Mass spectrometry was applied to identify metabolites and estimate the efficiency of phenanthrene biodegradation and transformation by rhizosphere bacteria Pseudomonas aureofaciens BS1393. Strains P. aureofaciens BS1393(pOV17) and P. aureofaciens BS1393(NPL-41) bearing various naphthalene biodegradation plasmids were used in the work. The strain BS1393(pOV17) contains the pOV17 wild type naphthalene biodegradation plasmid that determines the oxidation of naphthalene to Krebs cycle metabolites. The strain BS1393(NPL-41) contains the mutant plasmid NPL-41 governing the initial stages of naphthalene oxidation into salicylic acid. The limiting stages of phenanthrene biodegradation in bacteria with various plasmids have been identified according to the accumulation of intermediates. When bacteria were grown on phenanthrene, the main metabolites were as follows: (a) 2-hydroxy-2H-benzo[h]chromene-2-carboxylic acid/trans-4-(1-hydroxynaph-2-yl)-2-oxobut-3-enoic acid, (b) 1-hydroxy-2-naphthoic acid, and (c) salicylic acid. In the strain BS1393(pOV17), metabolite (а) was observed during 1–14 days of cultivation. Unlike it, in the strain BS1393(NPL-41), an insignificant amount of this metabolite was found after only 14 days. The availability of metabolite (b) in the growth of both strains was an evidence of the limited rate of its further decarboxylation. Metabolite (c) as a final product was found in the growth of the strain BS1393(NPL-41). Contrastingly, in the strain BS1393(pOV17), this metabolite was not found, which indicates the complete oxidation of phenanthrene.

Major metabolites formed by Pseudomonas bacteria bearing various naphthalene degradation plasmids were identified using high performance liquid chromatography and mass spectrometry in combination with electrospray ionization. During the growth of P. aureofaciens BS1393(NPL-41) bacteria bearing the plasmid NPL-41, partial oxidation of naphthalene was shown to occur to form the major exometabolite (salicylate) and minor exometabolite (phenol). The growth of P. aureofaciens BS1393(pOV17) bacteria bearing the plasmid pOV17 was accompanied by complete oxidation of naphthalene and formation of trace amounts of salicylate and epoxybenzene. Based on the data of molecular mass spectrometry, we determined the dynamics of CO2 production and oxygen uptake in the process of bacterial growth. The specific quantity of CO2 per unit of cell amount for P. aureofaciens BS1393(NPL-41) bacteria was shown to exceed fivefold that for P. aureofaciens BS1393(pOV17). The respiratory quotient (RQ) representing the ratio of the molar concentrations of metabolic CO2 to consumed oxygen depends on the type of plasmids that determine naphthalene degradation. The value of RQ during the growth of P. aureofaciens BS1393(pOV17) bacteria was almost 1.5 times greater than in the case of P. aureofaciens BS1393(NPL-41). The ratio of O2 uptake to CO2 formed by the first culture was 4.5 mol O2/mol CO2; by the second culture, 6.5 mol O2/mol CO2.

RATIONALE Recommendations of relevant international organizations controlling the quality of grape wines and beverages specify that only tartaric acids of grape origin can be introduced to achieve the required parameters. The development of methods for determining the origin of tartaric acid in grape wine is of great technological significance. METHODS Organic dicarboxylic oxyacids were extracted from wines as barium salts. Carbon dioxide, which included all the carbon atoms of the acids, was used to determine the carbon isotope ratios by Isotope Ratio Mass Spectrometry. The alkyl part of the oxyacids was burned at 560°C in the presence of air; BaCO3 containing the carboxyl carbon was left. This carbonate was used to measure the carbon isotope ratios in the carboxyl part of the acid. The carbon isotope ratios of the alkyl part of tartaric acid were found by isotope mass balance. RESULTS The carbon isotope composition of carboxyl groups (δ(13) С values) in tartaric acid of grape (biogenic origin) had a higher (13) С content than the carbon in the alkyl part of the molecule. Tartaric acid produced by chemical synthesis (abiogenic origin) was noted to have a different (13) С/(12) С distribution: the carboxyl group of tartaric acid produced by chemical synthesis contained a smaller than or equal amount of (13) С to the alkyl part. CONCLUSIONS This is the first determination of the site-specific distribution of the (13) С/(12) С isotopes in tartaric acids as evidence of their biogenic and abiogenic origins. The presented method for determining the origin of tartaric acid can be used for efficient control of the quality of grape wines and beverages.

