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     

Interference by amino acids during the determination of N ammonium in soil

In the study of terrestrial N cycling, NH₄⁺ concentration and ¹⁵N enrichment are routinely determined by colorimetric continuous flow analysis and microdiffusion methods. Amino acids can interfere in these determinations; consequently the aim of the present study was to evaluate the significance of the interference. Glycine and glutamine are key amino acids in soil and were therefore used as ‘models’. Both glycine and glutamine interfered during continuous flow analysis, whereas interference during microdiffusion was of little importance. The effects of interference can be significant, e.g. estimates of gross mineralisation rate were reduced up to 33%, where we allowed for amino acid interference during determination of NH₄⁺ concentration. The potential influence of amino acid interference emphasises that development of continuous flow analysis to increase NH₄⁺ specificity is needed.     

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.     

Plant species affect the concentration of free sugars and free amino acids in different types of soil

Substantial amounts of low molecular weight organic compounds (LMWs) such as sugars and amino acids are transferred from plant roots into soil. These substances are released due to decomposition processes or leaching (exudation). Afterwards they can be metabolized by soil microorganisms into different compounds, or they can be partially re‐absorbed by the plants. The aim of this study was to clarify the influence of five wild plant species on the composition and pool sizes of LMWs extractable from three different soils. Four of the five species caused significant changes in soil LMW pools. In Chernozem, the sugar concentrations of soil with plants were up to 60 % higher than those of the bulk reference soil, and amino acids increased by as much as 207 %. The relative abundance of free amino acids in roots did not correlate with the relative abundance of amino acids in soil after six weeks of plant growth. The relative abundance of soil amino acids, that increased after plant growth, was strongly dependent on the type of soil and on the plant species present. We suggest that rather than rhizodeposition being dependent on soil type, it reflects differential microbial metabolization of amino acids in the respective soils.     

Improved RP‐HPLC and anion‐exchange chromatography methods for the determination of amino acids and carbohydrates in soil solutions

In spite of their low concentrations in soil solutions, low–molecular weight organic substances (LMWOS) such as amino acids, sugars, and uronic acids play a major role in the cycles of C and N in soil. With respect to their low concentrations and to possible matrix interferences, their analysis in soil leachates is a challenging task. We established two HPLC (high‐performance liquid chromatography) methods for the parallel determination of amino acids and carbohydrates in soil leachates. The pre‐column derivatization of amino acids with an o‐phthaldialdehyde (OPA) mercaptoethanol solution yields quantitation limits between 0.03 and 0.44 µmol L^–1 and S_D values of <8.3% (n = 9). High‐performance anion‐exchange chromatography (HPAEC) on a Dionex CarboPac PA 20 column with a NaOH acetate gradient combined with pulsed amperometric detection (PAD) was used for the determination of carbohydrates. The calibration curves obtained for 11 carbohydrates showed excellent linearity over the concentration range from 0.02 to 50.0 mg L^–1. Recovery studies revealed good results for all analytes (89%–108%). Interferences from Hg(II) salts and chloroform used for stabilization of the leachates did not occur with both chromatographic methods. The optimized method was successfully used for quantitative determinations of amino acids and carbohydrates in soil leachates.     

Identification of free amino acids in peat by gas chromatography and mass spectrometry

The qualitative and quantitative composition of free amino acids in a typical Finnish peat bog at various depths down to 5.3 m below the surface was studied using capillary gas chromatography and mass spectrometry. Sixteen amino acids were identified at each depth: α-alanine, β-alanine, glycine, valine, leucine, proline, isoleucine, serine, threonine, glutamic acid, aspartic acid, phenylalanine, tyrosine, γ-aminobutyric acid, ornithine and lysine. Their amounts decreased markedly at a depth of 40–100 cm. The total amount of amino acids varied between 0.6 and 5.6 g kg^?1 dry matter (i.e. 0.06–0.56%) depending on the depth. The proportion of neutral amino acids was greatest at all depths studied, except at the surface layer where it ranged between 41 and 72% by mass. The acidic amino acids decreased with depth from 56 to 23% of the total. The proportion of aromatic amino acids was very small, 3.2–5.5% by mass. In samples from aerobic conditions, where the microbial production of free amino acids was the greatest, α-ala, gly, glu and asp were most abundant. In peat from anaerobic conditions, where the microbiological activity was low, the proportion of the most chemically stable amino acid was exceptionally high. This may have been because glycine was a degradation product of other amino acids or peptides. Peat type and degree of decomposition had a strong influence on the total amount of free amino acids and their qualitative composition.     

HPLC method for analysis of free amino acids in fish using o-phthaldialdehyde precolumn derivatization

Precolumn derivatization applying o-phthaldialdehyde (OPA) was used to analyze free lysine, histidine, and ornithine, precursors of the respective biogenic amines cadaverine, histamine, and putrescine, which are considered indicators of fish quality and safety. This method uses 75% methanol to eliminate the use of strong acids as the extraction solution. Each analysis took 35 min, was reproducible, and allowed separation of primary amino acids in fish samples. A binary solvent delivery system coupled with a fluorescence detector and an Ultrasphere ODS column were utilized for HPLC separation. Linearity of the calibration curves was very good (r(2) = 0.99) for the amino acids of interest. Minimum concentrations of detection were 40 pmol/mL for histidine and lysine and 70 pmol/mL for ornithine. Average recoveries were 72% for lysine, 93% for histidine, and 98% for ornithine. This method used solvent gradient elution to study the levels of these analytes in mahi-mahi, bigeye tuna, and flounder.     

