Mid Sweden University

Low molecular mass organic acids (LMMOAs) and hydroxamate siderophores (HS) are molecules secreted by microbes and have previously been found in soil solution and in cultures. Mycorrhizal fungi are suggested to be involved in the nutrient uptake processes of trees and weathering of minerals. In this study soil samples taken from the O and E horizons of a podzol were extracted with 10 mM potassium phosphate buffer at pH 7.2. Variable parameters included addition of methanol to the extraction buffer and the use of ultrasonication or rotary shaking during extraction. LMMOAs and HS content of the soil extracts were determined. Analysis of soil extracts were carried out by liquid chromatography mass spectrometry (LC-MS) and the extraction results compared to results for soil solution samples obtained by centrifugation of the soils sampled. The extraction yields were significantly increased by addition of methanol to the extraction buffer, especially for the O horizon samples. Rotary shaking of the samples for 90 min gave slightly higher yields than ultrasonication for 15 min but the reduction in extraction time makes ultrasonication an attractive option. Of the HSs determined, ferricrocin was found in all samples. Optimal extraction conditions showed citric acid and isocitric acid to be the most abundant organic acids in the O and E horizons, respectively.

A sensitive and selective method for the analysis of aliphatic low molecular mass organic acids (LMMOAs) in natural waters is presented. The method is based on separation with ion exclusion chromatography and detection with electrospray ionization tandem mass spectrometry (LC-MS/MS). The extra selectivity gained by applying MS/MS allows for a minimum of sample preparation and the use of a sub-optimal mobile phase regarding chromatographic resolution. Instead the mobile phase, comprising aqueous formic acid With methanol as organic modifier, was mainly optimized for maximum sensitivity and long term MS stability. Detection limits for malonic, fumaric, maleic, succinic, citraconic, glutaric, malic, alpha-ketoglutaric, tartaric, shikimic, trans-aconitic, cis-aconitic, isocitric and citric acid were in the range 1-50 nM, while the detection limits for pyruvic, oxalic and lactic acid were around 250 nM for an injection volume of 100 mu L. Due to their metal-chelating properties, these LMMOAs are all considered to affect the bioavailability of metals and to be involved in soil forming processes. It is thus of interest to be able to monitor their presence in natural waters, and the method developed within this work was successfully applied for the analysis of LMMOAs in soil solution and stream water samples.

Siderophores are low molecular mass organic compounds with high iron(III) stability constants. They are produced by many microorganisms, often in combination with low molecular mass organic acids (LMMOAs). Both siderophores and LMMOAs accelerate mineral weathering, thereby enhancing mineral nutrient availability. Siderophore concentrations in natural environments, however, are largely unknown. In the present work, analytical methods based on liquid chromatography (LC) electrospray ionization mass spectrometry (ESI‐MS) were developed and used for concentration determination of selected hydroxamate siderophores and LMMOAs. The use of capillary LC with on‐line pre‐concentration via column switching enabled siderophore quantification with detection limits of 0.1 ‐ 5 nM. Through solid phase extraction even lower detection limits were achieved. In Swedish podzol soil solutions concentrations up to 12 nM ferricrocin and 2 nM ferrichrome were found in the upper soil layes. Additionally, in axenic pine (Pinus sylvestris) seedling cultures, the ectomycorrhizal fungus Hebeloma crustuliniforme was shown to produce ferricrocin as the main siderophore. Moreover, ferricrocin was found in enriched Baltic Sea cyanobacterial populations at concentrations up to 1 nM, while concentrations up to 0.05 nM ferrioxamine B were determined in Baltic Sea surface waters during cyanobacterial blooms. Siderophores have strong affinities for some actinides and may affect radionuclide mobilization in deep subsurface nuclear wase repositories. Two subsurface bacterial strains were shown to produce siderophores under aerobic conditions, i.e. Pseudomonas stutzeri produced mainly ferrioxamine E and Pseudomonas fluorescens produced one pyoverdin siderophore. Siderophores produced by aerobically grown P. stutzeri were not observed either in anaerobic cultures or in deep subsurface water samples.

