Site-specific natural isotope fractionation measured by deuterium NMR (SNIF-NMR) was used for investigating the deuterium transfers occurring in the fermentation of sugars into ethanol. In contrast to carbon-13, which is usually assumed to be randomly distributed within the glucose skeleton, very large deviations with respect to a statistical repartition are determined for deuterium. By transforming glucose samples from different origins into acetates and nitrates, the absolute values of the D/H ratios in the nonexchangeable sites were obtained. The hydroxyl sites were considered to contribute to the isotope content of the starting water medium. The site-specific isotope parameters were determined for more than 50 ethanol samples obtained by fermenting glucose samples issued from different starches and sucroses in water media with different isotope ratios. Several concentrations of sugar, several strains of the same yeast, and different temperatures of fermentation were considered. The whole set of isotope parameters characterizing the nonexchangeable sites of glucose, the starting and end water media, and the methyl and methylene sites of ethanol was submitted to factor analysis. The results are satisfactorily reproduced on the basis of two main factors which may be associated with the independent contributions of the isotope ratios of glucose and water. The isotope parameters of the end products and of the starting materials were then considered to be related by a set of linear equations and the coefficients of the redistribution matrix were calculated by multidimensional analysis. Thus the isotope ratio of the methyl site of ethanol exhibits a strong sensitivity toward the nonexchangeable sites of glucose and depends to a lesser extent on the isotope content of the starting water medium. By contrast, no direct connection is found between glucose and the methylene site which is only sensitive, with a strong discriminating effect against deuterium, to the isotope content of water. A redistribution coefficient slightly less than unity (0.96 for a concentration of sugar of 100 g L-I) is found between the isotope ratio of the end and starting water media. Although the bioconversion of glucose into ethanol occurs with high fractionation effects which are the source of high deuterium depletions in the methyl and methylene sites of ethanol as compared to glucose, constant redistribution parameters can be obtained in appropriate conditions and the isotope parameters measured in alcohols may be used as a fingerprint for characterizing the sugar and aqueous juice from which they have been produced. The site-specific natural isotope-parameters of ethanol therefore constitute a faithful and powerful probe for investigating the physiological biochemical and climatological effects which have governed the photosynthesis of sugars in natural conditions.The first issue of this journal, which appeared in 1879, began with a paper by D. P. Ricketts' which was devoted to the characterization of sugars from different origins. More t...

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Site-specific natural isotope fractionation of hydrogen studied by deuterium NMR (SNIF-NMR) spectroscopy is a powerful source of information on hydrogen pathways occurring in biosyntheses in natural conditions. The potential of the carbon counterpart of this method has been investigated and compared. Three typical molecular species, ethanol, acetic acid, and vanillin, have been considered. Taking into account the requirements of quantitative 13C NMR, appropriate experimental procedures have been defined and the repeatability and reproducibility of the isotope ratio determinations have been checked in different conditions. It is shown that the carbon version of the SNIF-NMR method is capable of detecting small differences in the carbon-13 content of the ethyl fragment of ethanols from different botanical or synthetic origins. These results are in agreement with mass spectrometry determinations of the overall carbon isotope ratios. Deviations with respect to a statistical distribution of 13C have been detected in the case of acetic acid and vanillin. However, since the method is very sensitive to several kinds of systematic error, only a relative significance can be attached at present to the internal parameters directly accessible. Isotope dilution experiments have also been carried out in order to check the consistency of the results. In the present state of experimental accuracy, the 13C NMR method is of more limited potential than 2H SNIF-NMR spectroscopy. However it may provide complementary information. Moreover it is particularly efficient for detecting and quantifying adulterations that aim to mimic the overall carbon-13 content of a natural compound by adding a selectivity enriched species to a less expensive substrate from a different origin.

The site-specific natural hydrogen isotope ratios of plant metabolites determined by 2H nuclear magnetic resonance (SNIF-NMR method) can provide powerful criteria for inferring mechanistic and environmental effects on biosynthetic pathways. This work examines the potential of isotopic profiles for the main constituents of carbohydrates, glucose and fructose, to distinguish different photosynthetic pathways. An appropriate analytical strategy, involving three suitable isotopic probes, has been elaborated with a view to measuring simultaneously, in conditions devoid of isotopic perturbations, all (or nearly all) of the carbon-bound hydrogen isotope ratios. It is shown that the type of photosynthetic metabolism, either C3 (sugar beet, orange, and grape), C4 (maize and sugar cane), or CAM (pineapple), and the physiological status of the precursor plant exert strong influences on the deuterium distribution in the sugar molecules. Consequently, this isotopic fingerprint may be a rich source of information for the comparison of mechanisms in metabolic pathways. In addition, it can provide complementary criteria to ethanol as a probe for the origin of sugars.

Isotope fractionation phenomena occurring at the natural abundance level in the course of liquid-vapor transformation have been investigated by using the SNIF-NMR method (site-specific natural isotope fractionation studied by NMR) which has a unique capability of providing simultaneous access to fractionation parameters associated with different molecular isotopomers. This new appproach has been combined with the determination of overall carbon and hydrogen fractionation effects by isotope ratio mass spectrometry (IRMS). The results of distillation and evaporation experiments of alcohols performed in technical conditions of practical interest have been analyzed according to the Rayleigh-type model. In order to check the performance of the column, unit fractionation factors were measured beforehand for water and for the hydroxylic sites of methanol and ethanol for which liquid-vapor equilibrium constants were already known. Inverse isotope effects are determined in distillation experiments for the overall carbon isotope ratio and for the site-specific hydrogen isotope ratios associated with the methyl and methylene sites of methanol and ethanol. In contrast, normal isotope effects are produced by distillation for the hydroxylic sites and by evaporation for all the isotopic ratios. The effective fractionation factors obtained from least-squares treatment of the data have been analyzed in terms of liquid-vapor equilibrium fractionation factors and in terms of kinetic evaporative fractionation factors associated either with the whole set of isotopomers or with the individual isotopic species. Suitable conditions for an accurate determination of the relative values of these parameters are provided by the proposed approach which gives access to several fractionation factors in a one-pot experiment. From a practical point of view, the unit fractionation factors determined in this work provide a quantitative basis for correcting the isotope ratio of the ethanol isotopic probe for physical effects due to distillation and evaporation.