Effects of amino-acid chemistry and soil properties on the behavior of free amino acids in acidic forest soils

Free amino acids (FAAs) in soil solution are increasingly recognized as a potentially important source of nitrogen (N) for plants, yet we are just beginning to understand the behavior of FAAs in soil. I investigated the effects of amino-acid chemistry and soil properties on mineralization, microbial assimilation and sorption of amino-acid N in soils from three ecosystems representing the two endpoints and mid point of a temperate forest fertility gradient ranging from low mineral N availability/high FAA oak forests to high mineral N availability/low FAA maple-basswood forests. Soils were amended with six ¹⁵N-labeled amino-acid substrates that ranged widely in chemical properties, including molecular weight, C:N ratio, average net charge, hydrophobicity, and polarity: Arginine (Arg), Glutamine (Gln), Glutamate (Glu), Serine (Ser), Glycine (Gly) and Leucine (Leu). Mineralization of amino-acid N accounted for 7-45% (18% avg.) of the added label and was most strongly affected by soil characteristics, with mineralization increasing with increasing soil fertility. Mineralization of amino-acid N was unrelated to amino-acid C:N ratio, rather, I observed greater N mineralization from polar FAAs compared to non-polar ones. Assimilation of amino-acid N into microbial biomass accounted for 6-48% (29% avg.) of the added label, and was poorly predicted by either intrinsic amino-acid properties or soil properties, but instead appeared to be explicable in terms of compound-specific demand by soil micoorganisms. Sorption of amino-acid N to soil solids accounted for 4-15% (7% avg.) of the added label and was largely controlled by charge characteristics of individual amino acids. The fact that both positively- and negatively-charged amino acids were more strongly sorbed than neutral ones suggests that cation and anion exchange sites are an important factor controlling sorption of FAAs in these acid forest soils. Together, the findings from this study suggest that there may be important differences in the behavior of free amino acids in sandy, acidic forest soils compared to generalizations drawn from finer-textured grassland soils, which, in turn, might affect the availability of some FAAs in soil solution.     

Quantitative determination of short-chain free fatty acids in milk using solid-phase microextraction and gas chromatography

The objective was to establish a rapid, precise, and accurate methodology for the quantification of short-chain free fatty acids (FFA) (C(4)-C(12)) in milk by solid-phase microextraction and gas chromatography. Sampling conditions such as fiber type, pH, salt addition, temperature, volume, and time were investigated. FFA extraction consisted of placing 40 mL of milk containing 28% NaCl at pH 1.5 in a sealed vial and equilibrating for 30 min at 70 degrees C. A polyacrylate fiber was exposed to the sample headspace for 60 min and desorbed for 5 min into the gas chromatograph. Calibration curves for FFA followed linear relationships with highly significant (p < 0.001) correlation coefficients (R(2) = 0.99). Coefficients of variation of less than 7.7% for FFA concentrations indicated that the technique was reproducible. The limits of quantification for C(4)-C(10) were in the low parts per million level, which were below the concentration range found in fresh pasteurized milk (0.48-2.52 ppm) or rancid milk (4.73-32.31 ppm).     

Supercritical fluid extraction of bioavailable amino acids from soils and their liquid chromatographic determination with fluorometric detection

A new procedure with supercritical CO2 modified with 0.5 mL of water and 0.75 mL of 0.1 M HCl in situ and 0.75 mL of water on-line at 15 MPa and 50 degrees C for 45 min was applied for the extraction of bioavailable amino acids from soil samples. Total extraction time was 60 min, but more favorable conditions are even possible for selected groups of amino acids. All analytes were trapped into 20 mL of methanol with satisfactory recovery (94-104%) and determined using high-performance liquid chromatography with fluorometric detection on a Zorbax Eclipse column (4.6 x 75 mm, 3.5 microm) with Na2HPO4 and acetonitrile/methanol/water as a mobile phase. Linear calibration curves were obtained (r > 0.999 except 0.99823 for Ile) with lower limits of detection (S/N = 3) in the range from 1.54 pg (Gly) to 13.5 pg (Cy2) or from 18.6 fmol (Ser) to 64.8 fmol (Lys). Validation and repeatability data are also given. Comparable results were obtained with a robust, commonly used extraction method (0.5 M ammonium acetate, 60 min in shaker, followed by filtration and lyophilization). Limiting values of artificial release of amino acids were also determined for each soil sample to eliminate any false results to ensure that all extracted amino acids originate from soil solution and exchangeable bound positions of soil samples.     

