Soil Fatty Acid Research Articles

Determination of Free Fatty Acids in Soil and Bryophyte Plants by Precolumn Derivatization via HPLC with Fluorescence Detection and Tandem Mass Spectrometry (HPLC‐MS/MS)

A sensitive method for the determination of 30 kinds of free fatty acids (FFAs, C1‐C30) with 1‐[2‐(p‐toluenesulfonate)‐ethyl]‐2‐phenylimidazole‐[4,5‐f]‐9,10‐phenan‐ threne (TSPP) as labeling reagent and using high performance liquid chromatography with fluorescence detection and identification by online postcolumn mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive‐ion mode (HPLC/MS/APCI) has been developed. TSPP could easily and quickly label FFAs in the presence of K2CO3 catalyst at 90°C for 30 min in N,N‐dimethylformamide (DMF) solvent, and maximal labeling yields close to 100% were observed with a 5‐fold excess of molar reagent. Derivatives were stable enough to be efficiently analyzed by high performance liquid chromatography. TSPP was introduced into fatty acid molecules and effectively augmented MS ionization of fatty acid derivatives and led to regular MS and MS/MS information. The collision induced cleavage of protonated molecular ions formed specific fragment ions at m/z [MH]+(molecular ion), m/z [M′+CH2CH2]+(M′ was molecular mass of the corresponding FFA) and m/z 295.0 (the mass of protonated molecular core structure of TSPP). Fatty acid derivatives were separated on a reversed‐phase Eclipse XDB‐C8 column (4.6×150 mm, 5 µm, Agilent) with a good baseline resolution in combination with a gradient elution. Linear ranges of 30 FFAs are 2.441×10−3 to 20 µmol/L, detection limits are 3.24∼36.97 fmol (injection volume 10 µL, at a signal‐to‐noise ratio of 3, S/N 3:1). The mean interday precision ranged from 93.4 to 106.2% with the largest mean coefficients of variation (R.S.D.)<7.5%. The mean intraday precision for all standards was <6.4% of the expected concentration. Excellent linear responses were observed with correlation coefficients of >0.9991. Good compositional data could be obtained from the analysis of extracted fatty acids from as little as 200 mg of bryophyte plant samples. Therefore, the facile TSPP derivatization coupled with HPLC/MS/APCI analysis allowed the development of a highly sensitive method for the quantitation of trace levels of short and long chain fatty acids from biological and natural environmental samples.

Radiocarbon content and stable carbon isotopic ratios of individual fatty acids in subsurface soil: Implication for selective microbial degradation and modification of soil organic matter

A subsurface soil sample was studied for molecular composition and stable carbon isotopic ratios (δ13C) of fatty acids (FAs) using a capillary gas chromatography (GC) and GC/combustion/isotope ratio mass spectrometer, respectively. Compound specific radiocarbon analyses of FAs were also conducted using an off-line preparative capillary GC/accelerator mass spectrometer. Molecular distribution of FAs (C14-C32) is characterized by even carbon numbered predominance with two maxima at n-C16 and n-C28, being similar to that of plant leaf. However, branched chain C15 and C17 acids were abundant in soil, indicating bacterial degradation and modification of soil organic carbon. The δ13C of FAs (-35.2 to -23.0‰) are within the range of plant leaf δ13C (-36.4 to -31.2‰), except for heavy values (-23.0 to -28.1‰) of C14 to C18. The heavier δ13C values are most likely interpreted by microbial re-synthesis of shorter chain FAs in soil. We also report, for the first time, a significant diversity of Δ14C values (+17 to +127‰) in soil FAs, with higher values (+96 to +127‰) for saturated and unsaturated C18 and lower values (+16 to +19‰) for longer chain C28 and C30. The higher radiocarbon values can be explained by a combination of selective microbial decomposition of shorter chain FAs in subsurface soil, and the subsequent deposition and mixing of modern carbon with old carbon in soil. The modern carbon may be transported downward in the form of shorter chain FAs and other water-soluble organic compounds by meteoric water and/or tree roots, followed by microbial re-synthesis of lipids in soil. This study implies that microbial degradation and modification of soil organic matter play an important role in geochemical processes which control the carbon cycle on the Earth.

