Improving The Methods For Compound-Specific Oxygen And Carbon Isotope Analysis Of α-cellulosic And Hemicellulosic Sugars In Higher Plants

Compound specific isotopic analysis(CSIA)of 18O and 13C in plant sugars is an important tool for tracing water movement and partitioning in the plants,for understanding carbon and water cycling in ecosystems,and to potentially provide insights into reconstructing paleoclimates.This study focuses on optimizing the purification and derivatization processes of sugars(monosaccharides)obtained from α-cellulose and hemicellulose extracted from higher plants for CSIA)of oxygen and carbon.It was proposed to compare accepted methods for cellulose extraction,purification and examine various monosaccharide derivatisation methods to arrive at an optimal approach.(1)Firstly,the research focused on C3 grasses collected from the southern coastal part of China,Hainan.A series of well-established α-cellulose extraction methods including Jayme-Wise,Loader,Brendel and Zhou’s extraction procedures were compared.α-Cellulose was extracted from the grass samples using the Zhou method and hydrolyzed with 72%H2SO4.The hydrolysed sugars were identified by GC/MS(gas chromatography/mass spectrometry)after derivatisation to alditol acetates.Oxygen isotope analysis of the sugars was performed,after methylation,on a GC/Py/IRMS system.Two methods for sugar methylation were compared,a modified Pur die methylation with DMS/MeI/Ag2O and NaOH/MeI/DMSO methylation.The latter proved to be the most appropriate as it produced more precise results in less time with fewer degradation peaks.Compound-specific isotope analysis was performed on hydrolysates using GC/Pyrolysis/IRMS,comparing results with bulk isotope analysis of α-cellulose products obtained through EA/Py/IRMS.The results suggest that the Zhou technique produces α-cellulose with the highest degree of purity,exhibiting minimal amounts of residual lignin and the second-lowest levels of non-glucose sugars.In addition,it was determined through isotopic analysis that the O-2-O-6 moiety of the α-cellulose glucosyl units exhibited a depletion in 18O ranging from 0.0 mUr-4.3 mUr(with an average of 1.9 mUr)in a manner that was specific to each species,as compared to the α-cellulose products.The positive isotopic bias observed when utilising α-cellulose in lieu of glucosyl units can be attributed to residual hemicellulose derived pentoses.The results indicate that the Zhou technique produces α-cellulose with high purity and minimal impurities.(2)Aimed to reduce the amount of carbon added during derivatization for GC/C/IRMS(gas chromatography/combustion/isotope ratio mass spectrometry)analysis of sugars hydrolytically released from grass α-cellulose extracts and secondly,to determine the extent of enhancement in the accuracy and precision of the δ13C measurements.α-Cellulose was extracted from grass leaves collected from the southern part of China(Hainan)using the Zhou method.The extractedα-cellulose was hydrolysed and derivatized to methylboronates/TMS for δ13C measurements.The δ13C isotopic values for C3 grass α-celluloses varied between-33 mUr to-19 mUr which is very close to the reported values.These results were then compared with those generated from methylated samples.The results showed methylation is not useful for 13C analysis because of a large degree of fractionation,much more than observed with the methylboronate/TMS derivatives(MBA method).A comparison of averaged δ13C values between hydrolysed α-cellulose grass leaves that differed only slightly in their bulk carbon isotope signature(-29.70 mUr)and CSIA by GC/C/IRMS-calculated(-29.14 mUr)showed that the MBA/TMS derivatization method delivers significantly improved precision and accuracy compared to other methodologies.(3)Describes an isotopic analysis of the tree rings of Dahurian larch(Larix gmelinii),focusing on the comparison of α-cellulose and hemicellulose isotopic signatures.Both hemicellulose(alkali extraction)and α-cellulose(extracted with the Zhou method were extracted sequentially and subject to Saeman hydrolysis(72%H2SO4),followed by methylation prior to δ18O measurement.δ18O was measured for α-cellulose and hemicelluloses from three age groups;old(19281930);middle-aged(1948-1950)and young(1968-1970)tree growth rings.It is known that hemicellulose contaminants can bias the isotopic signature of αcellulose(glucose)hence it was proposed to quantify the effect by separate analysis of α-cellulose and hemicellulose.It was found the hemicellulose derived glucose is 1.04 mUr enriched compared with the α-cellulose derived glucose.It was also found that mannose and galactose,the major constituents of conifer hemicellulose are marginally depleted compared to cellulosic glucose.The study reveals significant differences in isotopic enrichment between the two(cellulose and hemicellulose)indicating a different biosynthetic origin for the glucose derived from α-cellulose and hemicellulose.