Effects of temperature and life stage on the fatty acid composition of Collembola

Fatty acid (FA) analysis is used as a promising tool to investigate trophic interactions in soil food webs. The FA profile of neutral lipids in consumers is affected by the diet, and the occurrence and amount of certain FAs can reflect feeding strategies. We investigated the lipid composition of the Collembola Folsomia candida, Heteromurus nitidus and Protaphorura fimata with the fungus Chaetomium globosum as food source. The impact of environmental temperature and life stage was assessed, with special respect to linoleic acid (18:2ω6,9) as a marker FA for fungal feeding. In all Collembola species the ratio of C16/C18 in neutral lipid fatty acids (NLFAs) increased with decreasing temperature. In the NLFAs of F. candida and H. nitidus the Unsaturation Index and the amount of 18:2ω6,9 decreased with temperature, whereas in P. fimata effects were the opposite. The composition of phospholipid fatty acids (PLFAs) differed between species, but was little affected by temperature. The degree of unsaturation in NLFAs increased with the age of Collembola, mainly due to higher amounts of 18:2ω6,9 and a lower proportion of 18:1ω9. The biomarker linoleic acid represented over 20% of FAs in all fungal feeding Collembola. Despite considerable influence of temperature and life stage on its proportion, the amount was always higher than in individuals reared on other diets. This suggests that linoleic acid can serve as marker for fungal feeding independent of such physiological variations in Collembola.     

Collembolan trophic preferences determined using fatty acid distributions and compound-specific stable carbon isotope values

The trophic preferences of soil invertebrates such as Collembola are often determined by the analysis of gut contents, or through visual observations of the location of individuals. As an alternative approach, two species of Collembola, Folsomia candida and Proisotoma minuta, were offered a choice of the soil fungus Cladosporium cladosporioides or the bacterial feeding nematode Panagrellus redivivus; each exhibited distinct fatty acid profiles and stable carbon isotopic compositions. Over 21 days, the fatty acids i15:0, i17:0, 18:1(n-7) and 18:2(n-6) all increased in abundance in both collembolan species consistent with direct routing from the nematode dietary choice which contained a high concentration of these components. Collembolan fatty acid δ¹³C values increased by between 5.7 and 21.6‰ over 21 days reflecting those of the nematode diet. Therefore, both fatty acid profiles and δ¹³C values were consistent with a strong feeding preference of F. candida and P. minuta for the nematodes over the offered fungi. In fact, neither collembolan species consumed any detectable amount of C. cladosporioides. Comparison of the δ¹³C values of the 16:0 and 18:0 fatty acids (which are biosynthesised by the Collembola as well as directly incorporated from the diet) and the 16:1(n-7) and 18:2(n-6) components (which are not biosynthesised by the Collembola) demonstrated that the input of distinct pools of C can lead to large shifts in δ¹³C values between diet and consumer. The fatty acids that were not biosynthesised by Collembola better reflected the δ¹³C values of the diet helping to differentiate between biosynthesised and directly incorporated compounds; an important prerequisite in the interpretation of compound-specific δ¹³C values in trophic behaviour tests. The combination of fatty acid distributions and δ¹³C values is a significant improvement on traditional methods of examining feeding preferences, since it determines directly the assimilated dietary carbon rather than relying on indirect observations, such as the proximity of individuals to a defined food source.     

Translocation of surface litter carbon into soil by Collembola

Soil invertebrates are important in nutrient cycling in soils, but the degree to which mesofauna such as Collembola are responsible for the direct movement of carbon (C) from the litter layer into soil has not yet been ascertained. We used naturally occurring stable C isotopic differences between a C₄ soil and alder leaves (C₃) to examine the effect of the collembolan Folsomia candida on C translocation into soil in laboratory microcosms. Collembolan numbers greatly increased in the presence of alder, but despite large collembolan populations there were no changes in decomposition rate (measured as litter mass loss, cumulative respired CO₂ and alder C:N ratios). Small changes in the δ¹³C values of bulk soil organic matter were detected, but could not be assigned to collembolan activity. However, mean δ¹³C values of soil microbial phospholipid fatty acids (PLFAs) were significantly lower in the presence of alder and Collembola together, demonstrating that collembolan activities resulted in greater availability of litter-derived C to the soil microbial community. Additionally, the presence of Collembola resulted in the translocation of alder-derived compounds (chlorophyll and its breakdown product pheophytin) into soil, demonstrating that Collembola modify soil organic matter at the molecular level. These results are consistent with deposition of collembolan faeces in underlying soil and demonstrate that despite their small size, Collembola contribute directly to C transport in the litter-soil environment.     

