Mass spectrometric characterization of amino sugar aldononitrile acetate derivatives used for isotope enrichment assessment of microbial residues

Identifying the transformation of amino sugars in soils is essential in understanding microbial contribution to soil organic matter turnover and sequestration. Using a recently developed method, combining gas chromatograph/mass spectrometry (GC/MS) with laboratory incubation of substrates containing ¹³C or ¹⁵N isotopes, we were able to trace isotopic changes in amino sugar compounds. This allows us to quantitatively evaluate C or N enrichment in amino sugars during transformation in soils using the fragment (F) abundance ratio of m/z F + n to F (n is original skeleton atom number in each fragment). However, there is still lack of detail structural and substitutional information for each ion fragment. In order to improve the interpretation and increase our ability to study amino sugar turnover, we grew labeled amino sugars in lab-cultured organisms. We spectrometrically investigated the ion structures and original skeleton C number (mass shift n) in major ion fragments based on applying multiple representative isotope labels. Our results categorically confirm that previously made assumptions were correct regarding the substitutional number “n” of the glucosamine (He et al., 2006). Our study also added valuable structural information for aldononitrile acetate derivatized glucosamine and muramic acid upon electron impact ionization in MS.     

Temporal responses of soil microorganisms to substrate addition as indicated by amino sugar differentiation

Amino sugars are one of the important microbial residue biomarkers which are associated with soil organic matter cycling. However, little is known about their transformation kinetics in response to available substrates because living biomass only contributes a negligible portion to the total mass of amino sugars. By using ¹⁵N tracing technique, the newly synthesized (labeled) amino sugars can be differentiated from the native portions in soil matrix, making it possible to evaluate, in quantitative manner, the transformation pattern of amino sugars and to interpret the past and ongoing changes of microbial communities during the assimilation of extraneous ¹⁵N. In this study, laboratory incubations of soil samples were conducted by using ¹⁵NH₄⁺ as nitrogen source with or without glucose addition. Both the ¹⁵N enrichment (expressed as atom percentage excess, APE) and the contents of amino sugars were determined by an isotope-based gas chromatography-mass spectrometry. The significant ¹⁵N incorporation into amino sugars was only observed in glucose plus ¹⁵NH₄⁺ amendment with the APE arranged as: muramic acid (MurN) > glucosamine (GlcN) > galactosamine (GalN). The dynamics of ¹⁵N enrichment in bacterial-derived MurN and fungal-derived GlcN were fitted to the hyperbolic equations and indicative for the temporal responses of different soil microorganisms. The APE plateau of MurN and fungal-derived GlcN represented the maximal extent of bacterial and fungal populations, respectively, becoming active in response to the available substrates. The different dynamics of the ¹⁵N enrichment between MurN and GlcN indicated that bacteria reacted faster than fungi to assimilate the labile substrates initially, but fungus growth was dominant afterward, leading to integrated microbial community structure over time. Furthermore, the dynamics of labeled and unlabeled portions of amino sugars were compound-specific and substrate-dependent, suggesting their different stability in soil. GlcN tended to accumulate in soil while MurN was more likely degraded as a carbon source when nitrogen supply was excessive.     

Land-use effects on amino sugars in particle size fractions of an Argiudoll

Identifying amino sugar pools from different land-use systems may advance our knowledge of land-use effects on the fate of microbially-derived substances. Surface soils (0–10 cm) from (1) native pasture, (2) a >80-years-arable site, and (3) a >80-years-afforested site were fractionated into clay, silt, fine-, and coarse-sand fractions. Then, soil organic carbon, N, glucosamine, galactosamine, mannosamine, and muramic acid were analyzed.Afforestation did not influence the amino sugar content in bulk soil, whereas cultivation reduced the content by 54%. The concentrations of amino sugars in g kg⁻¹ SOM declined after both long-term cropping and afforestation by 6% and 13%, respectively, relative to that in the grassland. The amino sugar depletion at the forest site occurred mainly from the silt fraction (by 25%), while that in the cultivated site was mainly due to preferential loss of amino sugars from clay (by 19% compared with the grassland). Both ratios of glucosamine to galactosamine and glucosamine to muramic acid increased when the prairie was converted to forest or cultivated land, suggesting that bacterial N especially is better preserved than fungal N under prairie conditions.     

