Isotopic detection of recent photosynthate carbon flow into grassland rhizosphere fauna

In this study, we measured the incorporation of recent photosynthate-C inputs into active rhizosphere fauna (earthworms, enchytraeids, mites and collembolans) in an upland grassland soil under natural environmental conditions. This was achieved by means of a ¹³CO₂ pulse-chase experiment made during the growing season, followed by a 20-day dynamic sampling of soil fauna for ¹²C/¹³C analysis by IRMS. The effect of post-¹³C labelling defoliation (cutting) on fauna ¹²C/¹³C ratios was also examined.Results showed that earthworms made up over 93% of the extracted fauna biomass, while mites, collembolans and enchytraeids together accounted for less than 7%. All fauna groups showed evidence of tracer ¹³C in their tissues within a week of ¹³CO₂ pulse labelling in both control and cut treatments. Cutting significantly increased the amount of tracer ¹³C entering the organisms (P=0.0002). Similarly, the fauna group also had a significant effect (P=0.0001). Time did not have any effect on fauna ¹³C content between groups as differences were only significant at the last sampling occasion. The interaction time×animal group, however, had a significant effect (P=0.0054).Collembolans accounted for most of the tracer ¹³C measured within the fauna biomass, i.e. mean±standard deviation of 44.78±12.75% and 44.29±14.69% of fauna ¹³C in control and cut treatments, respectively. Mites and earthworms contained between 22.13% and 28.45%, and enchytraeids less than 6% of the tracer ¹³C. We conclude that, during the growing season, there was a rapid incorporation of recent photosynthate-C into rhizosphere mesofauna. This carbon transfer was most significantly increased by defoliation in mites and collembolans (P<0.01). These results provide evidence that soil foodweb carbon dynamics are not solely underpinned by detrital decomposition but are also affected by short-term plant rhizodeposition patterns.     

Soil carbon release along a gradient of physical disturbance in a harvested northern hardwood forest

Changes in soil respiration associated with forest harvest could increase net loss of CO₂ to the atmosphere relative to pre-harvest values. By excavating quantitative soil pits across a gradient of physical disturbance in a harvested northern hardwood forest, this study examines C release from mineral soil. Mineral soil samples were analyzed for pH, percent organic matter (%OM), C and N concentration, δ¹³C, and total C per unit area. Results show a relationship between degree of disturbance and C concentration in soil 10-30 cm beneath the O-horizon. Highly disturbed sites show C depletion, with horizons from disturbed sites containing 25% less total C than the least disturbed sites. δ¹³C signatures of soil profiles at these sites show vertical mixing of plant-derived material into deeper mineral horizons. Mixing, as a result of physical disturbance, could have led to the observed C depletion by physical or chemical destabilization, or through the promotion of microbial respiration in deep mineral soil. Regardless of the mechanism, these results suggest elevated CO₂ emissions from soil following harvest, and, thus, have implications for the validity of wood biomass as a carbon neutral energy source.     

Carbon fluxes in soil food webs of increasing complexity revealed by C labelling and C natural abundance

Soil food webs are mainly based on three primary carbon (C) sources: root exudates, litter, and recalcitrant soil organic matter (SOM). These C sources vary in their availability and accessibility to soil organisms, which could lead to different pathways in soil food webs. The presence of three C isotopes (¹²C, ¹³C and ¹⁴C) offers an unique opportunity to investigate all three C sources simultaneously. In a microcosm experiment we studied the effect of food web complexity on the utilization of the three carbon sources. We choose an incomplete three factorial design with (i) living plants, (ii) litter and (iii) food web complexity. The most complex food web consisted of autochthonous microorganisms, nematodes, collembola, predatory mites, endogeic and anecic earthworms. We traced C from all three sources in soil, in CO₂ efflux and in individual organism groups by using maize grown on soil developed under C₃ vegetation and application of ¹⁴C labelled ryegrass shoots as a litter layer. The presence of living plants had a much greater effect on C pathways than food web complexity. Litter decomposition, measured as ¹⁴CO₂ efflux, was decreased in the presence of living plants from 71% to 33%. However, living plants increased the incorporation of litter C into microbial biomass and arrested carbon in the litter layer and in the upper soil layer. The only significant effect of food web complexity was on the litter C distribution in the soil layers. In treatments with fungivorous microarthropods (Collembola) the incorporation of litter carbon into mineral soil was reduced. Root exudates as C source were passed through rhizosphere microorganisms to the predator level (at least to the third trophic level). We conclude that living plants strongly affected C flows, directly by being a source of additional C, and indirectly by modifying the existing C flows within the food web including CO₂ efflux from the soil and litter decomposition.     