Fatty acid composition and change in Collembola fed differing diets: identification of trophic biomarkers

To assess the potential of fatty acid (FA) compositions to act as biomarkers in the soil food web, two species of Collembola, Folsomia candida and Proisotoma minuta, were switched to four possible diets: Cladosporium cladosporioides (a common soil fungus), Panagrellus redivivus (a bacteria feeding nematode), Zea mays (maize) and Alnus glutinosa (alder). The change in FA content of the Collembola was observed over the following 39 days. The four diets produced significant shifts in the FA compositions of the Collembola, with P. redivivus causing the most extreme changes; Collembola fed P. redivivus gained complex FA compositions similar to those of the nematode diet. Changes in the relative abundances of some FAs were found to follow negative exponential curves, as the components either accumulated in, or were removed from, the FA pool in the Collembola; abundance half-lives varied between 0.5 and 22.4 days, indicating that Collembolan FA compositions changed readily with the input of new exogenous components. The results demonstrate that Collembolan FA compositions are influenced by diet, and that the abundances of FAs such as i15:0, i17:0 and 18:1(n-7) may be used as biomarkers of nematode consumption by Collembola. In contrast, the C₂₀ polyunsaturated FAs cannot be used as biomarkers for nematode predation as Collembola possess the ability to biosynthesise high abundances of these compounds when not provided by the diet.     

The concentrations of fatty acids in organo-mineral particle-size fractions of a Chernozem

Fatty acids, the most abundant class of soil lipids, indicate pedogenetic processes and soil management. However, their quantitative distribution in organo‐mineral particle‐size fractions is unknown. The concentrations of n‐C_10:0 to n‐C_34:0 fatty acids both in whole soil samples and in the organo‐mineral particle‐size fractions of the Ap horizon of a Chernozem were determined (i) to evaluate the effects of long‐term fertilization and (ii) to investigate their influence on the aggregation of organo‐mineral primary particles. Quantification by gas chromatography/mass spectrometry (GC/MS) showed that long‐term fertilization with nitrogen, phosphorus and potassium (NPK) and farmyard manure (FYM) led to larger concentrations (25.8 µg g^?1) of fatty acids than in the unfertilized sample (22.0 µg g^?1). For particle‐size fractions of the unfertilized soil, the fatty acid concentrations increased from the coarse silt to the clay fractions (except for fine silt). Fertilization with NPK and FYM resulted in absolute enrichments of n‐C_21:0 to n‐C_34:0 fatty acids with a maximum at n‐C_28:0 in clay (×2.2), medium silt (×2.0), coarse silt (×1.8) and sand (×2.9) compared with the unfertilized treatment (the factors of enrichment are given in parentheses). New evidence for the aggregate stabilizing function of n‐C_21:0 to n‐C_34:0 fatty acids was shown by the characteristic pattern in size‐fractionated, disaggregated and aggregated samples. Highly significant correlations of fatty acid concentrations with organic C concentrations and specific surface areas are interpreted as indicators of (i) trapping of fatty acids in organic matter macromolecules and (ii) direct bonding to mineral surfaces. This interpretation was supported by the thermal volatilization and determination of fatty acids by pyrolysis‐field ionization mass spectrometry (Py‐FIMS).     

Tracking Collembola feeding strategies by the natural C signal of fatty acids in an arable soil with different fertilizer regimes

