use driven change in soil pH affects microbial carbon cycling processes.

Soil microorganisms act as gatekeepers for soil-atmosphere carbon exchange by balancing the accumulation and release of soil organic matter. However, poor understanding of the mechanisms responsible hinders the development of effective land management strategies to enhance soil carbon storage. Here we empirically test the link between microbial ecophysiological traits and topsoil carbon content across geographically distributed soils and land use contrasts. We discovered distinct pH controls on microbial mechanisms of carbon accumulation. Land use intensification in low-pH soils that increased the pH above a threshold (~6.2) leads to carbon loss through increased decomposition, following alleviation of acid retardation of microbial growth. However, loss of carbon with intensification in near-neutral pH soils was linked to decreased microbial biomass and reduced growth efficiency that was, in turn, related to trade-offs with stress alleviation and resource acquisition. Thus, less-intensive management practices in near-neutral pH soils have more potential for carbon storage through increased microbial growth efficiency, whereas in acidic soils, microbial growth is a bigger constraint on decomposition rates.

Insights into the fate of a 13C labelled phenol pulse for stable isotope probing (SIP) experiments.

Stable isotope probing (SIP) using DNA or RNA as a biomarker has proven to be a useful method for attributing substrate utilisation to specific microbial taxa. In this study we followed the transfer of a (13)C(6)-phenol pulse in an activated sludge micro-reactor to examine the resulting distribution of labelled carbon in the context of SIP. Most of the added phenol was metabolically converted within the first 100 min after (13)C(6)-phenol addition, with 49% incorporated into microbial biomass and 6% respired as CO(2). Less than 1% of the total (13)C labelled carbon supplied was incorporated into microbial RNA and DNA, with RNA labelling 6.5 times faster than DNA. The remainder of the added (13)C was adsorbed and/or complexed to suspended solids within the sludge. The (13)C content of nucleic acids increased beyond the initial consumption of the (13)C-phenol pulse. This study confirms that RNA labels more efficiently than DNA and reveals that only a small proportion of a pulse is incorporated into nucleic acids. Evidence of continued (13)C incorporation into nucleic acids suggests that cross-feeding of the SIP substrate was rapid. This highlights both the benefits of using a biomarker that is rapidly labelled and the importance of sampling within appropriate timescales to avoid or capture the effects of cross-feeding, depending on the goal of the study.

Land use not litter quality is a stronger driver of decomposition in hyperdiverse tropical forest.

In hyperdiverse tropical forests, the key drivers of litter decomposition are poorly understood despite its crucial role in facilitating nutrient availability for plants and microbes. Selective logging is a pressing land use with potential for considerable impacts on plant-soil interactions, litter decomposition, and nutrient cycling. Here, in Borneo's tropical rainforests, we test the hypothesis that decomposition is driven by litter quality and that there is a significant "home-field advantage," that is positive interaction between local litter quality and land use. We determined mass loss of leaf litter, collected from selectively logged and old-growth forest, in a fully factorial experimental design, using meshes that either allowed or precluded access by mesofauna. We measured leaf litter chemical composition before and after the experiment. Key soil chemical and biological properties and microclimatic conditions were measured as land-use descriptors. We found that despite substantial differences in litter quality, the main driver of decomposition was land-use type. Whilst inclusion of mesofauna accelerated decomposition, their effect was independent of land use and litter quality. Decomposition of all litters was slower in selectively logged forest than in old-growth forest. However, there was significantly greater loss of nutrients from litter, especially phosphorus, in selectively logged forest. The analyses of several covariates detected minor microclimatic differences between land-use types but no alterations in soil chemical properties or free-living microbial composition. These results demonstrate that selective logging can significantly reduce litter decomposition in tropical rainforest with no evidence of a home-field advantage. We show that loss of key limiting nutrients from litter (P & N) is greater in selectively logged forest. Overall, the findings hint at subtle differences in microclimate overriding litter quality that result in reduced decomposition rates in selectively logged forests and potentially affect biogeochemical nutrient cycling in the long term.

Seasonal variations in decomposition processes in a valley-bottom riparian peatland.

