Plant capture of free amino acids is maximized under high soil amino acid concentrations

Free amino acids (AA's) represent a significant source of available N for some plants and soil microorganisms. It can be expected, however, that significant competition will exist between plants and microorganisms for this organic N resource. Our study indicated that microbial capture and utilization of glycine was very rapid at a range of soil solution concentrations (0.1 μM to 10 mM) indicating that significant competition will exist between roots and soil microorganisms. Plant capture of free AA's was maximal at high soil solution concentrations where microbial utilization was slowest. Our results suggest that plant capture of soil dissolved organic N may primarily occur in organic rich patches in soil where concentrations of free AA's are high.     

Identification of amino acids in humic acid

Davidson, Sowden and Atkinson^l) in 1951 reported on the amino acids in the three soil organic matter fractions, and Stevenson, Marks, Varner and Martin~) in 1952 reported on the identification of eight amino acids from clay-adsorbed organic colloids in Brookstom slity clay loam. Also, some investigations on these amino acids have been carried out in our laboratory.     

Kinetics of soil microbial uptake of free amino acids

 Amino acids and proteins typically form the biggest input of organic-N into most soils and provide a readily available source of C and N for soil microorganisms. Amino acids can also be taken up directly by plant roots, providing an alternative source of available soil N. However, the degree to which plants can compete against the soil microbial population for amino acids in soil solution remains poorly understood. The aim of this study was to measure the rate of microbial uptake of three contrastingly charged ¹⁴C-labelled amino acids (glutamate^1–, glycine⁰, lysine^0.9+) over a wide concentration range (0.1–5 mM) and in two contrastingly managed soils varying in their degree of erosion, organic-C content and microbial biomass. Amino acid uptake was concentration dependent and conformed to a single Michaelis-Menten equation. The mean maximum amino acid uptake rate (V _max) for the non-eroded (control) soil (high organic-C, high biomass) was 0.13±0.02 mmol kg^–1 h^–1, while half maximal uptake occurred at a concentration (K _m) of 2.63±0.07 mM. Typically, V _max was fourfold lower and K _m twofold lower in the eroded soil (low available organic-C, low biomass) compared to the non-eroded (control) soil. Amino acid substrate concentration had little effect on the proportion of amino acid utilized in catabolic versus anabolic metabolism and was similar for both. While the results obtained here represent the summation of kinetics for a mixed soil population, they indicate that amino acid uptake is saturated at concentrations within the millimolar range. Because the affinity constants also were similar to those described for plant roots, we hypothesized that competition for amino acids between plants and microbes will be strong in soil but highly dependent upon the spatial distribution of roots and microbes in soil. Received: 2 March 2000     

Mineralization of soil organic nitrogen solubilized by aqua ammonia fertilizer

Organic N solubilized by NH_3(aq) was extracted from ¹⁵N-labelled or unlabelled soil, concentrated and added to non-extracted soil, which was incubated under aerobic conditions at 27±1°C. Gross N mineralization, gross N immobilization, and nitrification in soils with or without addition of unlabelled soluble organic N were estimated by models based on the dilution of the NH ₄ ⁺ or NO _inf3 ^sup- pools, which were labelled with ¹⁵N at the beginning of incubation. Mineralization of labelled organic N was measured by the appearance of label in the mineral N pool. Although gross N mineralization and gross N immobilization were increased in two soils between day 0 and day 7 following addition of unlabelled organic N solubilized by NH_3(aq), there was no increase in net N mineralization. Solubilization of ¹⁵N-labelled organic N increased and the ¹⁵N enrichment of the soluble organic N decereased as the concentration of NH_3(aq) added increased. A constant proportion of approximately one-quarter of the labelled organic N added at different rates to non-extracted soil was recovered in the mineral N pool after an incubation period of 14 days, and the availability ratios calculated from net N mineralization data were 1.1:1 and 2.1:1 for 111 and 186 mg added organic-N kg^-1 soil, respectively, indicating that the mineralization of organic N was increased by solubilization.     