Amino sugars as specific indices for fungal and bacterial residues in soil

Amino sugars are important indices for the contribution of soil microorganisms to soil organic matter. Consequently, the past decade has seen a great increase in the number of studies measuring amino sugars. However, some uncertainties remain in the interpretation of amino sugar data. The objective of the current opinion paper is to summarize current knowledge on amino sugars in soils, to give some advice for future research objectives, and to make a plea for the correct use of information. The study gives an overview on the origin of muramic acid (MurN), glucosamine (GlcN), galactosamine (GalN), and mannosamine (ManN). Information is also provided on measuring total amino sugars in soil but also on compound-specific δ¹³C and δ¹⁵N determination. Special attention is given to the turnover of microbial cell-wall residues, to the interpretation of the GlcN/GalN ratio, and to the reasons for converting fungal GlcN and MurN to microbial residue C. There is no evidence to suggest that the turnover of fungal residues generally differs from that of bacterial residues. On average, MurN contributes 7% to total amino sugars in soil, GlcN 60%, GalN 30%, and ManN 4%. MurN is highly specific for bacteria, GlcN for fungi if corrected for the contribution of bacterial GlcN, whereas GalN and ManN are unspecific microbial markers.     

Amino sugars and muramic acid—biomarkers for soil microbial community structure analysis

Characterizing functional and phylogenetic microbial community structure in soil is important for understanding the fate of microbially-derived compounds during the decomposition and turn-over of soil organic matter. This study was conducted to test whether amino sugars and muramic acid are suitable biomarkers to trace bacterial, fungal, and actinomycetal residues in soil. For this aim, we investigated the pattern, amounts, and dynamics of three amino sugars (glucosamine, mannosamine and galactosamine) and muramic acid in the total microbial biomass and selectively cultivated bacteria, fungi, and actinomycetes of five different soils amended with and without glucose. Our results revealed that total amino sugar and muramic acid concentrations in microbial biomass, extracted from soil after chloroform fumigation varied between 1 and 27 mg kg⁻¹ soil. In all soils investigated, glucose addition resulted in a 50-360% increase of these values. In reference to soil microbial biomass-C, the total amino sugar- and muramic acid-C concentrations ranged from 1-71 g C kg⁻¹ biomass-C. After an initial lag phase, the cultivated microbes revealed similar amino sugar concentrations of about 35, 27 and 17 g glucosamine-C kg⁻¹ TOC in bacteria, fungi, and actinomycetes, respectively. Mannosamine and galactosamine concentrations were lower than those for glucosamine. Mannosamine was not found in actinomycete cultures. The highest muramic acid concentrations were found in bacteria, but small amounts were also found in actinomycete cultures. The concentrations of the three amino sugars studied and muramic acid differed significantly between bacteria and the other phylogenetic microbial groups under investigation (fungi and actinomycetes). Comparison between the amino sugar and muramic acid concentrations in soil microbial biomass, extracted after chloroform fumigation, and total concentrations in the soil showed that living microbial biomass contributed negligible amounts to total amino sugar contents in the soil, being at least two orders of magnitude greater in the soils than in the soil inherent microbial biomass. Thus, amino sugars are significantly stabilized in soil.     

Amino sugars in soils of the North American cultivated prairie

Characterizing amino sugar dynamics in cultivated soils helps to further understand the influence of cultivation on soil organic matter turnover. This study was designed to evaluate accumulations and patterns of four amino sugars in 17 surface (0–10 cm) soil samples along a climosequence in the North American long-term cultivated prairie from Saskatoon, Candada, to Texas, USA. Mean annual temperature (MAT) ranged from 0.9 to 22.2°C and mean annual precipitation (MAP) from 300 to 1308 mm. Samples were analyzed for glucosamine, mannosamine, galactosamine, and muramic acid. Amino sugar contents (mg kg^?1 soil) varied markedly among the 17 sites and were controlled by mean annual temperature (MAT) and clay and silt contents, mainly. The relationship between amino sugar-N proportions to total N (%) and MAT followed parabolic regression models. Compared with native sites, amino sugars were depleted by 53% and the amino sugar-N by 18% of the total, on average, after long-term cropping. The intensity of amino sugar-N depletion correlated positively with MAT (r = 0.77^***). Bacterially-derived galactosamine and muramic acid declined preferentially to mainly chitin-derived glucosamine after long-term cropping. The glucosamine-to-galactosamine and glucosamine-to-muramic acid ratios can be used, therefore, as indicators for the identification of land use effects on microbially-derived SOM residues.     

