Andrew J. Midwood

Through the eye of the needle: a review of isotope approaches to quantify microbial processes mediating soil carbon balance

For soils in carbon balance, losses of soil carbon from biological activity are balanced by organic inputs from vegetation. Perturbations, such as climate or land use change, have the potential to disrupt this balance and alter soil-atmosphere carbon exchanges. As the quantification of soil organic matter stocks is an insensitive means of detecting changes, certainly over short timescales, there is a need to apply methods that facilitate a quantitative understanding of the biological processes underlying soil carbon balance. We outline the processes by which plant carbon enters the soil and critically evaluate isotopic methods to quantify them. Then, we consider the balancing CO(2) flux from soil and detail the importance of partitioning the sources of this flux into those from recent plant assimilate and those from native soil organic matter. Finally, we consider the interactions between the inputs of carbon to soil and the losses from soil mediated by biological activity. We emphasize the key functional role of the microbiota in the concurrent processing of carbon from recent plant inputs and native soil organic matter. We conclude that quantitative isotope labelling and partitioning methods, coupled to those for the quantification of microbial community substrate use, offer the potential to resolve the functioning of the microbial control point of soil carbon balance in unprecedented detail.

Phytoavailability of Cd and Zn in soil estimated by stable isotope exchange and chemical extraction

The distribution of labile Cd and Zn in two contrasting soils was investigated using isotopic exchange techniques and chemical extraction procedures. A sewage sludge amended soil from Great Billings (Northampton, UK) and an unamended soil of the Countesswells Association obtained locally (Aberdeen, UK) were used. 114Cd and 67Zn isotopes were added to a water suspension of each soil and the labile metal pool (E-value) determined from the isotope dilution. Samples were obtained at 13 time points from 1h to 50 days. For the sewage sludge amended soil, 29 μg Cd g−1 (86% of total) and 806 μg Zn g−1 (65% of total) were labile and for the Countesswells soil the value was 8.6 μg Zn g−1 (13% of total); limits of detection prevented a Cd E-value from being measured in this soil. The size of the labile metal pool was also measured by growing plants for 90 days and determining the isotopic content of the plant tissue (L-value). Thlaspi caerulescensJ. & C. Presl (alpine penny cress), a hyperaccumulator of Zn and Cd, Taraxacum officinale Weber (dandelion) and Hordeum vulgare L. (spring barley) were used. L-values were similar across species and lower than the E-values. On average the L-values were 23±0.8 μg Cd g−1 and 725±14 μg Zn g−1 for the Great Billings soil and 0.29±0.16 μg Cd g−1 and 7.3±0.3 μg Zn g−1 for the Countesswells soil. The extractable metal content of the soils was also quantified by extraction using 0.1 M NaNO3, 0.01 M CaCl2, 0.5 M NaOH, 0.43 M CH3COOH and 0.05 M EDTA at pH 7.0. Between 1.3 and 68% of the total Cd and between 1 and 50% of the total Zn in the Great Billings soil was extracted by these chemicals. For the Countesswells soil, between 6 and 83% of the total Cd and between 0.1 and 7% of the total Zn was extracted. 0.05 M EDTA and 0.43 M CH3COOH yielded the greatest concentrations for both soils but these were less than the isotopic estimates. On the whole, E-values were numerically closer to the L-values than the chemical extraction values. The use of isotopic exchange provides an alternative estimate of the labile metal pool within soils compared to existing chemical extraction procedures. No evidence was obtained that T. caerulescens is able to access metal within the soil not freely available to the other plants species. This has implications for long term remediation strategies using hyperaccumulating plant species, which are unlikely to have any impact on non-labile Cd and Zn in contaminated soil.

Challenges in measuring the δ13C of the soil surface CO2 efflux

The δ13C of the soil surface efflux of carbon dioxide (δ13CRS) has emerged as a powerful tool enabling investigation of a wide range of soil processes from characterising entire ecosystem respiration to detailed compound-specific isotope studies. δ13CRS can be used to trace assimilated carbon transfer below ground and to partition the overall surface efflux into heterotrophic and autotrophic components. Despite this wide range of applications no consensus currently exists on the most appropriate method of sampling this surface efflux of CO2 in order to measure δ13CRS. Here we consider and compare the methods which have been used, and examine the pitfalls. We also consider a number of analysis options, isotope ratio mass spectrometry (IRMS), tuneable diode laser spectroscopy (TDLS) and cavity ring-down laser spectroscopy (CRDS). δ13CRS is typically measured using chamber systems, which fall into three types: closed, open and dynamic. All are imperfect. Closed chambers often rely on Keeling plots to estimate δ13CRS, which may not be appropriate without free turbulent air mixing. Open chambers have the advantage of being able to maintain steady-state conditions but analytical errors may become limiting with low efflux rates. Dynamic chambers like open chambers are complex, and controlling pressure fluctuations caused by air movement is a key concern. Both open and dynamic chambers in conjunction with field portable TDLS and CRDS analysis systems have opened up the possibility of measuring δ13CRS in real time permitting new research opportunities and are on balance the most suited to this type of measurement.