Rapid extraction and analysis of volatile fatty acids in soil

Abstract

The concentrations of volatile fatty acids (VFA) in soils are important in studies involving phytotoxicity and fermentation processes. Concentrations of acetic, propionic, and butyric acids as low as 0.21, 0.14, and 0.10 mmol kg^‐1soil in water extracts were accurately determined. The extracts were filtered through 45 μm millipore disc filters and injected directly into a gas chromatograph following addition of purified formic acid. The formic acid eliminated ghosting of peaks. The gas chromatograph was equipped with a flame ionization detector and a 60/80 Carbopack C/0.3% Carbowax 20M/0.1% H₃PO₄packed precolumn (0.15 m) and column (1.83 m). The precolumn was changed after 150 to 200 sample injections when contaminated beyond acceptable limits. There was good separation of VFA with no interfering organic volatiles in extracts of soil containing glucose, cellulose or straw incubated anaerobically for as long as 4 weeks. The advantages of the procedure are relative rapidity and simplicity as well as improved sensitivity in measuring small quantities of volatile fatty acids in soil     

Acrylamide in French fries: influence of free amino acids and sugars

The free amino acid profile and sugar (fructose, glucose, and sucrose) composition were determined in potato samples selected to give a large range of variation (a total of 66 samples). From these samples French fries were produced in a laboratory-scale simulation of an industrial process followed by a finish fry at 180 degrees C for 3.5 min using a restaurant fryer. The final product was blast frozen and analyzed for acrylamide. Acrylamide was detected in all samples, but its concentration varied significantly from 50 to 1800 ng/g. For isotope dilution (13C3) acrylamide analysis, samples were extracted with water, cleaned up on HLB Oasis polymeric and Accucat mixed mode anion and cation exchange SPE columns, and analyzed by LC-MS/MS. Statistical analysis of the data indicates that the effect of sugars and asparagine on the concentration of acrylamide in French fries is positive and significant (p < 0.001). It appears that one of the ways acrylamide formation in French fries can be effectively controlled is by the use of raw products with low sugar (and to a lesser degree, asparagine) content.     

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.     

Reducing interferences by amino acids in the determination of ammonium by an automated indophenol method

Abstract

Positive interference by amino acids in the determination of ammonium in soil extracts by automated indophenol methods can be minimised by using the nitroprusside catalysed reaction. Hydrolysis of amino acids is virtually eliminated because this reaction enables the use of low sample volumes, reagent concentrations and reaction temperatures.     

Comparison of ceramic suction cups and KC1 extraction for the determination of nitrate in soil

Comparisons of nitrate concentrations in soil core extracts and ceramic suction cup isolates were made on a coarse sand and a sandy loam to evaluate the methods for estimating nitrate concentrations percolating through the root zone. On the coarse sand the comparisons were made at 80 cm on three dates during the winter of 1988/89. For each date, 103 soil samples and 101 or 102 suction cup isolates were taken. On the sandy loam the comparisons were made at 25 and 80 cm on two dates in March 1990. For each date and depth, 57 soil samples and 55–57 suction cup isolates were taken. On the coarse sand the mean nitrate concentrations were the same for the two methods. On the sandy loam the results obtained using the two methods differed significantly in only one of the four comparisons. However, the results indicated that the suction cup isolates differed slightly from the volume-averaged concentrations obtained from the soil samples. The type of frequency distribution, normal or lognormal, seemed to be influenced both by the mean nitrate concentration and by the soil structure, and thus by the pattern of percolation. The size of sample required to estimate the mean nitrate concentration with a probability of 95% and a precision of 10% varied from 7 to 106.     

Rapid and sensitive quantification of amino acids in soil extracts by capillary electrophoresis with laser-induced fluorescence

A growing body of research is arguing that amino acids are key components of the soil nitrogen cycle. For example, we now know that many plants can take up intact amino acids, even in competition with soil microbes. Our growing recognition of the importance of amino acids is not matched by knowledge of the amounts and type of amino acids in the soil, certainly not in comparison with our encyclopaedic knowledge of inorganic N. The primary reason that less is known about amino acids than inorganic N is that measuring the amounts of individual amino acids with conventional chromatographic techniques is slow and typically requires extensive sample clean-up if KCl extracts are analysed. The aim of this study was to develop capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) as a more rapid alternative for measuring individual amino acids in KCl extracts of soil. The CE-LIF method separated 17 common amino acids within 12 min, with detection limits between 7 and 250 nM. One molar KCl extracts could be analysed without any sample clean-up or de-salting, and spike and recovery tests indicated that the complex matrix of soil extracts did not affect quantification. Further evidence of the suitability and robustness of the method came from the repeated analysis (n=5) of the same soil KCl extract. The relative standard deviation of migration times for replicate analyses were <0.2% while relative standard deviations for peak areas were <5%. To demonstrate application of the CE-LIF method to real world problems it was used to analyse amino acids in 1 M KCl extracts from a sub-alpine grassland and a Eucalyptus regnans forest. The most abundant amino acids were Ala, Gly and Arg. Other amino acids present at smaller concentrations or in a minority of samples were Asn, Cit, GABA, Glu, His, Phe, Leu, and Lys. The proposed CE-LIF method offers significant advantages over chromatographic methods via its rapidity, reproducibility and, most importantly, its ability to analyse crude KCl extracts.