Eliminating amino acid interference during spectrophotometric NH4 analysis

Amino acids can interfere with NH₄⁺ in spectrophotometric NH₄⁺ determination hampering accurate quantification of the fate of NH₄⁺ and dissolved organic N in soils. Serious interference has been reported for soils rich in organic matter, and for soils that have been fumigated, oven-dried or fertilized where between 5 and 60% of the NH₄⁺ detected could be attributed to amino acid interference. We investigated whether a combination of gas diffusion and the classical Berthelot method can eliminate this interference, increasing the selectivity of the NH₄⁺ analysis. We tested this approach using synthetic amino acid solutions and a large set of terrestrial samples (n = 353), including pore water samples, (fumigated or dried) soil extracts, and extracts of manures, composts and crop residues. The evaluated method produced accurate (recovery > 99.7%) and reproducible (standard error = 2.2%) NH₄⁺ concentrations, eliminating any interference between amino acids and NH₄⁺ (interference < 0.3%). Interference from K₂SO₄ was also eliminated. The method is robust, broadly applicable and will improve our understanding of nitrogen cycling in various ecosystems, in particular those where high levels of amino acids occur.     

Determination of 15N abundance of amino acids in soil hydrolysates

¹⁵N abundance of amino acids in soil hydrolysates was determined by emission spectroscopic method.     

A method to assess whether ‘preferential use’ occurs after N ammonium addition; implication for the N isotope dilution technique

The robustness of the assumption of equilibrium between native and added N during ¹⁵N isotope dilution has recently been questioned by Watson et al. (Soil Biol Biochem 32 (2000) 2019-2030). We re-analyzed their raw data using equations that consider the added and native NH₄⁺ and NO₃⁻ pools as separate state variables. Gross mineralization rates and first-order rate constants for NH₄⁺ and NO₃⁻ consumption were obtained by combining analytical integration of the differential equations with a non-linear fitting procedure. The first-order rate constants for NH₄⁺ consumption and NO₃⁻ immobilization for the added NH₄⁺ and NO₃⁻ pool were used to estimate gross mineralization rates and first-order rate constants for nitrification of native NH₄⁺. The latter were 2-4 times lower than the first-order rate constants derived from the added N pool. This discrepancy between first-order rate constants for nitrification implies that one or more process rates estimated for the added N pools cannot be applied to the native N pools. Preferential use of the added N resulted in an overestimation of the gross mineralization by 1.5-2.5-fold, emphasizing the need for critical evaluation of the assumption of equilibrium before gross mineralization rates are calculated.     

Soil- and enantiomer-specific metabolism of amino acids and their peptides by Antarctic soil microorganisms

Most nitrogen (N) enters many Arctic and Antarctic soil ecosystems as protein. Soils in these polar environments frequently contain large stocks of proteinaceous organic matter, which has decomposed slowly due to low temperatures. In addition to proteins, considerable quantities of d-amino acids and their peptides enter soil from bacteria and lengthy residence times can lead to racemisation of l-amino acids in stored proteins. It has been predicted that climate warming in polar environments will lead to increased rates of soil organic N turnover (i.e. amino acids and peptides of both enantiomers). However, our understanding of organic N breakdown in these soils is very limited. To address this, we tested the influence of chain length and enantiomeric composition on the rate of breakdown of amino acids and peptides in three contrasting tundra soils formed under the grass, moss or lichen-dominated primary producer communities of Signy Island in the South Orkney Islands. Both d- and l-enantiomers of the amino acid monomer were rapidly mineralized to CO₂ at rates in line with those found for l-amino acids in many other terrestrial ecosystems. In all three soils, l-peptides were decomposed faster than their amino acid monomer, suggesting a different route of microbial assimilation and catabolism. d-peptides followed the same mineralization pattern as l-peptides in the two contrasting soils under grass and lichens, but underwent relatively slow decomposition in the soil underneath moss, which was similar to the soil under the grass. We conclude that the decomposition of peptides of l-amino acids may be widely conserved amongst soil microorganisms, whereas the decomposition of peptides of d-amino acids may be altered by subtle differences between soils. We further conclude that intense competition exists in soil microbial communities for the capture of both peptides and amino acids produced from protein breakdown.     