Measuring the 13C content of soil‐respired CO2 using a novel open chamber system

Carbon dioxide respired by soils comes from both autotrophic and heterotrophic respiration. 13C has proved useful in differentiating between these two sources, but requires the collection and analysis of CO2 efflux from the soil. We have developed a novel, open chamber system which allows for the accurate and precise quantification of the delta13C of soil-respired CO2. The chamber was tested using online analyses, by configuring a GasBench II and continuous flow isotope ratio mass spectrometer, to measure the delta13C of the chamber air every 120 s. CO2 of known delta13C value was passed through a column of sand and, using the chamber, the CO2 concentration stabilized rapidly, but 60 min was required before the delta13C value was stable and identical to the cylinder gas (-33.3 per thousand). Changing the chamber CO2 concentration between 200 and 900 micromol.mol(-1) did not affect the measured delta13C of the efflux. Measuring the delta13C of the CO2 efflux from soil cores in the laboratory gave a spread of +/-2 per thousand, attributed to heterogeneity in the soil organic matter and roots. Lateral air movement through dry sand led to a change in the delta13C of the surface efflux of up to 8 per thousand. The chamber was used to measure small transient changes (+/-2 per thousand) in the delta13C of soil-respired CO2 from a peaty podzol after gradual heating from 12 to 35 degrees C over 12 h. Finally, soil-respired CO2 was partitioned in a labelling study and the contribution of autotrophic and heterotrophic respiration to the total efflux determined. Potential applications for the chamber in the study of soil respiration are discussed.

Differences in soil water use by annual broomweed and grasses

The use of water in the upper 1 m of the soil profile by 3 common herbaceous species of the southern Great Plains was examined by labeling soil water with 2H2O and H2(18)O. Uptake of labeled water from the 15 cm depth was approximately equal for all species. However, water uptake from the 75 cm depth was significantly greater by annual broomweed [Amphiachyris dracunculoides (DC.) Nutt] than either sideoats grama [Bouteloua curtipendula (Michx.) Torr] or curlymesquite [Hilaria belangeri (Steud.) Nash]. Although both grasses had greater root length density than annual broomweed at the 75 cm depth, annual broomweeds rate of water extraction from the 75 cm depth was nearly twice that of sideoats grama or curlymesquite. Greater access to and more rapid utilization of deeper soil water by annual broomweed relative to the grass species may partially explain annual broomweeds success at invading grasslands and reducing grass production in semi-arid rangelands.

Amino Acid Composition of Grape (Vitis vinifera L.) Juice in Response to Applications of Urea to the Soil or Foliage

Applications of nitrogen to vineyard foliage or soil at veraison can improve grape juice yeast assimilable nitrogen concentrations and may prevent the excessive vine growth, delayed maturity, and adverse changes in fruit properties sometimes associated with high applications of N earlier in the growing season. However, the consequences of late-season foliar- and soil-applied nitrogen for grape juice yeast assimilable nitrogen (YAN) and, specifically, grape juice amino acid profiles have rarely been directly compared. Over two years in drip-irrigated Merlot and Pinot gris vineyards, grape juice amino acid concentrations were measured from vines to which urea had been applied three times around veraison at 3.8 g N/vine to either the foliage or the soil surface. Foliar-applied urea (applied as a 2% w/v solution) was usually more effective at boosting grape juice ammonium and amino acid concentrations, although soil-applied urea improved some grape juice amino acids at the Pinot gris site. Changes in the amino acid profiles of grape juice, observed in response to foliar N applications but not soil N applications, may have implications for wine quality. Applications of 15N-labeled urea at the Pinot gris site demonstrated that a greater percentage of fertilizer N was incorporated into grape juice amino acids when urea was applied to the foliage than when it was applied to the soil surface. Late-season foliar applications of urea are a reliable, efficient, and effective method of improving grape juice YAN. Further work is required to examine how treatment effects vary among sites and cultivars under different management practices and to understand the implications of altered grape juice amino acid profiles for wine quality.

Minimising methodological biases to improve the accuracy of partitioning soil respiration using natural abundance 13C.