Compound specific stable isotope analysis (¹³C/¹²C ratio of fatty acids) was used to assess the allocation of plant carbon in soil microbiota, and to identify the trophic links to microbial grazers in an arable field with long-term mineral and organic fertilizer amendments. The feeding strategy of two dominant Collembola species, epedaphic Isotoma viridis and euedaphic Willemia anophthalma was determined. The investigation was conducted following a shift to amaranth, a C₄ plant, after 27 years of continuous C₃ crop rotation. The influence of new C₄ plant carbon was observed in microbial phospholipids (PLFAs) with higher δ¹³C recorded in C₄ amaranth than in C₃ clover soils. The strongest enrichment occurred in the fungal PLFA 18:2ω6,9c and bacterial PLFA 18:1ω9t with 11.2‰ and 6.6‰, respectively. However, other bacterial PLFAs showed no isotopic change, suggesting that the microbial community simultaneously utilized “new” and “old” plant carbon. The δ¹³C of Collembola fatty acids displayed species specific lipid pattern, which was affected by crop type, but not fertilizer amendments. Isotopic separation of Collembola lipids from amaranth and clover plots was more distinct in I. viridis than W. anophthalma. With up to 18‰, the enrichment in Collembola lipids was stronger than in microbial PLFAs, pointing to a distinct incorporation of carbon resources originating from the actual plant residues. The δ¹³C pattern in I. viridis indicated trophic links with bacteria, saprotrophic fungi and plant tissues, while saprotrophic fungi and plant tissues were accountable for the patterns observed in W. anophthalma.     

Trophic shift of stable isotopes and fatty acids in Collembola on bacterial diets

Fatty acid (FA) analysis is a promising tool to study trophic relationships in soil food webs. We determined FA biomarkers to trace bacterial food sources (Bacillus megaterium, Pseudomonas putida, Enterobacter aerogenes) of Collembola (Heteromurus nitidus, Protaphorura fimata, Folsomia candida). In addition, δ¹⁵N, δ¹³C, C/N ratio, body weight and NLFA/PLFA ratio (neutral lipid/phospholipid fatty acids) of Collembola were assessed. These measures indicated that P. putida ranked first, B. megaterium second and E. aerogenes third in food quality. FAs specific for bacteria were found in the NLFAs of the Collembola reflecting the respective bacterial diet. Biomarker FAs for gram-positive bacteria were methyl branched i14:0, i15:0, a15:0 and i17:0. Consumption of gram-negative bacteria was reflected by the cyclic form cy17:0 (E. aerogenes, P. putida) and by 16:1ω5 (P. putida).     

The fat that matters: Soil food web analysis using fatty acids and their carbon stable isotope signature

Chemical taxonomy based upon the composition of lipids is widely applied to investigate microbial communities and fatty acids have recently been employed to connect soil microbial and faunal food webs as well as to elucidate functional groups at higher trophic levels. The additional use of compound-specific isotopic analysis of ¹³C/¹²C ratios in fatty acids allows assessing specific trophic links and belowground carbon fluxes. In this review systematic patterns and processes underlying variations in the composition of fatty acids and their ¹³C/¹²C ratio are described. The emphasis is on biomarker fatty acids, their incorporation and modification, effects of pool size, and analytical methods. Further development of the application of fatty acid profiling to soil ecology should include both advances in experimental research and growth of theory. Accordingly, areas in which future experimentation can lead to progress in soil food web analysis are identified. Overall, combining fatty acid biomarker and their isotopic ratios will allow detailed insight into belowground trophic interactions.     

Fungal-feeding habits of six nematode isolates in the genus Filenchus

The family Tylenchidae is a large group of soil nematodes but their feeding habits are not fully known. We studied the fungal-feeding abilities of nematodes in the genus Filenchus. We measured population growth rates (PGRs) of six nematode isolates, representing three Filenchus species, when feeding on seven fungal species on two types of culture media. On Potato Dextrose Agar (PDA) Filenchus misellus, Filenchus discrepans and an unidentified Filenchus sp. generally showed moderate to large PGRs on saprophytic fungi (Rhizoctonia solani, Chaetomium globosum, Coprinus cinereus, Flammulina velutipes) and low PGRs on plant-pathogenic fungi (Fusarium oxysporum, Pythium ultimum). In soil medium amended with chopped soybean plant material or wheat bran, the status of most of the fungi as food for the nematodes was similar to that on PDA, although PGRs tended to be lower in the soil medium. However, C. globosum, a good food on PDA, only supported low PGR in soil for each of the three nematodes. The PGRs of F. misellus on C. globosum in soil were still low even when types and amounts of organic matter amendments were varied. A nematophagous fungus, Pleurotus ostreatus (oyster mushroom), was determined to be a food for Filenchus on PDA or in soil, based on PGR measurements corrected for extraction efficiency. To determine whether fungal species and culture media affected nematode extraction efficiencies and, consequently, the apparent PGRs, we compared efficiencies between R. solani, C. globosum and C. cinereus, and between PDA and soil. The relatively low extraction efficiencies across fungal species in soil seemed responsible for the lower nematode PGRs in soil than on PDA. On PDA generally, fungal species did not affect the assessment. In soil, effects of fungal species on extraction were significant, but not consistent, across nematode species. Nevertheless, the extraction efficiency differences in soil were considered not to affect assessment of the three fungi as food for the nematodes. The confirmation that three Filenchus species reproduce by feeding on fungi in soil suggests that fungal-feeding is not an unusual habit in the field, in this genus. We believe that in community studies, nematodes in the genus Filenchus should be considered fungal feeders or root and fungal feeders, rather than only plant feeders. Our confirmation of fungal-feeding habits in the genus Filenchus supports the hypotheses that plant-feeding nematodes evolved from those feeding on fungi.     