Estimates of Gross Transformation Rates of Dairy Manure N Using 15N Pool Dilution

Abstract

Most measurements of dairy manure nitrogen (N) availability depend on net changes in soil inorganic N concentration over time, which overlooks the cycling of manure N in the soil. Gross transformations of manure N, including mineralization (m), immobilization (i), and nitrification (n), can be quantified using ¹⁵N pool dilution methods. This research measures gross m, n, and i resulting from application of four freeze‐dried dairy manures that had distinctly different patterns of N availability. A sandy loam soil (coarse‐loamy, mixed, frigid Typic Haplorthod) was amended with four different freeze‐dried dairy manures and incubated at 25°C with optimal soil water content. The dilution of ¹⁵ammonium (NH4⁺) during a 48‐h interval (7–9 d and 56–58 d after manure application) was used to estimate m, whereas the dilution of ¹⁵nitrate (NO₃ ^?) was used to estimate n. Gross immobilization was calculated as gross minus net mineralization. Gross mineralization in the unamended soil was similar at 7‐ to 9‐d and 56‐ to 58‐d intervals and was significantly increased by the application of manures. For both amended and unamended soil, m was much greater (i.e., three‐ to nine‐fold) than estimated net mineralization, illustrating the degree to which manure N can be cycled in soil. At the early interval, both m and i were directly related to the manure C input, demonstrating the linkage between substrate C availability and N utilization by soil microbes. This research clearly shows that the application of dairy manures stimulates gross N transformation rates in the soil, improving our understanding of the impact of manure application on soil N cycling.     

Dynamics of 15N-labeled ammonium sulfate in various inorganic and organic soil fractions of wetland rice soils

Summary The dynamics of basally applied ¹⁵N-labeled ammonium sulfate in inorganic and organic soil fractions of five wetland rice soils of the Philippines was studied in a greenhouse experiment. Soil and plant samples were collected and analyzed for ¹⁵N at various growth stages. Exchangeable NH₄ ⁺ depletion continued after 40 days after transplanting (DAT) and corresponded with increased nitrogen uptake by rice plants. Part of the applied fertilizer was fixed by 2:1 clay minerals, especially in Maligaya silty clay loam, which contained beidellite as the dominant clay mineral. After the initial fixation, nonexchangeable ¹⁵N was released from 20 DAT in Maligaya silty clay loam, but fixation delayed fertilizer N uptake from the soil. Part of the applied N was immobilized into the organic fraction. In Guadalupe clay and Maligaya silty clay loam, immobilization increased with time while the three other soils showed significant release of fertilizer N from the organic fraction during crop growth. Most of the immobilized fertilizer N was recovered in the nondistillable acid soluble (alpha-amino acid + hydrolyzable unknown-N) fraction at crop maturity. Between 61% and 66% of applied N was recovered from the plant in four soils while 52% of fertilizer N was recovered from the plant in Maligaya silty loam. Only 20% – 30% of the total N uptake at maturity was derived from fertilizer N. Nmin (mineral N) content of the soil before transplanting significantly correlated with N uptake. Twenty-two to 34% of applied N was unaccounted for possibly due to denitrification and ammonia volatilization.     

Carbohydrate and amino acid composition of dissolved organic matter leached from soil