Near‐infrared spectroscopy can predict the composition of organic matter in soil and litter

The usefulness and limitations of near‐infrared reflectance spectroscopy (NIRS) for the assessment of several soil characteristics are still not sufficiently explored. The objective of this study was to evaluate the ability of visible and near‐infrared reflectance (VIS‐NIR) spectroscopy to predict the composition of organic matter in soils and litter. Reflectance spectra of the VIS‐NIR region (400–2500 nm) were recorded for 56 soil and litter samples from agricultural and forest sites. Spectra were used to predict general and biological characteristics of the samples as well as the C composition which was measured by ¹³C‐CPMAS‐NMR spectroscopy. A modified partial least‐square method and cross‐validation were used to develop equations for the different constituents over the whole spectrum (1st to 3rd derivation). Near‐infrared spectroscopy predicted well the C : N ratios, the percentages of O‐alkyl C and alkyl C, the ratio of alkyl C to O‐alkyl C, and the sum of phenolic oxidation products: the ratios of standard deviation of the laboratory results to standard error of cross‐validation (RSC) were greater than 2, the regression coefficients (a) of a linear regression (measured against predicted values) ranged from 0.9 to 1.1, and the correlation coefficients (r) were greater than 0.9. Satisfactorily (0.8 ≤ a ≤ 1.2, r ≥ 0.8, and 1.4 ≤ RSC ≤ 2.0) assessed were the contents of C, N, and production of DOC, the percentages of carbonyl C and aromatic C and the ratio of alkyl C to aromatic C. However, the N‐mineralization rate and the microbial biomass were predicted unsatisfactorily (RSC < 1.4). The good and satisfactory predictions reported above indicate a marked usefulness of NIRS in the assessment of biological and chemical characteristics of soils and litter.     

Kinetic Parameters of Gross N Mineralization of Peat Soils as Related to the Composition of Soil Organic Matter

Peat land has been considered as an alternative type of land for agricultural development especially in the tropics. In the present study, the N-supplying capacity, one of the most important soil properties in terms of crop production, of peat soils was examined. Ten peat soil samples were collected from Indonesia, Malaysia, and Japan. Gross N mineralization in the soil samples was estimated using a zero-order model, and kinetic parameters of mineralization were determined using a simple type model. Soil organic matter composition was investigated using ¹³C CPMAS NMR. Mineralization potential ( N ₀), apparent activation energy ( E _a), and mineralization rate constant ( k ) ranged between 571–2,445 mg kg⁻¹, 281–8,181 J mol⁻¹, and 0.009–0.020 d⁻¹, respectively. Although none of the parameters showed a significant correlation with the soil C/N ratio, a negative correlation was observed between the k value and the ratio of the proportion of alkyl C in total C to that of O -alkyl C estimated by ¹³C CPMAS NMR. The latter suggested that the k values were higher in the peat soils relatively rich in readily decomposable organic matter including carbohydrates.     

Quantitative aspects of solid-state C-NMR spectra of humic substances from soils of volcanic systems

Cross-Polarisation Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance spectroscopy (CPMAS ¹³C-NMR) represents one of the most powerful tools to investigate soil organic matter (SOM) mainly because of its inherent capacity to provide a semi-quantitative evaluation of carbon distribution. A critical parameter during acquisition of CPMAS ¹³C-NMR spectra is the contact time required to obtain the cross-polarisation between proton and carbon nuclei. The procedure to evaluate the best contact time for the acquisition of a quantitative CPMAS ¹³C-NMR spectrum is to perform Variable Contact Time (VCT) experiments. In this work the structural features of a number of purified humic substances from Italian and Costarican volcanic soils were investigated by CPMAS ¹³C-NMR spectroscopy after having performed preliminary VCT experiments. The VCT experiments showed that the average contact times vary according to the origin and chemical structure of the humic material. The optimal contact times (OCT) for nine humic samples were between 250 and 800 μs These values were different from the time of 1000 μs that is commonly applied as the best average contact time for humic materials. Moreover, by comparing the NMR data to those obtained by elemental analysis (C/H ratio), it appeared that the efficiency of the cross-polarisation between protons and carbons, and hence the contact time, is affected not only by the number of protons, but also by their distribution over the molecules. The evaluation of errors in quantitative estimation of the different carbons revealed that the use of OCT generally reduced by half the loss of signals occurring when the average contact time of 1000 μs is used in CPMAS ¹³C-NMR spectra of humic substances.     