13C NMR spectra of soil organic fractions extracted with organic solvents

Soil organic fractions extracted in sequence with ethyl ether, acetone, benzene and dioxane have been investigated with ¹³C NMR spectroscopy. The spectra of ethyl ether and acetone fractions are identical and show the presence of signals assigned to normal long chain (C_23±2) fatty acids. A very similar spectral pattern is displayed by the benzene fraction which appears to consist of a mixture (50 ± 10)% of normal fatty acids and normal alkanes (C_21±3). A comparison with data in the literature indicates that the well developed signals at 14, 23, 30 (very intense) and 32 ppm are a common feature of the lipid fraction extracted from different soils.A completely different spectrum has been obtained from the dioxane fraction. Most signals appearing in the range 55–75 ppm can be attributed to oxygen bonded sp³ carbon atoms. The lack of aromatic signals seems to exclude the possibility that this fraction was derived from lignin residues.The comparison with ¹H NMR spectra and the occurrence of distinct and sharp signals indicate that ¹³C NMR is a valuable tool in the study of soil organic fractions extracted with organic solvents.     

Contrasting effects of nitrogen limitation and amino acid imbalance on carbon and nitrogen turnover in three species of Collembola

Soil animal detritivores play an important role in facilitating decomposition processes but little information is available on how the quality of dietary resources affects their stoichiometry of carbon (C) nitrogen (N) and phosphorus (P), and turnover of C and N. This study investigated how a fungal diet, Fusarium culmorum, with a low N content and imbalanced amino acid (AA) composition affected the physiology of three soil-dwelling collembolans (Folsomia candida, Protaphorura fimata and Proisotoma minuta) in comparison to a control diet, Saccharomyces cerevisiae, with a high N content and balanced AA composition. We compared the elemental composition of animals, their growth rates and tissue replacement of C and N. We also measured the individual AA δ¹³C to investigate the extent that Collembola may rely on endogenous sources to compensate for scarcity of essential AAs. The results showed that animal's N content tracked closely the composition of their diets, decreasing from around 10 to 7% N from the high to low N diet. They also had a significant increase of C and a decrease of P. P. fimata was less affected than F. candida and P. minuta. The total incorporation of C and N in the animals due to growth and tissue replacement decreased from 11-17 to 6-12% DM d⁻¹ on the high and low N diet respectively with P. fimata experiencing the smallest change. Essential AAs δ¹³C did not always match perfectly between Collembola species and their diets; particularly on the low N diet. Isotope patterns of AAs indicate that bacteria may have been the alternative source of essential AAs. While the results of this study cannot be extrapolated directly to the dynamics of Collembola populations in the field, they serve to demonstrate their flexibility in adapting physiologically to the temporal and spatial patchiness of the soil environment.     

Fate of fatty acids derived from biogas residues in arable soil

Biogas residues are rich in microbial biomass and contribute to organic matter formation when applied to soils. Here we present a detailed analysis of the fatty acids derived from ¹³C-labelled biogas residues applied to arable soil and incubated for 378 days. We applied a differential approach using phospholipid fatty acids and total fatty acids to evaluate the carbon dynamics in living biomass and non-living soil organic matter. Biogas residue addition increased the microbial biomass in soil. The sum of ¹³C-labelled phospholipid fatty acids decreased to ∼60% during incubation whereas the decrease of t-FA was higher (to 33%). Compound-specific fatty acid analysis showed fatty acid specific incorporation or loss of ¹³C, indicating hints for the carbon flow within the microbial food web. Overall, microbial biomass in biogas residues may be a significant contributor to soil organic matter formation.     