RATIONALE Microbial degradation of soil organic matter (heterotrophic respiration) is a key determinant of net ecosystem exchange of carbon, but it is difficult to measure because the CO2 efflux from the soil surface is derived not only from heterotrophic respiration, but also from plant root and rhizosphere respiration (autotrophic). Partitioning total CO2 efflux can be achieved using the different natural abundance stable isotope ratios (δ(13)C) of root and soil CO2. Successful partitioning requires very accurate measurements of total soil efflux δ(13)CO2 and the δ(13)CO2 of the autotrophic and heterotrophic sources, which typically differ by just 2-8‰. METHODS In Scottish moorland and grass mesocosm studies we systematically tested some of the most commonly used techniques in order to identify and minimise methodological errors. Typical partitioning methods are to sample the total soil-surface CO2 efflux using a chamber, then to sample CO2 from incubated soil-free roots and root-free soil. We investigated the effect of collar depth on chamber measurements of surface efflux δ(13)CO2 and the effect of incubation time on estimates of end-member δ(13)CO2. RESULTS (1) a 5 cm increase in collar depth affects the measurement of surface efflux δ(13)CO2 by -1.5‰ and there are fundamental inconsistencies between modelled and measured biases; (2) the heterotrophic δ(13)CO2 changes by up to -4‰ within minutes of sampling; we recommend using regression to estimate the in situ δ(13)CO2 values; (3) autotrophic δ(13)CO2 measurements are reliable if root CO2 is sampled within an hour of excavation; (4) correction factors should be used to account for instrument drift of up to 3‰ and concentration-dependent non-linearity of CRDS (cavity ringdown spectroscopy) analysis. CONCLUSIONS Methodological biases can lead to large inaccuracies in partitioning estimates. The utility of stable isotope partitioning of soil CO2 efflux will be enhanced by consensus on the optimum measurement protocols and by minimising disturbance, particularly during chamber measurements.

Two-year dynamics of foliage labelling in 8-year-old Pinus pinaster trees with 15N, 26Mg and 42Ca—simulation of Ca transport in xylem using an upscaling approach

Abstract• IntroductionAtmospheric deposition is an important input of major nutrients into forest ecosystems. The long-term goal of this work was to apply stable isotope methodology to assess atmospheric nutrient deposition in forest systems.• Materials and methodsA labelling experiment of foliage with stable isotopes of primary and secondary macro nutrients (15N, 26Mg and 42Ca injected into the stem sapwood) was carried on standing trees to monitor interactions between canopy and precipitations. 15N rapidly reached the foliage; however, Mg and Ca were not detected in foliage until more than a year after injection.• Results and discussionThe delay in mobilization of Mg and Ca prevented us from accurately modelling deposition contributions of these two elements. Nonetheless, an upscaling approach based on published results on Ca transport in shoots xylem was used to simulate our results. These simulations of Ca transport at the tree scale were consistent with our experimental data.• ConclusionThis consistency suggested that mechanisms of nutrient transport are the same at the different scales. Nitrogen was rapidly transported in the xylem to foliage, probably mainly by mass flow. Conversely, transport of Mg and particularly Ca was considerably delayed, probably due to successive cation exchanges along the xylem vessels.

Late-Season Foliar Urea Applications Can Increase Berry Yeast-Assimilable Nitrogen in Winegrapes (Vitis vinifera L.)

In the Okanagan Valley of British Columbia, low rates of nitrogen fertilizer are typically applied early in the growing season to prevent excessive vine growth, disease, and adverse changes in grape juice composition. As a consequence, grape juice yeast assimilable nitrogen (YAN) concentrations at harvest are often below the level considered sufficient to complete fermentation during winemaking and require augmentation with additional nitrogen. Over a three-year period at seven study sites planted to five winegrape varieties, late-season foliar applications of urea-N were investigated as a method for enhancing grape juice YAN. Foliar-applied solutions of urea at rates equivalent to 14–18 kg N/ha/yr (1% w/v, applied in one year only) or 28–36 kg N/ha/yr (2% w/v) caused significant improvements in grape juice YAN concentrations in six out of seven of the study sites each year, but there was no consistent pattern among years as to which study sites were most amenable to treatment. Little of the N applied in the foliar spray treatments appeared to be retained by the vines, and there were only few, small negative effects of treatment application on vine performance and juice quality. Thus, urea sprays applied to the foliage around the time of veraison show considerable promise as a supplement to more traditional soil fertilization programs on these and similar sites.

Sampling root-respired CO2 in-situ for 13C measurement

Background and aimsRoot-respired δ13CO2 can be useful for exploring plant carbon allocation and root respiratory fractionation as well as for partitioning soil-surface CO2 emissions into plant root and soil organic matter (SOM) sources, a necessary measure for calculating the contribution of heterotrophic respiration of soil carbon to net ecosystem exchange. Root CO2 is usually sampled from excised roots, however, excision alters respiration rate and isolates the root sample from aboveground plant processes.MethodsTo improve the integrity of root efflux δ13CO2 measurements, we designed a chamber for sampling root-respired CO2 in situ from minimally disturbed tree roots. We compared root δ13CO2 values from excised and attached roots in the field and we pruned mature Scots pine trees to induce a measureable change in root δ13CO2.ResultsExcised root samples containing root wounds gave more 13C-depleted measurements of root-respired δ13CO2 than intact roots by 1.8 ‰. Using chambers to sample CO2 from attached roots, we measured a diurnal change in root-respired δ13CO2 of 3–4 ‰, triggered by pruning foliage from the trees.ConclusionsThis chamber system permits high-frequency sampling of live root-respired δ13CO2 that enables greater insight into plant respiratory processes and more accurate partitioning of soil-surface CO2 emissions.