C and N natural abundance of the soil microbial biomass

Stable isotope analysis is a powerful tool in the study of soil organic matter formation. It is often observed that more decomposed soil organic matter is ¹³C, and especially ¹⁵N-enriched relative to fresh litter and recent organic matter. We investigated whether this shift in isotope composition relates to the isotope composition of the microbial biomass, an important source for soil organic matter. We developed a new approach to determine the natural abundance C and N isotope composition of the microbial biomass across a broad range of soil types, vegetation, and climates. We found consistently that the soil microbial biomass was ¹⁵N-enriched relative to the total (3.2 ‰) and extractable N pools (3.7 ‰), and ¹³C-enriched relative to the extractable C pool (2.5 ‰). The microbial biomass was also ¹³C-enriched relative to total C for soils that exhibited a C3-plant signature (1.6 ‰), but ¹³C-depleted for soils with a C4 signature (−1.1 ‰). The latter was probably associated with an increase of annual C3 forbs in C4 grasslands after an extreme drought. These findings are in agreement with the proposed contribution of microbial products to the stabilized soil organic matter and may help explain the shift in isotope composition during soil organic matter formation.     

A GC/MS method for the assessment of N and C incorporation into soil amino acid enantiomers

Identifying the transformation process of amino acid enantiomers was essential to probe into the fate, turnover and aging of soil nitrogen due to their important roles in the biogeochemical cycling. If this can be achieved by differentiating between the newly biosynthesized and the inherent compounds in soil, then the isotope tracer method can be considered most valid. We thereby developed a gas chromatography/mass spectrometry (GC/MS) method to trace the ¹⁵N or ¹³C isotope incorporation into soil amino acid enantiomers after being incubated with ¹⁵NH₄⁺ or U-¹³C-glucose substrates. The most significant fragments (F) as well as the related minor ions were monitored by the full scan mode and the isotope enrichment in amino acids was estimated by calculating the atom percentage excess (APE). ¹⁵NH₄⁺ incorporation was evaluated according to the relative abundance increase of m/z F+1 to F for neutral and acidic amino acids and F+2 to F (mass 439) for lysine. The assessment of ¹³C enrichment in soil amino acids was more complicated than that of ¹⁵N due to multi-carbon atoms in amino acid molecules. The abundance ratio increment of m/z F+n to F (n is the original skeleton carbon number in each fragment) indicated the direct conversion from the added glucose to amino acids, but the total isotope incorporation from the added ¹³C can only be calculated according to all target isotope fragments, i.e. the abundance ratio increment summation from m/z (Fa+1) through m/z (Fa+T) represented the total incorporation of the added ¹³C (Fa is the fragment containing all original skeleton carbons and T is the carbon number in the amino acid molecule). This method has a great advantage especially for the evaluation of high-abundance isotope enrichment in organic compounds compared with GC/C/IRMS. And in principle, this technique is also valid for amino acids besides enantiomers if stereoisomers are not concerned. Our assessment approach could shine a light on investigating the biochemical mechanism of microbial transformation of N and C in soils of terrestrial ecosystem.     