Accurate estimates of mycelial exudation in time and space are crucial for the assessment of ectomycorrhizal involvement in biogeochemical processes. Knowledge of exudation from mycelia of ectomycorrhizal fungi is still limited, especially for fungi in symbiosis with a host. Pinus sylvestris seedlings colonized by Hebeloma crustuliniforme were grown in aseptic multicompartment dishes. This novel system enabled identification of exudates originating only from extramatrical mycelium. At harvest, hyphal density and numbers were estimated using microscopic imaging. A fractal geometric approach was adopted for calculation of exudation rates. The main compounds identified were oxalate and ferricrocin. The exudation rate for oxalate was 19 +/- 3 fmol per hyphal tip h(-1) (mean +/- standard error of the mean) or 488 +/- 95 fmol hyphal mm(-2) h(-1). Ferricrocin rates were approx. 10 000 times lower. The fractal dimension (D) of the mycelia was 1.4 +/- 0.1, suggesting an explorative growth. Potassium nutrition was a significant regulatory factor for ferricrocin but not oxalate. The results suggest that hyphal exudation may alter the chemical conditions of soil microsites and affect mineral dissolution. Calculations also indicated that oxalate exudation may be a significant carbon sink.

Concentrations up to 2 and 12 nM of the hydroxamate siderophores ferrichrome and ferricrocin, respectively, were identified in soil solutions of podzolic forest soils at four sites in both northern and southern Sweden. No ferrichrysin was detected. As with the dissolved organic carbon and low molecular mass organic acids, the highest concentrations of the siderophores were found in the upper layers i.e. the mor layer, the eluvial and upper illuvial horizons. At the southern sites, the concentrations of ferrichrome and ferricrocin were both of similar magnitude and did not differ between the two sites. In contrast, soil solutions at the two northern sites contained more ferricrocin than ferrichrome; the ferricrocin concentrations were also higher at the northern sites than at the southern sites. Analyses were performed by high performance liquid chromatography with a porous graphitic carbon column on which ferrichrome, ferricrocin and ferrichrysin were separated. Detection by electrospray ionization mass spectrometry (ESI-MS) combined with on-line sample pre-concentration, by means of column-switching, enabled detection limits of 0.1-0.2 nM for ferrichrome, ferrichrysin and ferricrocin. The structural identities of the siderophores were further verified by MS/MS fragmentation. Fragmentation of ferrichrome, ferricrocin and ferrichrysin occured mainly via peptide cleavage. The most intense fragments were typified by the loss of one of the three iron(III) chelating hydroxamate residues, i.e N5-acyl-N5-hydroxy ornithine.

Centrifugation is a common technique to obtain soil solution. However, this method may disrupt living cells in the soil and release their contents into the soil solution. To examine this possibility, we compared leaching of low molecular mass organic acids (LMMOAs) and dissolved organic carbon (DOC) from the root of Scots pine (Pinus sylvestris) packed in quartz sand via extraction by centrifugation and milder procedures of water immersion and suction. Centrifugation enhanced leaching of shikimate appreciably; leaching of DOC, lactate, malate, and phosphate was also significantly enhanced. The concentration leached from the root was greater than 10 mu M for shikimate, and generally ranged between 0.5 and 5 mu M for the other acids. As for actual soil samples, this level of leaching could be appreciably large in upper mineral horizon(s) if the root density is as large as in our experiments and if the concentrations of LMMOAs in the soil matrix are relatively small.

Siderophores in soil solution of coniferous forest soils have been chemically identified. The siderophores ferrichrome and ferricrocin were identified in soil solution of the upper organic layer and also in the liquid medium of a cultivated ectomycorrhizal fungus by High Performance Luquid Chromatography Mass Spectrometry (HPLC-MS).