Land use effects on amino sugar signature of chromic Luvisol in the semi-arid part of northern Tanzania

 Characterizing amino sugar signature in particle size separates of tropical soils is important for further understanding the fate of microbial-derived compounds during the decomposition of soil organic matter (SOM) in tropical agroecosystems. We investigated the impact of land-use changes on the nature, amount and dynamics of amino sugars in soil of the semi-arid northern Tanzania. Samples were collected from the uppermost 10 cm of native woodland, degraded woodland, fields cultivated for 3 and 15 years and homestead fields fertilized with animal manure. The amount of glucosamine, galactosamine, mannosamine and muramic acid were determined in bulk soil and size separates. Compared to the native woodland, a 68% and 72% reduction in total amino sugar contents were found in the 3- and 15-year cultivated fields, respectively. Moreover, 39% of the total amino sugar was lost from the degraded woodland. This may be attributed to accelerated decomposition of amino sugars and/or decreasing microbial biomass input under the semi-arid environment following clear-cutting and cultivation. In contrast, only a 20% decline was found from the fields where animal manure had been applied. Most of the amino sugar depletion occurred from the coarse and fine sand-associated SOM. The decline from the silt and clay-bound amino sugar was relatively small, indicating the importance of organo-mineral associations in the stabilization of microbial-derived sugars in this tropical soil. After 15 years of continuous cultivation, the ratio of glucosamine:galactosamine increased from 1.44 to 2.23, while the ratio of glucosamine:muramic acid increased from 14.5 to 26.5 (P<0.05). These results suggest that cultivation may have led to preferential depletion of bacterial-derived amino sugars (muramic acid and galactosamine) compared with fungal-derived glucosamine. Received: 22 June 2000     

The automated determination of glucosamine,galactosamine, muramic acid,and mannosamine in soil and root hydrolysates by HPLC

Amino sugars make a significant contribution to soil organic N and are mainly of microbial origin. The most important amino sugars in soil are glucosamine, galactosamine, muramic acid, and mannosamine. A method was developed for the simultaneous determination of these four amino sugars by high‐performance reverse‐phase liquid chromatography in standard solutions, soil and root hydrolysates. Pre‐column derivatization with o‐phthaldialdehyde (OPA) was used in an automated sample injector with thermostatic regulation of the reagent at 4 °C. The separation of the four amino sugars was fully satisfactory and was not disturbed by other fluorescent components in the soil and root hydrolysates.     

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.     

Optimisation of amino sugar quantification by HPLC in soil and plant hydrolysates

Amino sugars are increasingly used as indicators for the accumulation of microbial residues in soil and plant material. A reverse-phase high-performance liquid chromatography method was improved for the simultaneous determination of muramic acid, mannosamine, glucosamine and galactosamine in soil and plant hydrolysates via ortho-phthaldialdehyde (OPA) pre-column derivatisation and fluorescence detection. The retention time was reduced, and the separation of muramic acid and mannosamine was optimised by modifying the mobile phase. The effects of excitation wavelength, OPA reaction time, tetrahydrofuran concentration and pH value of the mobile phase on the amino sugar separation were tested. Quantification limits were in the range of 0.13 to 0.90 μg ml⁻¹. No interferences exist from amino acids or other primary amines, occurring in soil and plant hydrolysates.     

Elevated CO2 does not favor a fungal decomposition pathway

We examined the effect of prolonged elevated CO₂ on the concentration of fungal- and bacterial-derived compounds by quantifying the soil contents of the amino sugars glucosamine, galactosamine and muramic acid. Soil samples were collected from three different terrestrial ecosystems (grassland, an aspen forest and a soybean/corn agroecosystem) that were exposed to elevated CO₂ under FACE conditions for 3-10 years. Amino sugars were extracted from bulk soil and analyzed by gas chromatography. Elevated CO₂ did not affect the size or composition of the amino sugar pool in any of the systems. However, high rates of fertilizer N applications decreased the amount of fungal-derived residues in the grassland system. We suggest that these results are caused by a decrease in saprophytic fungi following high N additions. Furthermore, our findings imply that the contribution of saprophytic fungi and bacteria to SOM in the studied ecosystems is largely unaffected by elevated CO₂.     