We compared data on the extent of bioremediation in soils polluted with oil. The data were obtained using conventional methods of hydrocarbon determination: extraction gas chromatography-mass spectrometry, extraction IR spectroscopy, and extraction gravimetry. Due to differences in the relative abundances of the stable carbon isotopes (13C/12C) in oil and in soil organic matter, these ratios could be used as natural isotopic labels of either substance. Extraction gravimetry in combination with characteristics of the carbon isotope composition of organic products in the soil before and after bioremediation was shown to be the most informative approach to an evaluation of soil bioremediation. At present, it is the only method enabling quantification of the total petroleum hydrocarbons in oil-polluted soil, as well as of the amounts of hydrocarbons remaining after bioremediation and those microbially transformed into organic products and biomass.

Проведен сравнительный анализ данных, полученных с использованием стандартных методов количественного определения углеводородов (экстракционный газо-хромато-масс-спектрометрический, экстракционный ИК-спектроскопический и экстракционно-гравиметрический), которые позволяют оценить степень биоремедиации почв, загрязненных нефтью. Различия в распределении стабильных изотопов углерода (13С/12) нефти и почвенного органического вещества были использованы в качестве природной изотопной “метки” этих веществ. Показано, что при оценке степени биоремедиации почв наиболее информативным методическим подходом является экстракционно-гравиметрический метод в сочетании с характеристиками изотопного состава углерода органических продуктов в почве до и после биоремедиации. В настоящее время этот подход можно рассматривать как единственный метод, который позволяет определить общее количество углеводородов и почвенного органического вещества в загрязненной нефтью почве, а также их содержание, остававшееся в почве после биоремедиации, и количество углеводородов, трансформированных микроорганизмами в органические продукты.

Distribution of 13C/12C isotopes in vegetative (roots, grapevine, leaves) and generative (berries) parts of vine plants of the West European genetically different varieties Cabernet Sauvignon and Aligoté growing on soils of Krasnodar krai and Rostov oblast, as well as autochthonous varieties Sibirkovy and Krasnostop Zolotovsky growing in Rostov oblast, has been studied using isotopic mass spectrometry methods. It has been shown that the variations of δ13C values in plant tissues and berries are related to the climatic conditions of plant growth: moisture (a sum of annual precipitation) and temperature (a sum of annual effective temperatures). The carbon isotope ratios of vegetative and generative parts of vine plants have been found to be noticeably affected by vine varieties. The different 13C contents in ethanol produced from wine of the Aligoté and Cabernet Sauvignon varieties grown in two Russian vineyard regions are due to vine growth conditions, variety attribution and wine production techniques. An analytically significant parameter determined as exemplified by the Aligoté and Cabernet Sauvignon varieties in fermentation of vine harvested in different seasons and in both vineyard regions was an increased 13C content in ethanol with respect to dry (non-volatile) residue in wine after distillation of ethanol. This characteristic has been determined by a systematic difference of about 1–2‰ between the δ13C value of ethanol and the dry residue. A relative constancy in the carbon isotope composition of ethanol and of the dry residue in the final product is the basis for determining the authenticity of grape wines by means of isotopic mass spectrometry irrespective of natural factors.

