Amino acids derivatization and analysis: Sediment slurries were centrifuged to separate the sediments from the medium and the sediments were freeze-dried and homogenized. 50 mg of samples spiked with the internal reference standard norleucine were decalcified and acid hydrolyzed (6M, 110℃) for 15 h. The hydrolysate was centrifuged to remove the sediments and subsequently defatted with hexane/ DCM mixture (V/V, 2:1) three times. The purification and derivatization of amino acids were performed by following the protocols described by Takano et al.53 and Chikaraishi et al.54, respectively. In brief, the hydrolysates were evaporated under N2 gas to dryness and purified using Dowex 50WX8 200 400 mesh cation exchange resin to eliminate the matrix effects. The purified amino acids were then isopropylated with a mixture of isopropanol and acetyl chloride (4/1, v/v) at 100 ℃ for 2 h. The solution was then evaporated to dryness and followed 3 times with DCM addition and evaporation to remove any remaining reagent. The AA isopropyl esters were then acylated using a mixture of pivaloyl chloride and DCM (1/1, v/v) at 100 ℃ for 2 h to obtain pivaloyl-isopropyl ester (Pv/AA/iPr). The Pv/AA/iPr solution was then evaporated to dryness and followed 3 times DCM addition and evaporation to remove any remaining reagent. Liquid-liquid extraction was performed by using MiliQ water and a hexane/DCM mixture (2/1, v/v). The Pv/AA/iPr were stored frozen (-20℃) and dissolved in ethyl acetate before analysis.
Incubation time was controlled based on the development of δ13C values of CO2 in the headspace. δ13C of CO2 was measured by Thermo Finnigan Trace GC coupled to a Thermo Finnigan Delta plus XP isotope ratio mass spectrometer (IRMS). Deviations of triplicate isotopic measurement of δ13C of CO2 were between ± 1‰ and ± 1,000‰ (for CO2 with label uptake of >10,000‰). Slurries were harvested at five time points for detailed analyses.
Polar lipid-derived fatty acids (PLFAs) and their stable carbon isotopes are frequently combined to characterize microbial populations involved in the degradation of organic matter, offering a link to biogeochemical processes and carbon sources used. However, PLFA patterns derive from multiple species and may be influenced by substrate types. Here, we investigated such dependencies by monitoring the transformation of position-specifically 13C-labeled amino acids (AAs) in coastal marine sediments dominated by heterotrophic bacteria. Alanine was assimilated into straight-chain FAs, while valine and leucine incorporation led to the characteristic production of even- and odd-numbered iso-series FAs. This suggests that identical microbial communities adjust lipid biosynthesis according to substrate availability. Transformation into precursor molecules for FA biosynthesis was manifested in increased 13C recoveries of the corresponding volatiles acetate, isobutyrate and isovalerate of up to 39.1%, much higher than for PLFAs (<0.9%). A significant fraction of 13C was found in dissolved inorganic carbon (up to 37.9%), while less was recovered in total organic carbon (up to 17.3%). We observed a clear discrimination against the carboxyl C, whereby C2 and C3 positions were preferentially incorporated into PLFAs. Therefore, position-specific labeling is an appropriate tool for reconstructing the metabolic fate of protein-derived AAs in marine environments.
DIC measurement: an aliquot of the slurry was filtered with a 0.2-µm filter to remove cells and other particles. After filtration, 1 mL of samples were acidified with 100 μL 45% phosphoric acid overnight in an Exetainer® vial pre-purged with CO2-free air before analysis. All samples were measured with a Delta Ray Isotope Ratio Infrared Spectrometer (IRIS) with URI Connect and autosampler (Thermo Fisher Scientific, Germany) with an analytical error of ±1‰. All isotopic values are reported in the delta notation as δ13C relative to the Vienna PeeDee Belemnite (VPDB) standard. Deviations of triplicate isotopic measurement of sample DIC were between ± 1‰ and ± 1,000‰ (for DIC with label uptake of >10,000‰). DIC concentration was calculated from the released amount of CO2 by calibration with sodium hydrogen carbonate solution.
Im Fokus dieses Projektes stehen die Charakterisierung und Visualisierung von Mikroorganismen in der Rhizosphäre von Paddy Soils. Mit Hilfe molekularbiologischer Methoden (CARD-FISH/DGGE) und mikropedologischer Anwendungen (Fluoreszenzmikroskopie/Rasterelektronenmikroskopie) soll die Populationsdynamik in der Rhizosphäre von Nassreis erforscht werden. Zudem soll der Aktivitätszustand der Mikroorganismen im Zusammenhang mit der Entwicklung der Wurzeln untersucht werden. Während die Reispflanze verschiedene Wachstumsstadien durchläuft, ändern sich sowohl chemische als auch physikalische Charakteristika der Rhizosphäre. Um den Akti-vitätszustand und die Substratspezifität von Mikroorganismen zu erforschen, sollen Methoden wie Mikroautoradiographie (MAR) und Stable Isotope Probing (SIP) mit CARD-FISH kombiniert werden. Kooperationspartner: Hainan Institute for Tropical Agricultural Resources (HITAR), Sanya, PR China.