Determination of Free Fatty Acids from Soil and Bryophyte by HPLC with Fluorescence Detection and Identification with Mass Spectrometry

A simple and sensitive method for the determination of free fatty acids using acridone-9-ethyl- p-toluenesulfonate (AETS) as tagging reagent by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed. Studies on derivatization conditions indicate that free fatty acids react rapidly and smoothly with AETS in the presence of K 2CO 3 catalyst in N, N-dimethylformamide (DMF) solvent at 85°C to give the corresponding sensitive fluorescence derivatives. The maximal derivatization yields are close to 100% with a six fold molar reagent excess. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Separation of the derivatized fatty acids exhibits a good baseline resolution in combination with a gradient elution on a reversed-phase Eclipse XDB-C 8 column. The identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode. The contents of 19 free fatty acids in soil and three kinds of bryophytes ( Tree Moss, Entodon macropodus, Dicranum caesium) were determined. The results indicate that the bryophyte plants enrich an abundance of free fatty acids from soil. The fluorescence excitation and emission wavelengths of derivatives were 404nm of λ ex and 440 nm of λ em, respectively. Excellent linear responses were observed with the linear correlation coefficients over 0.9996, and detection limits (at signal-to-noise of 3:1) are 12.3–43.7 fmol. The proposed method is sensitive and reproducible for the determination of free fatty acids from real samples with satisfactory results.

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.

Impact of coloniser plant species on the development of decomposer microbial communities following deglaciation

The effects of coloniser plant species on microbial community growth and composition were investigated on recently deglaciated terrain at Glacier Bay, south-east Alaska. Analysis of microbial communities using phospholipids fatty acid analysis (PLFA) revealed that Alnus and Rhacomitrium had the greatest impact on microbial growth, increasing total PLFA by some 6–7 fold relative to bare soil, whereas Equisetum led to a 5.5 fold increase in total PLFA relative to bare soil. These coloniser species also had significant effects on the composition of their associated microbial communities. Rhacomitrium, Alnus, and Equisetum increased bacterial PLFA, a measure of bacterial biomass, relative to bare soil. Rhacomitrium and Alnus also dramatically increased the concentration of the fungal fatty 18:2ω6 in soil relative to bare soil, by 12-fold and 8-fold, respectively. The net effect of the above changes was a significant increase in the ratio of fungal: bacterial fatty acids in soil associated with Alnus and Rhacomitrium, but not Equisetum. Possible reasons for these effects of particular plants on microbial communities are discussed, as is their significance in relation to the development of microbial communities in relatively sterile, recently deglaciated ground.

Characterization of soil microbial communities under different potato cropping systems by microbial population dynamics, substrate utilization, and fatty acid profiles

The effects of 11 different 2- and 3-yr potato crop rotations on soil microbial communities were characterized over three field seasons using several techniques. Assessments included microbial populations determined by soil dilution plate counts on various general and selective culture media, microbial activity by fluorescein diacetate (FDA) hydrolysis, single carbon source substrate utilization (SU) profiles, and fatty acid methyl ester (FAME) profiles. Potato rotation crops evaluated in research plots at Newport, ME, included barley/clover, canola, green bean, millet, soybean, sweet corn, and a continuous potato control. Soil populations of culturable bacteria and overall microbial activity tended to be highest following barley, canola, and sweet corn rotations, and lowest with continuous potato. Differences among rotations were less apparent during the potato phase of the rotations. Populations of actinomycetes and fluorescent pseudomonads tended to be greater in barley rotations than in most other rotations. SU profiles derived from BIOLOG GN2 plates indicated that certain rotations, including barley, canola, and sweet corn tended to have higher overall microbial activity, and barley and sweet corn rotations averaged higher substrate richness and diversity. Soybean and potato rotations tended to have lower substrate richness and diversity. Principal component analyses of SU data revealed differences among rotation soil communities in their utilization of individual carbon sources and substrate guilds, including carbohydrates, carboxylic acids, amines/amides, and amino acids. Analyses of soil FAME profiles demonstrated distinct differences among all the rotation soils in their relative composition of fatty acids, indicating differences in their microbial community structure. Fatty acids most responsible for differentiation among rotation soils included 16:1 ω5c, 16:1 ω7c, 18:2 ω6c, 18:1 ω9c, 12:0, and 13:0 anteiso, with 16:1 ω5c being the single greatest determinant. Overall, monounsaturated fatty acids, particularly 16:1 ω5c, were most prevalent in sweet corn rotations and polyunsaturates were highest in barley and millet rotations. Straight chain saturated fatty acids comprised the greatest proportion of fatty acids in soils under continuous potato. FAME biomarkers for microorganism groups indicated barley and millet rotations had the highest ratio of fungi to bacteria, and soybean and continuous potato had the lowest ratio. This research has demonstrated that different crop rotations have distinctive effects on soil microbial communities that are detectable using a variety of techniques. Further studies will identify more specific changes associated with particular rotations and relate these changes to potential effects on disease management, crop health, and crop productivity.