By the example of glucose uptake by the soil bacteria Pseudomonas aureofaciens BS1393(pBS216) and Rhodococcus sp. 3–30 immobilized on a solid-phase surface (quartz sand), their growth parameters were determined: growth rate (doubling time), total CO2 production, CO2 production per cell, lag period with respect to substrate uptake, respiratory quotient. The growth of P. aureofaciens and Rhodococcus sp. on glucose revealed (1) differences of the lag period with respect to substrate (lag time of ∼4 h for P. aureofaciens and ∼26 h for Rhodococcus sp.); (2) differences between the maximal rates of CO2 production (∼50 μg C-CO2 g−1 sand h−1 for P. aureofaciens and ∼8.5 μg C-CO2 g−1 sand h−1 for Rhodococcus sp.); (3) differences in CO2 production per cell (∼1.94 × 10−9 μM CO2/CFU for P. aureofaciens and more than ∼3.4 × 10−9 μM CO2/CFU for Rhodococcus sp.). The kinetics of the metabolic CO2 isotopic composition was shown to be determined by the difference in the carbon isotopic characteristics of products in the cell. Upon introduction of glucose into the medium (the preparatory stage of the metabolism), the uptake of intracellular 13C-depleted products (lipids) is noted; at the stage of the maximal cell growth rate, introduced glucose is mainly metabolized; and at the final stage, upon exhaustion of substrate, the “stored” products—the lipid fraction—get involved in the metabolism. At the maximal rate of glucose uptake, the CO2 carbon isotopic fractionation coefficient relative to organic products of microbial biosynthesis was determined to be α = 1.009 ± 0.002.

Using molecular and isotopic mass spectrometry, we investigated the toxic effect of naphthalene as a representative of polycyclic aromatic hydrocarbons (PAHs) on plants growing under sterile conditions and plants inoculated with microorganisms capable and incapable of naphthalene degradation. Tobacco plants of the Samsun variety were grown in a closed gas-nutrient system on a mineral medium with sucrose as a carbon source. Naphthalene used as a toxicant at a concentration of 5.2 × 10 −4 % contained 13 C isotope whose amount was characterized by the value δ 13 C = +281.4 ± 0.6‰ relative to the PDB standard and differed from that of sucrose, the main source of carbon (δ 13 C = −12.0 ± 0.1‰). Degradation of naphthalene was determined by the inclusion of its carbon in metabolic CO 2 and plant tissues (the root, stem, leaves). The effect of naphthalene on plants was indicated by the rates of O 2 production and CO 2 uptake during the light period as compared with the dark period of exposure. A decrease of the toxic effect of naphthalene on plants was observed only at the inoculation of plants with Pseudomonas aureofaciens BS1393 rhizosphere bacteria bearing plasmid pBS216, which controls the naphthalene biodegradation ability. The occurrence of other heterotrophic microorganisms incapable of naphthalene degradation had no similar protective effect.

На примере потребления глюкозы почвенными бактериями Pseudomonas aureofaciens BS1393(pBS216) и Rhodococcus sp. 3-30, иммобилизованными на твердофазной поверхности (кварцевый песок), определены их ростовые показатели: скорость роста бактерий (время удвоения клеток), суммарная продукция СО2 и продукция из расчета на бактериальную клетку, лаг-период относительно потребления субстрата, дыхательный коэффициент. При росте бактерий P. aureofaciens и Rhodococcus sp. на глюкозе выявлены различия штаммов по параметрам: (1) лаг-периодов относительно внесенного субстрата (лаг-период около 4 ч для P. aureofaciens и около 26 ч для Rhodococcus sp.); (2) максимальной скорости продукции СО2 (около 50 мкг СО2 г-1 песка ч-1 для P. aureofaciens и около 8.5 мкг СО2 г-1 песка ч-1 для Rhodococcus sp.); (3) продукции СО2 из расчета на одну клетку (около 1.94 ? 10-9 мкмоль СО2 для P. aureofaciens и около 3.4 ? 10-9 мкмоль СО2 для Rhodococcus sp.). Показано, что кинетика изотопного состава метаболической СО2 определяется различием в изотопных характеристиках углерода продуктов в клетке: после внесения глюкозы в среду (подготовительная стадия метаболизма) отмечено использование внутриклеточных обедненных 13 продуктов (липидов), на стадии максимальной скорости роста клеток метаболизируется, главным образом, внесенная глюкоза, и на завершающей стадии после исчерпания субстрата в обмен включаются “запасенные” продукты – липидная фракция. При максимальной скорости потребления глюкозы определен коэффициент фракционирования изотопов углерода СО2 относительно органических продуктов микробного биосинтеза, который составляет величину = 1.009 ± 0.002.