Organic C and N stabilization in a forest soil: Evidence from sequential density fractionation

In mineral soil, organic matter (OM) accumulates mainly on and around surfaces of silt- and clay-size particles. When fractionated according to particle density, C and N concentration (per g fraction) and C/N of these soil organo-mineral particles decrease with increasing particle density across soils of widely divergent texture, mineralogy, location, and management. The variation in particle density is explained potentially by two factors: (1) a decrease in the mass ratio of organic to mineral phase of these particles, and (2) variations in density of the mineral phase. The first explanation implies that the thickness of the organic accumulations decreases with increasing particle density. The decrease in C/N can be explained at least partially by especially stable sorption of nitrogenous N-containing compounds (amine, amide, and pyrrole) directly to mineral surfaces, a phenomenon well documented both empirically and theoretically. These peptidic compounds, along with ligand-exchanged carboxylic compounds, could then form a stable inner organic layer onto which other organics could sorb more readily than onto the unconditioned mineral surfaces (“onion” layering model).To explore mechanisms underlying this trend in C concentration and C/N with particle density, we sequentially density fractionated an Oregon andic soil at 1.65, 1.85, 2.00, 2.28, and 2.55 g cm⁻³ and analyzed the six fractions for measures of organic matter and mineral phase properties.All measures of OM composition showed either: (1) a monotonic change with density, or (2) a monotonic change across the lightest fractions, then little change over the heaviest fractions. Total C, N, and lignin phenol concentration all decreased monotonically with increasing density, and ¹⁴C mean residence time (MRT) increased with particle density from ca. 150 years to >980 years in the four organo-mineral fractions. In contrast, C/N, ¹³C and ¹⁵N concentration all showed the second pattern. All these data are consistent with a general pattern of an increase in extent of microbial processing with increasing organo-mineral particle density, and also with an “onion” layering model.X-ray diffraction before and after separation of magnetic materials showed that the sequential density fractionation (SDF) isolated pools of differing mineralogy, with layer-silicate clays dominating in two of the intermediate fractions and primary minerals in the heaviest two fractions. There was no indication that these differences in mineralogy controlled the differences in density of the organo-mineral particles in this soil. Thus, our data are consistent with the hypothesis that variation in particle density reflects variation in thickness of the organic accumulations and with an “onion” layering model for organic matter accumulation on mineral surfaces. However, the mineralogy differences among fractions made it difficult to test either the layer-thickness or “onion” layering models with this soil. Although SDF isolated pools of distinct mineralogy and organic-matter composition, more work will be needed to understand mechanisms relating the two factors.     

Carbon dynamics determined by natural C abundance in microcosm experiments with soils from long-term maize and rye monocultures

Understanding carbon dynamics in soil is the key to managing soil organic matter. Our objective was to quantify the carbon dynamics in microcosm experiments with soils from long-term rye and maize monocultures using natural ¹³C abundance. Microcosms with undisturbed soil columns from the surface soil (0-25 cm) and subsoil (25-50 cm) of plots cultivated with rye (C₃-plant) since 1878 and maize (C₄-plant) since 1961 with and without NPK fertilization from the long-term experiment ‘Ewiger Roggen’ in Halle, Germany, were incubated for 230 days at 8 °C and irrigated with 2 mm 10⁻² M CaCl₂ per day. Younger, C₄-derived and older, C₃-derived percentages of soil organic carbon (SOC), dissolved organic carbon (DOC), microbial biomass (C_mic) and CO₂ from heterothropic respiration were determined by natural ¹³C abundance. The percentage of maize-derived carbon was highest in CO₂ (42-79%), followed by C_mic (23-46%), DOC (5-30%) and SOC (5-14%) in the surface soils and subsoils of the maize plots. The percentage of maize-derived C was higher for the NPK plot than for the unfertilized plot and higher for the surface soils than for the subsoils. Specific production rates of DOC, CO₂-C and C_mic from the maize-derived SOC were 0.06-0.08% for DOC, 1.6-2.6% for CO₂-C and 1.9-2.7% for C_mic, respectively, and specific production rates from rye-derived SOC of the continuous maize plot were 0.03-0.05% for DOC, 0.1-0.2% for CO₂-C and 0.3-0.5% for C_mic. NPK fertilization did not affect the specific production rates. Strong correlations were found between C₄-derived C_mic and C₄-derived SOC, DOC and CO₂-C (r≥0.90), whereas the relationship between C₃-derived C_mic and C₃-derived SOC, DOC and CO₂-C was not as pronounced (r≤0.67). The results stress the different importance of former (older than 40 years) and recent (younger than 40 years) litter C inputs for the formation of different C pools in the soil.