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Nitrogen sources of oligoporus placenta and trametes versicolor evaluated in a 2³ experimental plan
(2012)
Four full-factorial 2³ experimental plans were applied to evaluate the nitrogen (N) sources of Oligoporus placenta and Trametes versicolor and their interaction with the atmospheric N2-assimilating bacterium Beijerinckia acida. The effects of N from peptone, of sapwood and of N from gaseous N2 on fungal, bacterial and fungal–bacterial activity were investigated. The activities were determined by quantification of biomass, formation of CO2, consumption of O2 and laccase activity. The significance of each effect was tested according to t-test recommendation.
The activity of both fungi was enhanced by peptone rather than sapwood or gaseous N2. Nevertheless, comparative studies under an N2-free gas mixture as well as under air revealed that the presence of N2 affected bacterial growth and bacterial–fungal cocultivations. Elemental analysis isotope ratio mass spectrometry (IRMS) of the bacterial and fungal biomass enabled estimation of N transfer and underlined gaseous N2 as requisite for fungal–bacterial interactions. Combining full-factorial experimental plans with an analytical set-up comprising gas chromatography, IRMS and enzymatic activity allowed synergistic effects to be revealed, fungal N sources to be traced, and symbiotic fungal–bacterial interactions to be investigated.
Isotope ratio mass spectrometry (IRMS) is an advanced method to investigate carbon (C) and nitrogen (N) in organic samples. In particular, the N content, its isotope signature and the C/N ratio reveal important facts of nutrient cycling, niche separation and ecological food webs. In this study, the characteristics of N turnover of wood decomposing microorganisms were investigated.
The growth of the white rot causing basidiomycete Trametes versicolor is enhanced after addition of ammonia or urea, whereas the brown rot causing Oligoporus placenta is not accelerated. In addition, an interaction of each fungus with atmospheric N2 assimilating (diazotrophic) bacteria was investigated. Cultivation experiments with a gas mixture of 15N2/O2 and subsequent IRMS analysis of dry biomass of the diazotrophs Azotobacter croococcum, Beijerinckia acida and Novosphingobium nitrogenifigens revealed that they assimilated up to 12% of their N from N2. The experiments reflected N availability as a prerequisite for efficient growth of decomposing basidiomycetes and diazotrophs. Fungal–bacterial co-cultivation experiments showed that depending on the growth characteristics and bacterial N2 assimilation activity N is transferred from certain bacteria into fungal biomass. Thus, the experiments gave a first indication of an interaction between wood decomposing basidiomycetes and diazotrophs, which is a novel pathway of fungal N acquisition.
The white-rot fungus Hypholoma fasciculare coexists with a bacterial community that uses low-molecular weight carbon sources provided by fungal, extracellular enzyme activities. Since fungal development on wood is limited by the availability of nitrogen (N), bacteria could contribute to the N supply. To prove or disapprove an interaction in terms of N transfer, N sources of the fungus and the coexisting bacterial isolates were investigated, and the bacterial N2 fixation was quantified. Fungal, fungal—bacterial and bacterial wood decomposition was analysed by Fourier transform infrared spectroscopy (FTIR), mass loss and surface pH. Microbial N preferences were investigated by elemental analysis isotope ratio mass spectrometry (IRMS). In addition, diazotrophic activity was explored after cultivation under a 15N2/O2 atmosphere. Decomposition was similar with and without bacteria and both H. fasciculare and coexisting bacteria preferred reduced N species, such as urea, ammonium and organic N. In most of the bacteria, the 15N abundance in the biomass increased significantly but to a low extent if they were cultivated under a 15N2/O2 atmosphere. This effect is considered an artefact and attributed to adsorption rather than to bacterial N2 fixation activity. Hence, the bacteria coexisting with H. fasciculare rather competed for the same N sources than supported fungal N supply by diazotrophic activity.
Isotope reference materials are needed to calibrate and validate analytical procedures used for the determination of isotope amount ratios, procedurally defined isotope ratios or so-called δ values. In contrast to the huge analytical progress in isotope ratio analytics, the production of isotope reference materials has not kept pace with the increasing needs of isotope analysts. Three representative isotope systems are used to explain the technical and non-technical difficulties and drawbacks, on one hand, and to demonstrate what can be achieved at its best, on the other hand. A clear statement is given that new isotope reference materials are needed to obtain traceable and thus comparable data, which is essential for all kinds of isotope research. The range of available isotope reference materials and δ reference materials should be increased and matrix reference materials certified for isotope compositions or δ values, which do not exist yet, should be provided.
Boron isotope studies on technical materials and geological and environmental samples are one hot topic in nowadays isotope research. Provenance studies of artefacts, food and other products might become another one, as several recent studies demonstrate. Typically boron isotope determinations have been carried out by TIMS. To open up this research field to a broader scientific community we developed two analytical procedures for boron isotope determinations based on single collector SF-ICPMS combined with a sample preparation procedure consisting of dry-ashing and a three step ion chromatographic boron-matrix separation. The developed procedures consist of one low resolution (LR) and one medium resolution (MR) procedure. The repeatability for the δ11B determination in three independently measured aliquots lies between 0.2 and 0.8‰ for the LR procedure and between 0.3 and 1.5‰ for the MR procedure. The expanded uncertainties with a coverage factor of k=2 range between 1.4 and 1.6‰ for the LR procedure and between 2.9 and 3.2‰ for the MR procedure. The accuracy, expressed as average deviation from the reference values, is 0.43‰ for the LR procedure and 0.33‰ for the MR procedure. To test the practicability of the procedures the matrix tolerance has been investigated as well and was found to be up to 2 mg kg-1 of alkaline and alkaline earth elements in the final measurement solution containing 100 µg kg-1 boron. Thus a highly efficient matrix separation for SF-ICPMS boron isotope determinations is required, similar to TIMS.
Isotope reference materials are essential to enable reliable and comparable isotope data. This article reviews the work in this field within the past years. The focus is on all stable elements, except for classical stable isotopes (H, C, N, O, S) and for radioactive elements. Currently available isotope reference materials are listed. The limitations of synthetic isotope mixtures being used to characterize these materials are discussed, as well as the limitations of the isotope reference materials, such as uncertainty and homogeneity. The needs for present research on isotope variations are being considered and are compared to the limitations of current isotope reference materials. This disagreement between both can only be solved by providing isotope reference materials defining a δ-scale for each element of interest. Such materials should be provided with additional data on isotope abundances whenever possible. As an outlook a possible outline for a new program on isotope reference materials is discussed.
The variation of isotope abundance ratios is increasingly used to unravell natural and technical questions. In the past the investigation and interpretation of such variations was the field of a limited number of experts. With new upcoming techniques and research topics in the last decades, such as provenance and authenticity of food, the number of published isotope data strongly increased. The development of inductively coupled plasma mass spectrometers (ICPMS) from an instrument for simple quantitative analysis to highly sophisticated isotope abundance ratio machines influenced this process significantly. While in former times only experts in mass spectrometry were able to produce reproducible isotope data, nowadays many laboratories, never been in touch with mass spectrometry before, produce isotope data with an ICPMS. Especially for such user isotope reference materials (IRM) are indispensible to enable a reliable method validation. The fast development and the broad availability of ICPMS also lead to an expansion of the classical research areas and new elements are under investigation. Here all users require IRM to correct for mass fractionation or mass discrimination or at least to enable isotope data related to a common accepted basis. Despite this growing interest suitable IRM are still lacking for a number of isotope systems such as magnesium.