B52A-01 INVITED 10:20h
Probing the Activities of Soil Invertebrates Using Stable Isotope Approaches
Soil dwelling invertebrates play a vital role in determining the physical properties and nutrient cycling in soil. Their diverse behaviours influence organic litter, water and gas transport. They impact on other soil biota, e.g. microbes, plants, other invertebrates, etc. via their various grazing and predatory activities, and their role in the comminution of litter influences the activities of other decomposer organisms. However, major challenges exist in the study of the activities of such invertebrates due to the small sizes of many of the key organisms and the opaque nature of soil. This paper will provide an overview of a number of new approaches that have been developed to investigate the behaviours of soil invertebrates. The techniques we employ are based on the use of stable isotopes, exploiting both natural abundance labelling and artificially isotopically enriched tracers. Experiments range from simple feeding and choice experiments in laboratory arenas to pot-based microcosm studies, and field experiments (Chamberlain et al., 2004; Black et al. in press). The philosophy underpinning this research is to exploit fundamental biochemical information to determine the activities of organisms. Thus, compound-specific stable isotope determinations are one of our major goals since these yield high specificity stable isotopic information, often at the biochemical building block level. Compound-specific approaches also have the virtue of enhancing analytical sensitivity, such that the $\delta$$^{13}$C values of the biochemical components of individual specimens of low microgram-sized organisms, i.e. mesoinvertebrates, can be recorded their behaviours investigated (Evans et al., 2003; Black et al. in press).
http://www.chm.bris.ac.uk/org/evershed/
B52A-02 10:50h
Molecular Investigation of the Short-term Sequestration of Natural Abundance $^{13}$C\$\-$labelled Cow Dung in the Surface Horizons of a Temperate Grassland Soil
An adequate understanding of the carbon (C) sequestration potential of grasslands requires that the quantity and residence times of C inputs be measured. Herbivore dung is largely comprised of plant cell wall material, a significant source of stable C in intensively grazed temperate grassland ecosystems that contributes to the soil carbon budget. Our work uses compound-specific isotope analysis to identify the pattern of input of dung-derived compounds from natural abundance $^{13}$C$/-$labelled cow dung into the surface horizons of a temperate grassland soil over one year. C4 dung ($\delta$$^{13}$C $\-$12.6 $\permil$) from maize fed cows was applied to a temperate grassland surface ($\delta$$^{13}$C $\-$29.95 $\permil$) at IGER-North Wyke (Devon, UK), and dung remains and soil cores beneath the treatments collected at {\t} = 7, 14, 28, 56, 112, 224 and 372 days. Bulk dung carbon present in the 0$\-$1 cm and 1$\-$5 cm surface horizons of a grassland soil over one year was estimated using $\Delta$$^{13}$C between C$_{4}$ dung and C$_{3}$ dung, after Bol {\et al.} (2000). The major biochemical components of dung were quantified using proximate forage fibre analyses, after Goering and Van Soest (1970) and identified using `wet' chemical and GC-MS methods. Plant cell wall polysaccharides and lignin were found to account for up to 67 {%} of dung dry matter. Hydrolysed polysaccharides were prepared as alditol acetates for analyses (after Docherty {\et al.}, 2001), and a novel application of an off-line pyrolysis method applied to measure lignin-derived phenolic compounds (after Poole & van Bergen, 2002). This paper focuses on major events in the incorporation of dung carbon, estimated using natural abundance $^{13}$C{\-}labelling technique. This revealed a major bulk input of dung carbon after a period of significant rainfall with a consequent decline in bulk soil $\delta$$^{13}$C values until the end of the experiment (Dungait {\et al.}, submitted). Findings will be presented revealing contribution of plant cell wall polysaccharides and lignin to these bulk $\delta$$^{13}$C values, and their potential for sequestration considered. References: Bol, R., Amelung, W., Friedrich, C. Ostle, N. (2000). Tracing dung-derived carbon in temperate grassland using $^{13}$C natural abundance measurements. Soil Biology and Biochemistry, 32, 1337-1343. Goering and Van Soest (1970). Forage fibre analysis (apparatus, reagents, procedures and some applications). In: USDA-ARS Agricultural Handbook, 379. U. S. Government Printing Office, Washington D.C. Docherty, G., Jones, V. and Evershed, R.P. (2001). Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry $\delta$$^{13}$C analysis of small polyfunctional compounds. Rapid Communications in Mass Spectrometry, 15, 730-738. Poole, I. & van Bergen, P. F. (2002). Carbon isotope ratio analysis of organic moieties from fossil mummified wood: establishing optimum conditions for off-line pyrolysis extraction using gas chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry, 16, 1976-1981. Dungait, J. A. J., Bol, R. and Evershed, R.P. (submitted). The Fate of Dung Carbon in Temperate Grassland Soil: 1. Preliminary Findings Based on Bulk Stable Carbon Isotope Determinations. Isotopes in Health and Environmental Studies
http://www.chm.bris.ac.uk/org/evershed/
B52A-03 INVITED 11:05h
The Stable Soil Organic Carbon Pool: Small Molecules In Small Pores?