Low molecular weight organic substances (LMWOS) in soil and soil solution include mainly amino acids, carboxylic acids, and carbohydrates. Due to their high bioavailability they play a crucial role in the cycles of C and nutrients in soils. The variety of soil processes that involve LMWOS requires identifying their composition to elucidate reactions and transformations. In most studies, LMWOS are extracted under artificial conditions, e.g. batch experiments, which may overestimate the actual concentrations. This study measures the composition of carbohydrates and amino acids in solution of a Haplic Luvisol leached in a column experiment. A combined system for simultaneous leaching and blowout of CO₂ was used to estimate LMWOS decomposition. ¹⁴C-labeled glucose was added as a highly sensitive tracer to control the efficiency of the LMWOS extraction by leaching and to estimate LMWOS decomposition during leaching. High performance liquid chromatography (HPLC), optimized for soil extracts, was used to analyze LMWOS composition. For HPLC optimization, different preparations of leached solutions (filtration vs. centrifugation, and drying vs. no-drying) were compared. For sugar determination, drying had no influence on the solution concentrations. In contrast, amino acid concentrations significantly decreased by drying LMWOS eluted substances. Combining the HPLC identification of eluted substances with ¹⁴C tracer application revealed that about 5% of the glucose could be leached unchanged within 786 min (13.1 h), whereas about 84% remained in the soil, 9% were decomposed to CO₂, and 2% were transformed to other LMWOS and recovered in the soil solution. The total amino acid concentration (TAC) in soil solution was about 8.2 μmol l⁻¹, dominated by alanine (14.4% of TAC), glycine (13.4%), glutamic acid (9.9%), serine (9.4%), and leucine (9.3%). The total carbohydrate concentration was about 2.4 μM, dominated by glucose (29.9%), glucuronic acid (26.8%), and galacturonic acid (17.3%). Ratios of hexoses to pentoses, amino sugars glucosamine to galactosamine, and neutral sugars to uronic acids were determined. All three parameters pointed to the dominant influence of plants as the source of LMWOS in the leached soil solution. Within the small contribution of microorganisms, bacteria dominated over fungi. These used biomarker ratios as well as LMWOS concentrations differed widely from the ones obtained with conventional batch extraction. More research is necessary to evaluate the application of these biomarkers to soil solutions.     

Natural abundance of 15N in nitrate,ureides, and amino acids from plant tissues

The natural ¹⁵N abundances (δ¹⁵N values) were measured for nitrate and free and bound amino acids from the leaves of field-grown spinach (Spinacia oleracea L.) and komatsuna (Brassica campestris L.), as well as ureides and free and bound amino acids in the leaves and roots of hydroponically grown soybean (Glycine max L.) totally depending on dinitrogen. Nitrate from the spinach and komatsuna leaves and ureides from leaves and roots of soybean showed higher δ¹⁵N values than the total tissue N and N in free or bound amino acid fractions. The δ¹⁵N values of individual free and bound amino acids, determined by GC/C/MS using their acetylpropyl derivatives, were similar in leaf tissues except for proline but varied in soybean root tissues. The order of ¹⁵N enrichment was similar in the four samples: aspartic acid > glutamic acid > threonine, proline, valine > glycine + alanine +serine, γ-amino butyric acid, and phenylalanine.     

Gross N transformation rates and net N mineralisation rates related to the C and N contents of soil organic matter fractions in grassland soils of different age

We investigated the relationship between soil organic matter (SOM) content and N dynamics in three grassland soils (0-10 and 10-20 cm depth) of different age (6, 14 and 50 y-old) with sandy loam textures. To study the distribution of the total C and N content the SOM was fractionated into light, intermediate and heavy density fractions of particulate macro-organic matter (150-2000 μm) and the 50-150 μm and <50 μm size fractions. The potential gross N transformation rates (mineralisation, nitrification, NH₄⁺ and NO₃⁻ immobilization) were determined by means of short-term, fully mirrored ¹⁵N isotope dilution experiments (7-d incubations). The long-term potential net N mineralisation and gross N immobilization rates were measured in 70-d incubations. The total C and N contents mainly tended to increase in the 0-10 cm layer with increasing age of the grassland soils. Significant differences in total SOM storage were detected for the long-term (50 y-old) conversion from arable land to permanent grassland. The largest relative increase in C and N contents had occurred in the heavy density fraction of the macro-organic matter, followed by the 50-150 and <50 μm fractions. Our results suggest that the heavy density fraction of the macro-organic matter could serve as a good indicator of early SOM accumulation, induced by converting arable land to permanent grassland. Gross N mineralisation, nitrification, and (long-term) gross N immobilization rates tended to increase with increasing age of the grasslands, and showed strong, positive correlations with the total C and N contents. The calculated gross N mineralisation rates (7-d incubations) and net N mineralisation rates (70-d incubations) corresponded with a gross N mineralisation of 643, 982 and 1876 kg N ha⁻¹ y⁻¹, and a net N mineralisation of 195, 208 and 274 kg N ha⁻¹ y⁻¹ in the upper 20 cm of the 6, 14 and 50 y-old grassland soils, respectively. Linear regression analysis showed that 93% of the variability of the gross N mineralisation rates could be explained by variation in the total N contents, whereas total N contents together with the C-to-N ratios of the <50 μm fraction explained 84% of the variability of the net N mineralisation rates. The relationship between long-term net N mineralisation rates and gross N mineralisation rates could be fitted by means of a logarithmic equation (net m=0.24Ln(gross m)+0.23, R²=0.69, P<0.05), which reflects that the ratio of gross N immobilization-to-gross N mineralisation tended to increase with increasing SOM contents. Microbial demand for N tended to increase with increasing SOM content in the grassland soils, indicating that potential N retention in soils through microbial N immobilization tends to be limited by C availability.     