Net microbial amino sugar accumulation process in soil as influenced by different plant material inputs

Identifying the impact of plant material inputs on soil amino sugar synthesis may advance our knowledge of microbial transformation processes in soils. In a 12-week laboratory microcosm incubation, 1, 2, 4, and 6% (w/w) soybean leaf or maize stalk were initially added to soil, respectively, whereas soil without plant addition was used as a control. The results showed that adding organic materials to the soil led to a net accumulation of amino sugars, because of greater microbial synthesis. The ratios of glucosamine to galactosamine and of glucosamine to muramic acid, two indicators differentiating the relative contribution to soil organic matter of fungi and bacteria, showed substantial variance across the gradient of substrate addition. Our results suggest that the amount of nutrients in a given substrate is the primary attribute determining microbial net accumulation of soil amino sugars, especially in the relatively short term, whereas the composition of nutrients might be more important in the relatively long term when nutrients are not sufficient. The use of the two ratios (glucosamine to galactosamine and glucosamine to muramic acid) reflects different dynamics of galactosamine and muramic acid during the decomposition of organic substrates in soils. Muramic acid, compared with galactosamine, is more likely to accumulate in the soil active organic fraction under abundant nutrient conditions, whereas it would be decomposed along with active organic matter when the nutrients are scarce and remain in minor quantities in the clay fraction without being attacked by microbes.     

尿素向氨基糖的转化以及对土壤氨基糖库动态的影响

采用13CO(NH2)2为底物进行黑土培养实验,利用气相色谱/质谱技术测定土壤中三种氨基糖含量以及同位素富集比例,根据其微生物标识物作用探讨土壤中不同微生物群落对于尿素碳的同化利用特征及黑土氨基糖库对于尿素添加的响应。研究结果表明,尿素碳可以被土壤微生物同化利用,但是可利用性显著低于葡萄糖。氨基葡萄糖中13C富集比例显著高于胞壁酸,表明真菌对尿素碳的同化能力高于细菌。尿素添加使土壤有机碳含量有所下降,同时土壤氨基糖总量及其与有机碳的相对比例也显著降低,说明在碳源严重受限条件下,氨基糖可被优先分解利用以补充碳源供给。胞壁酸含量虽低,但其调节并平衡碳氮元素供给与需求的能力较强;氨基葡萄糖稳定性高于胞壁酸,但在碳源缺乏时也可部分分解。土壤氨基糖的动态与土壤碳氮的可利用性及其耦合作用密切相关,在平衡土壤碳氮需求方面具有一定的调节作用。     

Kinetics of amino sugar formation from organic residues of different quality

Amino sugars are key compounds of microbial cell walls, which have been widely used as biomarker of microbial residues to investigate soil microbial communities and organic residue cycling processes. However, the formation dynamics of amino sugar is not well understood. In this study, two agricultural Luvisols under distinct tillage managements were amended with uniformly ¹³C-labeled wheat residues of different quality (grain, leaf and root). The isotopic composition of individual amino sugars and CO₂ emission were measured over a 21-day incubation period using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS) and trace gas IRMS. Results showed that, the amount of residue derived amino sugars increased exponentially and reached a maximum within days after residue addition. Glucosamine and galactosamine followed different formation kinetics. The maxima of residue derived amino sugars formation ranged from 14 nmol g⁻¹ dry soil for galactosamine (0.8% of the original concentration) to 319 nmol g⁻¹ dry soil for glucosamine (11% of the original concentration). Mean production times of residue derived amino sugars ranged from 2.1 to 9.3 days for glucosamine and galactosamine, respectively. In general, larger amounts of amino sugars were formed at a higher rate with increasing plant residue quality. The microbial community of the no-till soil was better adapted to assimilate low quality plant residues (i.e. leaf and root). All together, the formation dynamics of microbial cell wall components was component-specific and determined by residue quality and soil microbial community.     

Shifts in amino sugar and ergosterol contents after addition of sucrose and cellulose to soil

An incubation experiment with organic soil amendments was carried out with the aim to determine whether formation and use of microbial tissue (biomass and residues) could be monitored by measuring glucosamine and muramic acid. Living fungal tissue was additionally determined by the cell-membrane component ergosterol. The organic amendments were fibrous maize cellulose and sugarcane sucrose adjusted to the same C/N ratio of 15. In a subsequent step, spherical cellulose was added without N to determine whether the microbial residues formed initially were preferentially decomposed. In the non-amended control treatment, ergosterol remained constant at 0.44 μg g⁻¹ soil throughout the 67-day incubation. It increased to a highest value of 1.9 μg g⁻¹ soil at day 5 in the sucrose treatment and to 5.0 μg g⁻¹ soil at day 33 in the fibrous cellulose treatment. Then, the ergosterol content declined again. The addition of spherical cellulose had no further significant effects on the ergosterol content in these two treatments. The non-amended control treatment contained 48 μg muramic acid and 650 μg glucosamine g⁻¹ soil at day 5. During incubation, these contents decreased by 17% and 19%, respectively. A 33% increase in muramic acid and an 8% increase in glucosamine were observed after adding sucrose. Consequently, the ratio of fungal C to bacterial C based on bacterial muramic acid and fungal glucosamine was lowered in comparison with the other two treatments. No effect on the two amino sugars was observed after adding cellulose initially or subsequently during the second incubation period. This indicates that the differences in quality between sucrose and cellulose had a strong impact on the formation of microbial residues. However, the amino sugars did not indicate a preferential decomposition of microbial residues as N sources.     