A year-long field survey was carried out at a valley-bottom riparian peatland site in North Wales, UK from January 2002 to December 2002 to examine the seasonal variation of decomposition processes and dissolved organic carbon (DOC) concentrations. Peat temperature, physicochemistry, organic carbon pools, basal CO(2) respiration and extracellular enzyme activities (beta-glucosidase, phosphatase, sulphatase and phenol oxidase) were monitored monthly. The results of a principle component eigenanalysis of field data show that concentrations of basal CO(2) respiration, soil solution DOC and phenolics were positively correlated to soil temperature (P<0.01, F=12.25; P<0.001, F=59.8; P<0.001, F=141.27) with Q(10) responses of 2.29, 6.42 and 14.42, respectively. Extracellular enzyme activities, however, were more strongly associated with seasonal changes in ion concentrations and did not correspond significantly to temperature alone suggesting limitations attributable to a combination of continuous anaerobiosis and/or the suppressive compounds. Restraints on soil enzyme activities may limit the loss of CO(2) from the microbial community that is dependent on soil enzyme activities for nutrient availability. The seasonal effect of temperature on DOC may be explained by increased plant rhizodeposition and microbial activity. These results do not imply that the long-term increasing trend in DOC export is explainable by temperature increase but suggest that temperature may be a key factor regulating the seasonal variation in DOC concentrations. Thus, seasonal temperature effects on DOC may represent an important component of long-term models of DOC export.

Root Herbivores Drive Changes to Plant Primary Chemistry, but Root Loss Is Mitigated under Elevated Atmospheric CO2.

Above- and belowground herbivory represents a major challenge to crop productivity and sustainable agriculture worldwide. How this threat from multiple herbivore pests will change under anthropogenic climate change, via altered trophic interactions and plant response traits, is key to understanding future crop resistance to herbivory. In this study, we hypothesized that atmospheric carbon enrichment would increase the amount (biomass) and quality (C:N ratio) of crop plant resources for above- and belowground herbivore species. In a controlled environment facility, we conducted a microcosm experiment using the large raspberry aphid (Amphorophora idaei), the root feeding larvae of the vine weevil (Otiorhynchus sulcatus), and the raspberry (Rubus idaeus) host-plant. There were four herbivore treatments (control, aphid only, weevil only and a combination of both herbivores) and an ambient (aCO2) or elevated (eCO2) CO2 treatment (390 versus 650 ± 50 µmol/mol) assigned to two raspberry cultivars (cv Glen Ample or Glen Clova) varying in resistance to aphid herbivory. Contrary to our predictions, eCO2 did not increase crop biomass or the C:N ratio of the plant tissues, nor affect herbivore abundance either directly or via the host-plant. Root herbivory reduced belowground crop biomass under aCO2 but not eCO2, suggesting that crops could tolerate attack in a CO2 enriched environment. Root herbivory also increased the C:N ratio in leaf tissue at eCO2, potentially due to decreased N uptake indicated by lower N concentrations found in the roots. Root herbivory greatly increased root C concentrations under both CO2 treatments. Our findings confirm that responses of crop biomass and biochemistry to climate change need examining within the context of herbivory, as biotic interactions appear as important as direct effects of eCO2 on crop productivity.

Harmonising conflicts between science, regulation, perception and environmental impact: the case of soil conditioners from bioenergy.

As the global population is expected to reach 9 billion by 2050, humanity needs to balance an ever increasing demand for food, energy and natural resources, with sustainable management of ecosystems and the vital services that they provide. The intensification of agriculture, including the use of fertilisers from finite sources, has resulted in extensive soil degradation, which has increased food production costs and CO2 emissions, threatening food security. The Bioenergy sector has significant potential to contribute to the formation of a circular economy. This paper presents the scientific, regulatory and socioeconomic barriers to the use of the nutrient waste streams from biomass thermal conversion (ash) and anaerobic digestion (digestate) as sustainable soil amendments for use in place of traditional fertilisers. It is argued that whilst the ability of combined ash and digestate to remedy many threats to ecosystems and provide a market to incentivise the renewable bio-energy schemes is promising, a step-change is required to alter perceptions of 'waste', from an expensive problem, to a product with environmental and economic value. This can only be achieved by well-informed interactions between scientists, regulators and end users, to improve the spread and speed of innovation with this sector.

Soil Methane Sink Capacity Response to a Long-Term Wildfire Chronosequence in Northern Sweden.

Boreal forests occupy nearly one fifth of the terrestrial land surface and are recognised as globally important regulators of carbon (C) cycling and greenhouse gas emissions. Carbon sequestration processes in these forests include assimilation of CO2 into biomass and subsequently into soil organic matter, and soil microbial oxidation of methane (CH4). In this study we explored how ecosystem retrogression, which drives vegetation change, regulates the important process of soil CH4 oxidation in boreal forests. We measured soil CH4 oxidation processes on a group of 30 forested islands in northern Sweden differing greatly in fire history, and collectively representing a retrogressive chronosequence, spanning 5000 years. Across these islands the build-up of soil organic matter was observed to increase with time since fire disturbance, with a significant correlation between greater humus depth and increased net soil CH4 oxidation rates. We suggest that this increase in net CH4 oxidation rates, in the absence of disturbance, results as deeper humus stores accumulate and provide niches for methanotrophs to thrive. By using this gradient we have discovered important regulatory controls on the stability of soil CH4 oxidation processes that could not have not been explored through shorter-term experiments. Our findings indicate that in the absence of human interventions such as fire suppression, and with increased wildfire frequency, the globally important boreal CH4 sink could be diminished.