For a long-term sequestration of carbon in soil the stable soil organic carbon pool is most important. Sorption to mineral phases is considered as a key process in the formation of this pool. This paper aims at presenting evidence on the location where stable carbon is found in soil, the type of bonding by which organic matter is stabilized, and the possible source of the stabilized organic matter. To approach this a combination of 14C AMS measurements, microspectroscopic techniques, BET surface measurements, stable isotope analysis, and molecular techniques was employed. Analyses were carried out on the mineral-associated heavy fraction of the soil, and soil and mineral samples used for dissolved organic matter sorption/desorption experiments and treatment with sodium hypochloride. The results suggest that parts of the mineral-associated organic matter is located on plain mineral surfaces while others can be found associated with meso- and micropores of minerals, in particular of high-porosity Fe- and Al-oxi/hydroxides. In these pore mouths, the organic matter is stabilized by strong chemisorptive bondings (ligand exchange) which may act three dimensionally. The mean turnover time of this type of organic carbon is in the range of several thousand years. There is evidence that (dissolved) organic matter sorbed into these pore mouths soon after formation of these pedogenic minerals will be barely accessible for microbial decomposition during the life time of the minerals themselves. This intimate association of organic matter with minerals may be responsible for the slow turnover times. But likewise this complicates extremely the characterization of the type of organic matter sorbed. So results obtained from the structural characterization of the dissolv ed organic matter being sorbed using IR, NMR, AFM, and degradative techniques are often not in concordance with the structure of the organic matter sorbed into the pore mouths of the minerals. However, results gave rise to the concept that small, flexible and highly charged molecules are primarily involved in this type of stabilization.
B52A-04 11:35h
Distribution of Organic Matter in Nano- and Micropores of Soil Microaggregates
The processes underlying the sequestration of organic matter (OM) in soil microaggregates are being studied using ultra-small small angle x-ray scattering (USAXS) and nitrogen adsorption to evaluate the pore size distribution of the total- and OM-filled porosity within microaggregates. Soil microaggregates (50 to 250 um) are particularly crucial to long-term sequestration because they protect C against decomposition, resulting in much longer residence times. Systematic changes in the pore-size distribution of OM were evaluated at two long-term field manipulations: a chronosequence of tallgrass prairie restoration (Fermilab, Batavia, IL, USA; Mollisol) and a 30-year comparison of till/no-till cultivation at two levels of N inputs (University of Kentucky, USA; Alfasol). The soil OM levels increased over time after restoration of cultivated soils to a prairie, and with decreased tillage and increased fertilizer inputs. The distribution of pores in microaggregates was measured in microaggregates before and after the OM was removed by combustion at 350oC. The total porosity was determined using data from the combusted, OM-free microaggregates. The distribution of the OM within the pores was determined by USAXS from differences in the porosity estimates for the combusted and intact samples. USAXS advantages include a very wide range of length scales measured (1 nm to 5 um) and not requiring connectivity of the pores to a probe molecule. The USAXS data reflect differences in the composition and density of pores, OM and minerals. There is a strong change in x-ray contrast when an entirely OM-filled void is cleared by combustion, compared to the case when a coating of OM in an air-filled pore is removed. This latter property was exploited to evaluate the protection of OM residing in pores with reduced access of microbes or microbial exoenzymes. The conversion of a cultivated soil to a prairie results in restructuring of the OM distribution, with new OM migrating to increasingly fill pores. Additionally, although the volume fraction of the total porosity increases with the pore diameter, there is a sharp decline in the abundance of OM-filled pores at length scales greater than 1 um. This pattern suggests that OM accumulation and preservation occurs via limiting physical access of microorganisms to OM within filled pores. Similar, but less strongly developed patterns were seen for the Kentucky site, but it is likely that strong adsorption to the Fe-oxide rich Alfasols could also contribute to OM accumulation at that site.
B52A-05 11:50h
Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions
Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.
B52A-06 12:05h
Presence and Expression of Microbial Genes Regulating Soil Nitrogen Dynamics Along the Tanana River Successional Sequence
We report on work to assess the functional gene sequences for soil microbiota that control nitrogen cycle pathways along the successional sequence (willow, alder, poplar, white spruce, black spruce) on the Tanana River floodplain, Interior Alaska. Microbial DNA and mRNA were extracted from soils (0-10 cm depth) for amoA (ammonium monooxygenase), nifH (nitrogenase reductase), napA (nitrate reductase), and nirS and nirK (nitrite reductase) genes. Gene presence was determined by amplification of a conserved sequence of each gene employing sequence specific oligonucleotide primers and Polymerase Chain Reaction (PCR). Expression of the genes was measured via nested reverse transcriptase PCR amplification of the extracted mRNA. Amplified PCR products were visualized on agarose electrophoresis gels. All five successional stages show evidence for the presence and expression of microbial genes that regulate N fixation (free-living), nitrification, and nitrate reduction. We detected (1) nifH, napA, and nirK presence and amoA expression (mRNA production) for all five successional stages and (2) nirS and amoA presence and nifH, nirK, and napA expression for early successional stages (willow, alder, poplar). The results highlight that the existing body of previous process-level work has not sufficiently considered the microbial potential for a nitrate economy and free-living N fixation along the complete floodplain successional sequence.