New trophic biomarkers for Collembola reared on algal diets

We used fatty acid (FA) analysis to investigate green algae and cyanobacteria as food sources for Collembola. We studied the effects of food quality on body mass and on neutral lipid (NLFA) and phospholipid (PLFA) fatty acid patterns of Collembola. Folsomia candida, Heteromurus nitidus and Protaphorura fimata were fed with common green algae (Chlorella vulgaris), filamentous soil algae (Klebsormidium flaccidum), cyanobacteria (Nostoc commune) and baker's yeast (Saccharomyces cerevisiae). Body mass of F. candida and H. nitidus was highest when reared on C. vulgaris and S. cerevisiae. P. fimata gained the most weight when fed baker's yeast. K. flaccidum and N. commune as resources resulted to low biomass in all Collembola. The four diets caused significant differences in the NLFA and PLFA composition of Collembola after six weeks of feeding. Two new trophic biomarker FAs indicating algal diets were assigned with 16:3ω3,6,9 and 16:2ω6,9, which were only present in NLFAs of Collembola consuming C. vulgaris and K. flaccidum. The amount of FAs from the ω7 family was high in Collembola lipids with cyanobacteria and yeast as food sources, whereas only trace amounts occurred in the NLFA fraction with algae as the resource. In summary, common soil algae and cyanobacteria differed in food quality for Collembola, depending on their growth form (unicellular versus filamentous) and/or secondary metabolites (e.g. cyanobacteria). The new FA biomarkers detected will allow further investigation of these trophic interactions under field conditions; for example, assessing the role of collembolan grazers in the formation of biological soil crusts.     

Dynamics of soil microbial populations involved in 2,4-D biodegradation revealed by FAME-based Stable Isotope Probing

Stable Isotope Probing (SIP) is a powerful tool for analysing the fate of pesticides in soil. Together with FAME (Fatty Acid Methyl Esters), it can help identify biodegradation pathways and recycling into the microbial biomass. The fate of ring-labelled ¹³C-2,4-dichlorophenoxyacetic acid or 2,4-D (C_2,4-D) was determined in soil during a 6-month incubation. The distribution of ¹³C among the microbial biomass, the CO₂ respired, the water, methanol and dichloromethane soluble fractions, and the residual non-extracted bulk soil was measured. Molecular analyses were carried out on the lipid and the non-extractable fractions. After 8 days, about half of the initial amount of C_2,4-D was mineralised; the other half remained in soil as non-extractable residues (NER). C_2,4-D continued to be mineralised, suggesting that NER were still bioavailable. Analysis of C_2,4-D-enriched FAME contained in the lipid fraction suggested that a succession of microbial populations was involved in 2,4-D biodegradation. This is possibly due to the change of 2,4-D availability. The C_2,4-D yield coefficient and degrader diversity evolved during the incubation, providing corroboratory evidence that different physiological groups were active during the incubation. The ¹³C-labelled microbial community was always less diverse than the total community, even at the end of the incubation, suggesting that the cross-feeding community is also a specific part of the total community. This work shows that molecular analysis of ¹³C-labelled pesticides is a useful tool for understanding both chemical and biological aspects of their fate in soil.     

Fatty acid patterns as biomarker for trophic interactions: Changes after dietary switch and starvation

Fatty acid (FA) analysis is becoming increasingly important for investigating trophic interactions in soil food webs. FA profiles of neutral lipids are affected by diet, and the occurrence and amount of certain FAs can reflect feeding strategies. However, to draw conclusions on feeding strategies in the field it is necessary to know physiological parameters of fatty acid metabolism such as the detection time and storage period of FAs. In this study we investigated the chronological change of FA biomarkers in the Collembola Heteromurus nitidus when switched between different food sources: leaves (Tilia europaea), a fungus (Chaetomium globosum) and two bacteria (Stenotrophomonas maltophilia, Bacillus amyloliquefaciens). Additionally, we followed the change of bacterial FA biomarkers during starvation. After 14 days of food deprivation bacterial FAs were still detectable in a sufficient amount to use them as dietary markers. Switching diet experiments demonstrated that FAs typical for a specific diet are already present after one day and are still detectable after 14 days of feeding on a different food source, suggesting that FA analysis can integrate the food choice of Collembola over a longer period of time, in contrast to snapshot methods such as gut content analysis.     