Исследован углеводородокисляющий потенциал почвенной микробиоты и интродуцированных в почву углеводородокисляющих микроорганизмов на основе количественных и изотопных характеристик углерода продуктов, образующихся при микробной деградации нефти. Из сравнения скоростей продукции СО2 в нативной почве и почве, загрязненной сырой нефтью, обнаружено, что интенсивность микробной минерализации почвенного органического вещества (ПОВ) в присутствии нефти выше по сравнению с незагрязненной почвой, т.е., обнаруживается затравочное влияние (прайминг-эффект) углеводородов нефти. Показано, что количество углерода вновь синтезированных органических продуктов за счет потребленной нефти (биомасса клеток и экзометаболиты) значительно превосходит количество ПОВ, израсходованное на продукцию СО2. Обнаружено, что в результате микробиологических процессов в почве, загрязненной нефтью, наблюдается мощный поток углекислоты, поступающей в атмосферу.

The hydrocarbon-oxidizing potential of soil microbiota and hydrocarbon-oxidizing microorganisms introduced into soil was studied based on the quantitative and isotopic characteristics of carbon in products formed in microbial degradation of oil hydrocarbons. Comparison of CO2 production rates in native soil and that polluted with crude oil showed the intensity of microbial mineralization of soil organic matter (SOM) in the presence of oil hydrocarbons to be higher as compared with non-polluted soil, that is, revealed a priming effect of oil. The amount of carbon of newly synthesized organic products (cell biomass and exometabolites) due to consumed petroleum was shown to significantly exceed that of SOM consumed for production of CO2. The result of microbial processes in oil-polluted soil was found to be a potent release of carbon dioxide to the atmosphere.

Using a theoretical model and mass isotopic balance, biogas (methane and CO2) released from buried products at their microbial degradation was analysed in the landfill of municipal and non-toxic industrial solid organic waste near Kaluga city, Russia. The landfill contains about 1.34×106 tons of waste buried using a ‘sandwich technique’ (successive application of sand–clay and waste layers). The δ13C values of biogenic methane with respect to CO2 were−56.8 (±2.5) ‰, whereas the δ13C of CO2 peaked at+9.12‰ (+1.4±2.3‰ on average), reflecting a virtual fractionation of carbon isotopes in the course of bacterial CO2 reduction at the landfill body. After passing through the aerated soil layers, methane was partially oxidised and characterised by δ13C in the range of−50.6 to−38.2‰, evidencing enrichment in 13C, while the released carbon dioxide had δ13C of−23.3 to−4.04‰, respectively. On the mass isotopic balance for the δ13C values, the methane production in the landfill anaerobic zone and the methane emitted through the aerated landfill surface to the atmosphere, the portion of methane oxidised by methanotrophic bacteria was calculated to be from 10 to 40% (averaged about 25%). According to the theoretical estimation and field measurements, the annual rate of methane production in the landfill reached about 2.9(±1.4)×109 g C CH4 yr−1 or 5.3(±2.6)×106 m3 CH4 yr−1. The average rates of methane production in the landfill and methane emission from landfill to the atmosphere are estimated as about 53 (±26) g C CH4 m−2 d−1 (or 4 (±2) mol CH4 m−2 d−1) and 33 (±12) g C CH4 m−2 d−1 (or 2.7 (±1) mol CH4 m−2 d−1), respectively. The calculated part of methane consumed by methanotrophic bacteria in the aerated part of the landfill was 13(±7) g C CH4 m−2 d−1 (or 1.1(±0.6) mol CH4 m−2 d−1) on average.