Spore-forming, Desulfosporosinus-like sulphate-reducing bacteria from a shallow aquifer contaminated with gasoline.

Previous studies on the geochemistry of a shallow unconfined aquifer contaminated with hydrocarbons suggested that the degradation of some hydrocarbons was linked to bacterial sulphate reduction. There was attenuation of naphthalene, 1,3,5-trimethylbenzene (TMB), toluene, p-xylene and ethylbenzene in the groundwater with concomitant loss of sulphate. Here, the recovery of eight strains of sulphate-reducing bacteria (SRB) from the contaminated site is reported. All were straight or curved rod-shaped cells which formed endospores. Amplification and sequencing of the 16S rDNA indicated that the strains were all sulphate reducers of the Gram-positive line of descent, and were most closely related to Desulfosporosinus (previously Desulfotomaculum) orientis DSM 8344 (97-98.9% sequence similarity). The strains clustered in three phylogenetic groups based on 16S rRNA sequences. Whole cell fatty acid compositions were similar to those of D. orientis DSM 8344, and were consistent with previous studies of fatty acids in soil and groundwater from the site. Microcosms containing groundwater from this aquifer indicated a role for sulphate reduction in the degradation of [ring-UL-14C]toluene, but not for the degradation of [UL-14C]benzene which could also be degraded by the microcosms. Adding one of the strains that was isolated from the groundwater (strain T2) to sulphate-enriched microcosms increased the rate of toluene degradation four- to 10-fold but had no effect on the rate of benzene degradation. The addition of molybdate, an inhibitor of sulphate reduction, to the groundwater samples decreased the rate of toluene mineralization. There was no evidence to support the mineralization of [UL-14C]benzene, [ring-UL-14C]toluene or unlabelled m-xylene, p-xylene, ethylbenzene, TMB or naphthalene by any of the strains in pure culture. Growth of all the strains was completely inhibited by 100 micromol l-1 TMB.

Relationship between N immobilization and volatile fatty acids in soil after application of pig and cattle slurry

A laboratory study was performed to determine decomposition of fatty acids and mineralization of C and N from slurries in soil. Fatty acids present in slurries decomposed within 1–2 days at 25°C in soil. Parallel to the fatty acid decomposition, immobilization of N was measured in soil. The correlation between the initial fatty acid concentrations in the slurries and the amounts of N immobilized were found to be highly significant (R 2=0.97). It was concluded that fatty acids act as an easily decomposable C source for microorganisms and cause immobilization of N. Immobilization of N was followed by a curvilinear mineralization of N in all slurrytreated soils. Despite mineralization, only fresh pig slurry and anaerobically digested pig slurry showed a net release of N over 70 days whereas cattle slurry and anaerobically fermented pig slurry did not. The percentage of slurry C evolved during 70 days was fresh pig slurry, 65%; anaerobically fermented pig slurry, 48%; anaerobically digested pig slurry, 45%; and anaerobically fermented cattle slurry, 42%.

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...

Occurrence of Curtobacterium sp. possessing ?-cyclohexyl fatty acids in soil with zinc added

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed ω-cyclohexyl fatty acids. ω-Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of ω-cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.

Determination of soluble carbohydrates, lactic acid and volatile fatty acids in soils and biological media

Methods for extraction, concentration and determination of minute quantities of soluble carbohydrates, lactic acid and volatile fatty acids have been described. Different factors affecting the accuracy of the determinations have been studied and corrections, where necessary, have been suggested.