Carbon allocation in mycelia of arbuscular mycorrhizal fungi during colonisation of plant seedlings

The direction of carbon (C) allocation in mycorrhizal mycelia is of fundamental importance to coexistence of individual plants. We therefore investigated the transfer of C from established plants to plant seedlings through fungal mycelia. C allocation by the arbuscular mycorrhizal (AM) fungus Glomus intraradices, from ‘donor’ plants to mycelia in soil and two different species of introduced ‘receiver’ seedlings, was investigated in a pot experiment using ¹³C labelling and fatty acid analysis. After ¹³CO₂ application to the shoots of Trifolium subterraneum or Plantago lanceolata, used as donor plants, T. subterraneum and P. lanceolata receiver seedlings were introduced. Samples were collected 4-20 days after ¹³CO₂ application and analysed regarding ¹³C and the fatty acid 16:1ω5, the signature of AM fungi. ¹³C transfer from T. subterraneum donor plants was demonstrated by ¹³C enrichment of the roots of the receiver seedlings, but not from the P. lanceolata donor plants. ¹³C allocation to the neutral lipid fatty acid 16:1ω5 was only 1 ng in each receiver seedling, but 2 μg of the fatty acid in whole soil. The results indicate that C allocation through mycelial networks is influenced by the donor plant species, but is not directed towards receiver seedlings to any higher degree than towards other directions. The importance of the extraradical AM fungal mycelium as a C sink was demonstrated.     

Contribution of primary organic matter to the fatty acid pool in agricultural soils

Fatty acids as major compounds of soil lipids may affect many soil properties, but the input and turnover rates in soil are largely unknown. The objective of this study was to identify and quantify fatty acids in soils as a result of input from primary sources such as plant residues, farmyard manure and soil organisms, and to evaluate the corresponding turnover- and stabilization processes. The concentrations of n-C_10:0 to n-C_34:0 fatty acids were determined in the Ap horizon of a Phaeozem with long-term cropping of rye and maize and the treatments ‘Unfertilized’ (‘U’) and fertilized with ‘Farmyard manure’ (‘FYM’). The most important primary sources of fatty acids such as rye and maize stubble and roots, soil micro- and mesofauna, and the applied FYM were also investigated. The quantification of fatty acids by gas chromatography/mass spectrometry (GC/MS) showed that long-term FYM application led to larger concentrations of n-alkyl fatty acids in the plots grown with rye (‘U’: 48.1 μg g⁻¹, ‘FYM’: 57.7 μg g⁻¹, **P≤0.01, n=3) and maize (‘U’: 17.0 μg g⁻¹, ‘FYM’: 23.4 μg g⁻¹, ***P≤0.001, n=3). The observed bimodal fatty acid distribution in soils from n-C_10:0 to n-C_21:0 and from n-C_21:0 to n-C_34:0 with a predominance at n-C_16:0 and at n-C_28:0 was apparently due to input from crop residues, soil organisms and FYM. The short-chain lengths may have originated from the investigated primary sources. The major contributors to the long-chain lengths, with a maximum at n-C_28:0, were rye stubble and FYM. A change in mono-culture from rye to maize, 38 years prior to sampling, led to a decrease in fatty acid concentrations by factors of about 2.8 (‘U’) and 2.5 (‘FYM’). Therefore, rye-derived fatty acids and soil tillage had a larger impact on fatty acid pools than the input of primary organic matter. The changes in fatty acid distributions and pools under the consideration of the quantified input of primary organic matter led to the conclusion that the short-chained fatty acids were more rapidly decomposed than the long-chains.     

Streptomyces sp: A source of odorous substances in potable water

Growth medium containing an aquatic actinomycete Streptomyces sp. was analyzed by gas-liquid chromatography and mass spectrometry for the purpose of identifying substances that may represent potential odorous contaminants of water. Twenty-one odorous and nonodorous compounds were identified. Phenyl, C₂, and C₄ structures were predominant. Free fatty acids from C₁₃ to C₁₆ were detected and in some cases accompanied by their ethyl (C₁₆) and butyl (C₁₅ and C₁₆) esters. The long-chain acids and esters had no detectable odor. Although goesmin, an earthy smelling substance with a low odor threshold, was detected, butyric acid was the principal metabolite under the growth conditions employed and represented the greatest potential as an odorous water pollutant under natural conditions.     