Commonly used clinical and biochemical parameters, such as the content of glucose, insulin, somatotropic hormone, triglycerides, lactate, pyruvate, and free fatty acids (FFA) in blood of practically healthy subjects and in patients with insulin-independent diabetes mellitus (IIDM), were compared with the parameters obtained by mass-spectrometric analysis of 13CO2 in expired air after 13C-glucose loading. It was shown that, as opposed to healthy subjects, the content of blood glucose and free fatty acids in patients with IIDM increased, the level of glucose dropped in progression upon short-term fasting, and the concentration of lactate changed both upon fasting and after the administration of small test doses of glucose. The use of the 13C-glucose breathing test (13C-GBT), which presupposes the loading of safe small doses of glucose enriched in 13C-isotope permitted one to reveal a number of novel quantitative diagnostic criteria for the evaluation of glucose metabolism in patients with IIDM: a decrease in the rate of 13C withdrawal as a constituent of expired carbon dioxide after the administration of 13C-glucose; a reduction in the amount of exogenous glucose metabolized to carbon dioxide; and increased oxidation of endogenous substrates participating in carbon dioxide formation. Small glucose loads proposed by the authors in 13C-GBT are safe for patients with diabetes mellitus and have no effect on the level of blood glucose in healthy persons. The parameters determined by noninvasive 13C-GBT are more sensitive for diagnosis than commonly used biochemical characteristics of blood in patients with IIDM. The diagnostic criteria obtained allow the prediction of the maximum prohibited glucose loading for every patient.

During alcohol fermentation, the carbon isotope composition of ethyl alcohol produced depended on the substrate used and was characterized by the value of delta 13C equal to -24.7 +/- 0.8/1000 (wheat grain), -22 +/- 0.1/1000 (rye grain), -22 +/- 0.5/1000 (products of wood hydrolysis), -15.3 +/- 0.3/1000 (maize grain) and -10 +/- 0.1/1000 (sugar cane). The isotope composition of carbon of ethyl alcohol obtained during catalytic hydroxylation of ethylene has a delta 13C of -30.6 +/- 0.3/1000. The possibility of quantitative determination of specific components in mixtures of ethanol samples with various isotope compositions (chemical synthesis and alcohol fermentation of raw material from C3- or C4-plants) was shown.

Fractionation of carbon isotopes by the bacterium Ectothiorhodospira shaposhnikovii was studied during its photomixotrophic growth on acetate (delta(13)C = -36.6 parts per thousand) and sodium bicarbonate (delta(13)C = -8.2 parts per thousand). It was shown that the carbon isotope composition of bacterial biomass essentially inherited that of acetate as the main carbon source. The content of C-13 in bicarbonate tended to decrease during mixotrophic growth of the bacterium due to the production of CO2 in the course of acetate metabolism. The metabolically produced carbon dioxide, as well as the cell biomass, had a carbon isotope composition close to that of acetate. Distribution of carbon isotopes in the main cellular fractions followed the pattern previously described for the photoautotrophically grown bacterium: the lipids were depleted of C-13 by -2 parts per thousand relative to the biomass and -4 parts per thousand relative to the protein and carbohydrate fractions.

The yeast Candida lipolytica grown on the glucose-containing medium with limited nitrogen source synthesizes citric acid. Schematically biosynthesis of citric acid involves condensation of oxaloacetate and acetyl-CoA, produced from pyruvate by carboxylation and decarboxylation respectively. We found that when C. lipolytica was grown on the glucose-containing mineral medium with limited nitrogen source at pH 4.5 the amount of exogenous CO2 incorporated into one of the citrate carboxyls was 20%, whereas it was only 8% at pH 6.0.