Sample preparation for D isotopomer measurements
Dry moss biomass formed during the incubations was ground to a fine powder at 30 Hz for 2 min using a MM 400 ball mill (Retsch®, Haan, Germany), and 200-700 mg portions were used as starting material for the following sample preparation for D isotopomer measurements. Glucose-containing structural polymers were hydrolyzed to glucose and converted to 1,2-O-isopropylidene-α-D-glucofuranose according to established protocols (Betson et al ., 2006). To remove contamination by a mannose derivative, which has overlapping signals with the NMR spectrum of the glucose derivative, an oxidation step was introduced. Each sample was dissolved in 20 mL 0.187 M phosphate buffer (NaH2PO4, pH 4), 15.6 mg NaClO2 was added per 100 mg sample and the pH was adjusted to 3.5 with H3PO4. The NaClO2 addition and pH adjustment was repeated five times over a period of 3 days (every 12 h). The complete reaction procedure was performed under the absence of light. 12 h after the last addition the reaction was quenched by adding a molar equivalent amount of Na2SO3 to the total amount of NaClO2 added, then the mixture was neutralized with CaCO3, vacuum-filtered and evaporated under reduced pressure. To remove remaining salts the sample was extracted three times with 20 mL ethanol. The derivative was subsequently converted to 3,6-anhydro-1,2-O-isopropylidene-α-D-glucofuranose following Schleucheret al . (1999). The derivative was purified by flash chromatography using silica gel and diethyl ether. Pure fractions were identified by thin-layer-chromatography and pooled. Diethyl ether was evaporated, the sample was washed with amylene-stabilized chloroform and purity was checked by 1H-NMR.