Turnover of soil organic matter and of microbial biomass under C3-C4 vegetation change: Consideration of C fractionation and preferential substrate utilization

Two processes contribute to changes of the δ¹³C signature in soil pools: ¹³C fractionation per se and preferential microbial utilization of various substrates with different δ¹³C signature. These two processes were disentangled by simultaneously tracking δ¹³C in three pools - soil organic matter (SOM), microbial biomass, dissolved organic carbon (DOC) - and in CO₂ efflux during incubation of 1) soil after C₃-C₄ vegetation change, and 2) the reference C₃ soil.The study was done on the Ap horizon of a loamy Gleyic Cambisol developed under C₃ vegetation. Miscanthus giganteus - a perennial C₄ plant - was grown for 12 years, and the δ¹³C signature was used to distinguish between ‘old’ SOM (>12 years) and ‘recent’ Miscanthus-derived C (<12 years). The differences in δ¹³C signature of the three C pools and of CO₂ in the reference C₃ soil were less than 1‰, and only δ¹³C of microbial biomass was significantly different compared to other pools. Nontheless, the neglecting of isotopic fractionation can cause up to 10% of errors in calculations. In contrast to the reference soil, the δ¹³C of all pools in the soil after C₃-C₄ vegetation change was significantly different. Old C contributed only 20% to the microbial biomass but 60% to CO₂. This indicates that most of the old C was decomposed by microorganisms catabolically, without being utilized for growth. Based on δ¹³C changes in DOC, CO₂ and microbial biomass during 54 days of incubation in Miscanthus and reference soils, we concluded that the main process contributing to changes of the δ¹³C signature in soil pools was preferential utilization of recent versus old C (causing an up to 9.1‰ shift in δ¹³C values) and not ¹³C fractionation per se.Based on the δ¹³C changes in SOM, we showed that the estimated turnover time of old SOM increased by two years per year in 9 years after the vegetation change. The relative increase in the turnover rate of recent microbial C was 3 times faster than that of old C indicating preferential utilization of available recent C versus the old C.Combining long-term field observations with soil incubation reveals that the turnover time of C in microbial biomass was 200 times faster than in total SOM. Our study clearly showed that estimating the residence time of easily degradable microbial compounds and biomarkers should be done at time scales reflecting microbial turnover times (days) and not those of bulk SOM turnover (years and decades). This is necessary because the absence of C reutilization is a prerequisite for correct estimation of SOM turnover. We conclude that comparing the δ¹³C signature of linked pools helps calculate the relative turnover of old and recent pools.     

Quantification of long‐chain fatty acids in dissolved organic matter and soils

The objective was to develop and adapt a versatile analytical method for the quantification of solvent extractable, saturated long‐chain fatty acids in aquatic and terrestrial environments. Fulvic (FA) and humic (HA) acids, dissolved organic matter (DOM) in water, as well as organic matter in whole soils (SOM) of different horizons were investigated. The proposed methodology comprised extraction by dichloromethane/acetone and derivatization with tetramethylammonium hydroxide (TMAH) followed by gas chromatography/mass spectrometry (GC/MS) and library searches. The C_10:0 to C_34:0 methyl esters of n‐alkyl fatty acids were used as external standards for calibration. The total concentrations of C_14:0 to C_28:0 n‐alkyl fatty acids were determined in DOM obtained by reverse‐osmosis of Suwannee river water (309.3 μg g^—1), in freeze‐dried brown lake water (180.6 μg g^—1), its DOM concentrate (93.0 μg g^—1), humic acid (43.1 μg g^—1), and fulvic acid (42.5 μg g^—1). The concentrations of the methylated fatty acids (n‐C_16:0 to n‐C_28:0) were significantly (r² = 0.9999) correlated with the proportions of marker signals (% total ion intensity (TII), m/z 256 to m/z 508) in the corresponding pyrolysis‐field ionization (FI) mass spectra. The concentrations of terrestrial C_10:0 to C_34:0 n‐alkyl fatty acids from four soil samples ranged from 0.02 μg g^—1 to 11 μg g^—1. The total concentrations of the extractable fatty acids were quantified from a Podzol Bh horizon (26.2 μg g^—1), Phaeozem Ap unfertilized (48.1 μg g^—1), Phaeozem Ap fertilized (57.7 μg g^—1), and Gleysol Ap (66.7 μg g^—1). Our results demonstrate that the method is well suited to investigate the role of long‐chain fatty acids in humic fractions, whole soils and their particle‐size fractions and can be serve for the differentiation of plant growth and soil management.