On the chemical purity and oxygen isotopic composition of a-cellulose extractable from higher plants and the implications for climate, metabolic and physiological studies

The ¹⁸O/¹⁶O ratio of a-cellulose in land plants has proved of interest for climate, environmental, physiological and metabolic studies. Reliable application of such ratio may be compromised by the presence of hemicellulose impurities in the a-cellulose product obtainable with current extraction methods, as the impurities are known to be isotopically different from that of the a-cellulose. We firstly compared the quality of hydrolysates of “a-cellulose products” obtained with four repre-sentative extraction methods (JAYME and WISE; BRENDEL; ZHOU; LOADER) and quantified the hemicellulose-derived non-glucose sugars in the “a-cellulose products” from 40 land grass species using GC/MS. Secondly, we performed com-pound-specific oxygen isotope analysis for the hydrolysates using GC/Pyrolysis/IRMS. These results were then compared with the bulk isotope analysis using EA/Pyrolysis/IRMS of the “a-cellulose products”. We found that the ZHOU method afforded overall the highest purity a-cellulose as judged by the minimal presence of lignin, and the second lowest presence of non-glucose sugars in the investigated grass species. Isotopic analysis then showed that the O-2~O-6 of the a-cellulose glucosyl units were all depleted in ¹⁸O by 0.0-4.3 mUr (with an average of 1.9 mUr) in a species-dependent manner relative to the a-cellulose products. The positive isotopic bias of using a-cellulose product instead of the glucosyl units stems mainly from the fact that the pentoses that dominate hemicellulose contamination in the a-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 bio-chemical precursor of pentoses and hexoses in cellulose, and are further enriched in ¹⁸O by the (incomplete) hydrolysis. Failure to prepare a-cellulose with the highest possible purity, free from lignin and hemicellulose contamination, can bias predictions based on mechanistic models linking a-cellulose oxygen isotope composition to plant growth conditions.