Evaluation of N2 fixation by applying 15N labeled plant material and ammonium sulfate

Effect of different ¹⁵N labeled sources on the estimation of N₂ fixation was investigated. The combination of ¹⁵N labeled ammonium sulfate, ¹⁵N labeled plant material, and ¹⁵N labeled ammonium sulfate with unlabeled plant material, was examined in pot experiments. Two cultivars of soybean (Glycine max) and one of mungbean (Vigna radiata) were used. No significant difference was observed among the treatments for the estimation of N₂ fixation. This was due to the homogeneity and stability of the ¹⁵N abundance in soil which resulted in a similar N uptake from the soil by the N² fixing and reference crops. The plant yield, total N uptake and amount of N₂ fixed were higher in the Yellow Soil than in the Andosol. The amount of N₂ fixed was strongly influenced by the plant growth and consequently it affected the plant yield. The slow decomposition of plant material in the Andosol resulted in a low yield in both the N₂ fixing and reference crops. Thus, the artificial decrease of the available N content in soil, by application of plant material, did not stimulate N, fixation but suppressed plant growth and N₂ fixation.     

Cold storage and laboratory incubation of intact soil cores do not reflect in-situ nitrogen cycling rates of tropical forest soils

Measurements of N transformation rates in tropical forest soils are commonly conducted in the laboratory from disturbed or intact soil cores. On four sites with Andisol soils under old-growth forests of Panama and Ecuador, we compared N transformation rates measured from laboratory incubation (at soil temperatures of the sites) of intact soil cores after a period of cold storage (at 5 °C) with measurements conducted in situ. Laboratory measurements from stored soil cores showed lower gross N mineralization and NH₄⁺ consumption rates and higher gross nitrification and NO₃⁻ immobilization rates than the in-situ measurements. We conclude that cold storage and laboratory incubation change the soils to such an extent that N cycling rates do not reflect field conditions. The only reliable way to measure N transformation rates of tropical forest soils is in-situ incubation and mineral N extraction in the field.     

Effects of repeated fertilizer and cattle slurry applications over 38 years on N dynamics in a temperate grassland soil

The effects of repeated synthetic fertilizer or cattle slurry applications at annual rates of 50, 100 or 200 m³ ha⁻¹ yr⁻¹ over a 38 year period were investigated with respect to herbage yield, N uptake and gross soil N dynamics at a permanent grassland site. While synthetic fertilizer had a sustained and constant effect on herbage yield and N uptake, increasing cattle slurry application rates increased the herbage yield and N uptake linearly over the entire observation period. Cattle slurry applications, two and four times the recommended rate (50 m³ ha⁻¹ yr⁻¹, 170 kg N ha⁻¹), increased N uptake by 46 and 78%, respectively after 38 years. To explain the long-term effect, a ¹⁵N tracing study was carried out to identify the potential change in N dynamics under the various treatments. The analysis model evaluated process-specific rates, such as mineralization, from two organic-N pools, as well as nitrification from NH₄⁺ and organic-N oxidation. Total mineralization was similar in all treatments. However, while in an unfertilized control treatment more than 90% of NH₄⁺ production was related to mineralization of recalcitrant organic-N, a shift occurred toward a predominance of mineralization from labile organic-N in the cattle slurry treatments and this proportion increased with the increase in slurry application rate. Furthermore, the oxidation of recalcitrant organic-N shifted from a predominant NH₄⁺ production in the control treatment, toward a predominant NO₃⁻ production (heterotrophic nitrification) in the cattle slurry treatments. The concomitant increase in heterotrophic nitrification and NH₄⁺ oxidation with increasing cattle slurry application rate was mainly responsible for the increase in net NO₃⁻ production rate. Thus the increase in N uptake and herbage yield on the cattle slurry treatments could be related to NO₃⁻ rather than NH₄⁺ production. The ¹⁵N tracing study was successful in revealing process-specific changes in the N cycle in relationship to long-term repeated amendments.     