Natural abundance δN and δC of DNA extracted from soil

We report the first simultaneous measurements of δ¹⁵N and δ¹³C of DNA extracted from surface soils. The isotopic composition of DNA differed significantly among nine different soils. The δ¹³C and δ¹⁵N of DNA was correlated with δ¹³C and δ¹⁵N of soil, respectively, suggesting that the isotopic composition of DNA is strongly influenced by the isotopic composition of soil organic matter. However, in all samples DNA was enriched in ¹³C relative to soil, indicating microorganisms fractionated C during assimilation or preferentially used ¹³C enriched substrates. Enrichment of DNA in ¹⁵N relative to soil was not consistently observed, but there were significant differences between δ¹⁵N of DNA and δ¹⁵N of soil for three different sites, suggesting microorganisms are fractionating N or preferentially using N substrates at different rates across these contrasting ecosystems. There was a strong linear correlation between δ¹⁵N of DNA and δ¹⁵N of the microbial biomass, which indicated DNA was depleted in ¹⁵N relative to the microbial biomass by approximately 3.4‰. Our results show that accurate and precise isotopic measurements of C and N in DNA extracted from the soil are feasible, and that these analyses may provide powerful tools for elucidating C and N cycling processes through soil microorganisms.     

Nitrogen isotope analysis of free glycine in soil using isotope dilution mass spectrometry

To enable the estimation of production and consumption rates of free glycine in soils through ¹⁵N isotope dilution experiments, an isotope dilution mass spectrometric method was developed. The method, which enabled high precision N isotope ratio determination of glycine in soil extracts at δ¹⁵N levels up to 4000‰ and concentrations from approximately 2 μM, is based on the following steps: (i) addition of glycine spike to the soil extract, (ii) removal of humic substances and pre-concentration of glycine using solid phase extraction, (iii) derivatization of amino acids, (iv) separation of the derivatives using gas chromatography (GC), (v) their combustion to yield sample N₂ gas, and (vi) finally the use of N isotope ratio mass spectrometry (IRMS). Judging by uncertainty budget calculations, the precision obtained (SD=0.01-0.06 at% ¹⁵N) is sufficient for detecting differences in N isotopic ratios obtained in ¹⁵N isotope dilution experiments.     

外源氮添加对土壤微生物残体积累动态的影响－基于Meta分析

微生物残体是土壤有机碳库的重要贡献者。为明确外源氮添加对土壤微生物残体积累动态的影响,本文收集整理了1980—2020年已发表的文献,共选取122组试验观测数据,利用整合分析方法（Meta-analysis）,以微生物残体标识物－氨基糖为目标组分,定量分析了不同种类和数量的外源氮添加对土壤中微生物来源细胞残体积累数量和组成比例的影响,并系统解析其主要影响因素。结果表明:外源氮添加（0 ~ 6000 kg hm?1）对微生物细胞残体的积累有显著的促进作用,并能引起土壤中真菌和细菌来源细胞残体相对比例发生明显变化。与不加氮对照相比,氮添加使土壤氨基糖总量增加27%,其中氨基葡萄糖、氨基半乳糖和胞壁酸含量分别增加22.5%、29.8%和19.0%。同时,不同种类外源氮素添加对氨基糖积累特征的影响也有所不同,表现为有机氮（如动物厩肥）比无机氮添加对氨基糖积累的促进作用更大。此外,氮添加对氨基糖的影响程度还与土壤自身的碳氮比、土地利用类型和自然降雨量等环境因子密切相关。其中是否添加碳源对微生物残体的响应有较大影响,表现为:无碳源添加会降低土壤氨基糖葡萄糖和胞壁酸对氮添加的响应,削弱了微生物残体对土壤有机质的贡献比例;而氮源同时配合碳源添加条件下,土壤氨基糖积累量显著高于单一氮源添加的处理,说明氮添加对微生物残体积累的影响存在着碳氮耦合效应。     

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.     

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.