13C pulse-labeling assessment of the community structure of active fungi in the rhizosphere of a genetically starch-modified potato (Solanum tuberosum) cultivar and its parental isoline.

• The aim of this study was to gain understanding of the carbon flow from the roots of a genetically modified (GM) amylopectin-accumulating potato (Solanum tuberosum) cultivar and its parental isoline to the soil fungal community using stable isotope probing (SIP). • The microbes receiving (13)C from the plant were assessed through RNA/phospholipid fatty acid analysis with stable isotope probing (PLFA-SIP) at three time-points (1, 5 and 12 d after the start of labeling). The communities of Ascomycota, Basidiomycota and Glomeromycota were analysed separately with RT-qPCR and terminal restriction fragment length polymorphism (T-RFLP). • Ascomycetes and glomeromycetes received carbon from the plant as early as 1 and 5 d after labeling, while basidiomycetes were slower in accumulating the labeled carbon. The rate of carbon allocation in the GM variety differed from that in its parental variety, thereby affecting soil fungal communities. • We conclude that both saprotrophic and mycorrhizal fungi rapidly metabolize organic substrates flowing from the root into the rhizosphere, that there are large differences in utilization of root-derived compounds at a lower phylogenetic level within investigated fungal phyla, and that active communities in the rhizosphere differ between the GM plant and its parental cultivar through effects of differential carbon flow from the plant.

A versatile method for stable carbon isotope analysis of carbohydrates by high-performance liquid chromatography/isotope ratio mass spectrometry.

We have developed a method to analyze stable carbon isotope ((13)C/(12)C) ratios in a variety of carbohydrates using high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS). The chromatography is based on strong anion-exchange columns with low strength NaOH eluents. An eluent concentration of 1 mM resulted in low background signals and good separation of most of the typical plant neutral carbohydrates. We also show that more strongly bound carbohydrates such as acidic carbohydrates can be separated by inclusion of NO(3) (-) as an inorganic pusher ion in the eluent. Analyses of neutral carbohydrate concentrations and their stable carbon isotope ratios are shown for plant materials and marine sediment samples both at natural abundance and for (13)C-enriched samples. The main advantage of HPLC/IRMS analysis over traditional gas chromatography based methods is that no derivatization is needed resulting in simple sample treatment and improved accuracy and reproducibility.

Stable isotopes and biomarkers in microbial ecology.

The use of biomarkers in combination with stable isotope analysis is a new approach in microbial ecology and a number of papers on a variety of subjects have appeared. We will first discuss the techniques for analysing stable isotopes in biomarkers, primarily gas chromatography-combustion-isotope ratio mass spectrometry, and then describe a number of applications in microbial ecology based on 13C. Natural abundance isotope ratios of biomarkers can be used to study organic matter sources utilised by microorganisms in complex ecosystems and for identifying specific groups of bacteria like methanotrophs. Addition of labelled substrates in combination with biomarker analysis enables direct identification of microbes involved in specific processes and also allows for the incorporation of bacteria into food web studies. We believe that the full potential of the technique in microbial ecology has just started to be exploited.

Bacterial populations and processes involved in acetate and propionate consumption in anoxic brackish sediment.

Bacterial populations and pathways involved in acetate and propionate consumption were studied in anoxic brackish sediment from the Grosser Jasmunder Bodden, German Baltic Sea. Uptake of acetate and propionate from the porewater was studied using stable carbon isotope-labeled compounds. Labeled acetate was not produced as an intermediate during propionate uptake experiments, and propionate consumption was not affected by the addition of acetate. In parallel, incorporation of labeled acetate and propionate into phospholipid-derived fatty acids (PLFA) was studied to indicate bacterial populations involved in the consumption of these substrates. The (13)C-acetate label was mainly recovered in even-numbered PLFA (16:1omega7c, 16:0 and 18:1omega7c). In contrast, primarily odd-numbered PLFA (a15:0, 15:0, 17:1omega6 and 17:0) and the even-numbered i16:0 were labeled after incubation with (13)C-propionate. Although single PLFA labeled with propionate are commonly found in sulfate reducers, the complete PLFA-labeling pattern does not resemble any of the know strains. However, the acetate-labeling pattern is similar to Desulfotomaculum acetoxidans and Desulfofrigus spp., two acetate-consuming, sulfate reducers. In conclusion, our data suggest that acetate and propionate were predominantly consumed by different, specialized groups of sulfate-reducing bacteria.