Determination of the carbon isotopic composition of whole/intact biological specimens using at-line direct thermal desorption to effect thermally assisted hydrolysis/methylation.

In this paper, we discuss the use of a direct thermal desorption (DTD) interface as an alternative to Curie-point flash pyrolysis system as an inlet technique in gas chromatography-combustion isotope-ratio mass spectrometry (GC/C-IRMS) analysis of whole/intact phytoplankton and zooplankton specimens. The DTD in combination with a combipal auto-injector is programmed to perform the injection, evaporation of solvents, transport of capped programmed-temperature vaporizer (PTV) liners to the PTV injector and chemical derivatisation (thermally assisted hydrolysis/methylation; THM) such that a profile of a cellular fatty acids is obtained. Flow-cytometric sorted microalgae and handpicked zooplankton are used as samples with trimethylsulfonium hydroxide (TMSH) as methylating reagent. A major advantage of this novel approach over the Curie-point technique is the automation of the total procedure, which allows unattended analysis of large sample series. The profiles and delta(13)C carbon isotopic signatures of the fatty acid methyl esters (FAMEs) produced are very similar to those obtained using the Curie-point flash pyrolysis method. It is shown that algal samples must be kept no longer than 48 h in the DTD sample tray prior to the THM-analysis in order to maintain the integrity of their FAME profile.

Differential effects of nitrogenous fertilizers on methane-consuming microbes in rice field and forest soils.

The impact of environmental perturbation (e.g., nitrogenous fertilizers) on the dynamics of methane fluxes from soils and wetland systems is poorly understood. Results of fertilizer studies are often contradictory, even within similar ecosystems. In the present study the hypothesis of whether these contradictory results may be explained by the composition of the methane-consuming microbial community and hence whether methanotrophic diversity affects methane fluxes was investigated. To this end, rice field and forest soils were incubated in microcosms and supplemented with different nitrogenous fertilizers and methane concentrations. By labeling the methane with 13C, diversity and function could be coupled by analyses of phospholipid-derived fatty acids (PLFA) extracted from the soils at different time points during incubation. In both rice field and forest soils, the activity as well as the growth rate of methane-consuming bacteria was affected differentially. For type I methanotrophs, fertilizer application stimulated the consumption of methane and the subsequent growth, while type II methanotrophs were generally inhibited. Terminal restriction fragment length polymorphism analyses of the pmoA gene supported the PLFA results. Multivariate analyses of stable-isotope-probing PLFA profiles indicated that in forest and rice field soils, Methylocystis (type II) species were affected by fertilization. The type I methanotrophs active in forest soils (Methylomicrobium/Methylosarcina related) differed from the active species in rice field soils (Methylobacter/Methylomonas related). Our results provide a case example showing that microbial community structure indeed matters, especially when assessing and predicting the impact of environmental change on biodiversity loss and ecosystem functioning.