13CO2 pulse labelling of plants in tandem with stable isotope probing: methodological considerations for examining microbial function in the rhizosphere.

Recently developed 13CO2 pulse labelling and stable isotope probing (SIP) methods offer the potential to track 13C-labelled plant photosynthate into phylogenetic groups of microbial taxa in the rhizosphere, permitting an examination of the link between soil microbial diversity and carbon flow in situ. We tested the feasibility of this approach to detect functional differences in microbial communities utilising recently fixed plant photosynthate in moisture perturbed grassland turfs. Specifically, we addressed two questions: (1) How does moisture perturbation (three treatments; continual wetting, drying, and drying followed by rewetting) affect the assimilation of 13C-labelled exudates carbon into the soil microbial community?; (2) Can 13C deposited in soil from pulse-labelled plants be used to identify microbes utilising plant exudates using SIP methodologies? Net CO2 fluxes showed that prior to 13CO2 pulse labelling, all treatments were photosynthetically active, but differences were observed in night time respiration, indicating moisture treatments had impacted on net CO2 efflux. Measurements of pulse-derived 13C incorporated into soil RNA over 2 months showed that there was only evidence of 13C enrichment in the continuously wetted treatments. However, isotopic values represented only a 0.1-0.2 13C at.% increase over natural abundance levels and were found to be insufficient for the application of RNA-SIP. These findings reveal that in this experimental system, the microbial uptake of labelled carbon from plant exudates is low, and further optimisation of methodologies may be required for application of SIP to natural plant-soil systems where 13C tracer dilution is a consideration.

Long term changes in the distribution and delta(15)N values of individual soil amino acids in the absence of plant and fertiliser inputs.

The long-term 'biodegradation' on soil amino acids was examined in the control plots of '42 parcelles' experiment, established in 1928 at INRA, Versailles (France). None of the plots is cultivated, but is kept free of weeds, and mixed to a depth of 25 cm twice yearly. Topsoil (0-10 cm depth) samples collected in 1929, 1963 and 1997 were subjected to acid hydrolysis (6 N HCl) for comparison. The distribution and delta(15)N natural abundance of 20 individual amino acids in the soils were determined, using ion chromatography (IC) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The total N and amino acid-N (AA-N), respectively, decreased by 54 % and 73 % in the period from 1929 to 1997. The average N loss was comparable for 1929-1963 (period 1) and 1963-1997 (period 2), but AA-N loss was three times faster in the former period. This significant reduction in total AA-N content was mirrored in the individual amino acids, which decreased by 74 % +/- 1 % (ranging 58-89 %) between 1929 and 1997. The bulk delta(15)N values generally increased from 1929 to 1997, mainly associated with comparable or even higher increase of delta(15)N of the non-AA-N in the soil. The residence time (t(1/2), time in which half of N was lost from a specific soil pool) was ca. 65 +/- 5 years for the bulk soil, and comparable for periods 1 and 2. However, between periods 1 and 2 it decreased from 128 to 41 years in the non-AA pool, but increased from 59 to 92 years in the AA-N pool. Proline and amino acids that appear early in soil microbial metabolic pathways (e.g. glutamic acid, alanine, aspartic acid and valine) had relatively high delta(15)N values. Phenylalanine, threonine, glycine and leucine had relatively depleted delta(15)N values. The average delta(15)N value of the individual amino acids (IAAs) increased by 1delta unit from 1929 to 1997, associated with a similar rise from 1929 to 1963, and no change thereafter till 1997. However, the delta(15)N values of phenylalanine decreased by more than 7delta(15)N units between 1929 and 1997. The delta(15)N shift of IAAs from 1929 to 1963 and from 1929 to 1997 was not influenced by the relative amount of N remaining compared with the 1929 soil concentrations. The only exception was phenylalanine which showed decreasing delta(15)N associated with its decreasing concentration in the soil. We conclude therefore that in the absence of plant and fertiliser inputs, no change in the delta(15)N value of individual soil amino acids occurs, hence the original delta(15)N values are preserved and diagnostic information on past soil N (cycling) is retained. The exception was phenylalanine, its delta(15)N decreased with decreasing concentration from 1929 to 1997, hence it acted as a 'potential' marker for the land use changes (i.e. arable cropping to a fallow). The long term biological processing and reworking of residual amino acids resulted in a (partial) stabilisation in the soil, evidenced by reduced N loss and increased residence time of amino acid N during the period 1963-1997.