On the Chemical Purity and Oxygen Isotopic Composition of -Cellulose Extractable from Higher Plants and the Implications for Climate, Metabolic, and Physiological Studies

The 18O/16O ratio of alpha-cellulose in land plants has proved of interest for climate, environmental, physiological, and metabolic studies. Reliable application of such a ratio may be compromised by the presence of hemicellulose impurities in the alpha- cellulose product obtainable with current extraction methods, as the impurities are known to be isotopically different from that of the alpha-cellulose. We first compared the quality of hydrolysates of "alpha- cellulose products" obtained with four representative extraction methods (Jayme and Wise; Brendel; Zhou; Loader) and quantified the hemicellulose-derived non-glucose sugars in the alpha-cellulose products from 40 land grass species using gas chromatography- mass spectrometry (GC/MS). Second, we performed compound specific isotope analysis of the hydrolysates using GC/Pyrolysis/ IRMS. These results were then compared with the bulk isotope analysis using EA/Pyrolysis/IRMS of the alpha-cellulose products. We found that overall, the Zhou method afforded the highest purity alpha-cellulose as judged by the minimal presence of lignin and the second-lowest presence of non-glucose sugars. Isotopic analysis then showed that the O-2-O-6 of the alpha-cellulose glucosyl units were all depleted in 18O by 0.0-4.3 mUr (average, 1.9 mUr) in a species-dependent manner relative to the alpha-cellulose products. The positive isotopic bias of using the alpha-cellulose product instead of the glucosyl units stems mainly from the fact that the pentoses that dominate hemicellulose contamination in the alpha-cellulose product are relatively enriched in 18O (compared to hexoses) as they inherit only the relatively 18O-enriched O-2-O-5 moiety of sucrose, the common precursor of pentoses and hexoses in cellulose, and are further enriched in 18O by the (incomplete) hydrolysis.