Temporal changes in distribution and composition of N from labeled fertilizer in soil organic matter fractions

Variations in the amount and composition of immobilized nitrogen (N) in major soil organic matter fractions were investigated in a 730-day soil incubation experiment using ¹⁵N-labeled urea and ¹⁵N nuclear magnetic resonance spectroscopy with the cross polarization/magic angle spinning (¹⁵N CPMAS NMR) method. After 730 days, 24.7% of the applied N was recovered from the soil as organic N. The urea-derived N recovered from humic acids and humin decreased from 11.2 and 33.8% of the applied amount after 14 days to 1.6 and 20.4% after 730 days, respectively. When these values were corrected for the microbial biomass (MB) N, they ranged from 9.0 to 1.2% and 28 to 18%, respectively. The proportion of urea-derived N recovered from fulvic acids was low, ranging between 0.4 and 5.8% (with MB N) or 5.6% (without MB N) of the applied amount, whereas that from water-soluble nonhumic substances (WS-NHS; NHS in the fulvic acid fraction) remained high, 28–33% of the applied amount after correction for the contribution of MB N up to day 365, and decreased to 0.9% thereafter. The ¹⁵N CPMAS NMR spectra of humic acids, fulvic acids, and humin showed the largest signal at −254 to −264 ppm, corresponding to peptide/amide N. The proportions of heterocyclic, peptide/amide, guanidine/aniline, and free amino N in the urea-derived humic acid N were 3–7, 83–90, 5–7, and 2–4%, respectively. More than 80% loss of the urea-derived humic acid N did not markedly alter their composition. No time-dependent variations were also observed for the proportions of respective N functional groups in humin N, which were 3–5, 71–78, 12–17, and 6–10% in the same order as above. These results suggest the greater importance of physical stability than structural variation for the initial accumulation of organic N in soil.     

Technique of soil sample pretreatment for analysis of 15N:14N isotope ratio

Abstract

The technique of simultaneous quantitative determination of mineral N soil forms (nitrates, exchangeable and non‐exchangeable ammonium, and total amount of these compounds) and sample pretreatment for the analysis of ¹⁵N:¹⁴N ratio is suggested. The technique is based on the selective association of NH₄ ⁺‐ions into indophenol complex and subsequent ethyl‐acetate extraction of this complex from solution. The mineralization of indophenol is carried out in alkaline medium with simultaneous NH₃ distillation into H₂SO₄ titrant. The application of given technique allows us to shorten significantly the time required for analysis and to increase the accuracy of analytical determination.     

Interference by magnesium in the determination of nitrate in 2 M KCl extracts of soil by steam distillation

Abstract

Steam distillation of 2 M KCl extracts of soil showed low recovery of NO₃‐N when compared with an automated method for NO₃‐N determination. The low recoveries were more pronounced in extracts where a soil:solution ratio of 1:2.5 had been used. In extracts where the Mg²⁺ content was in excess of 0.02 M Mg, recoveries of added NO₃‐N could be as low as 25%. Increasing the amount of Devarda's alloy or using a 1:10 soil:solution extraction ratio overcame the problem of low NO₃‐N recovery. Calcium salts did not interfere in the recovery of added NO₃‐N.