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.

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.

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.

Methylobacterium dichloromethanicum was found to be able to utilize dichloromethane (DCM) as the source of carbon and energy with the production of biomass, CO 2 , and HCl. A comparative analysis of the abundances of the major DCM isotopomers 35 Cl 2 12 C 1 H 2 , 35 Cl 37 Cl 12 C 1 H 2 , and 37 Cl 2 12 CH 2 1H 2 made it possible to estimate the fractionation of chlorine isotopes during the bacterial metabolism of DCM. The kinetic chlorine isotope effects for 35 Cl 37 Cl 12 C 1 H 2 ( m / z 86) and 37 Cl 2 12 C 1 H 2 ( m / z 88) relative to 35 Cl 2 12 C 1 H 2 ( m / z 84) were characterized by α 86/84 = 1.006 ± 0.002 and α 88/84 = 1.023 ± 0.003, respectively. The inference is made that the growth of M. dichloromethanicum on DCM is accompanied by the mass-independent fractionation of the DCM isotopomers.

The study deals with a comparative analysis of the relative abundances of the carbon isotopes 12 C and 13 C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic ( n -hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n -hexadecane (δ 13 C = –44.6 ± 0.2‰) were characterized by the values of δ 13 C CO 2 = –50.2 ± 0.4‰, δ 13 C biom = –46.6 ± 0.4‰, and δ 13 C exo = –41.5 ± 0.4‰, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (δ 13 C = –21 ± 0.4‰) were characterized by the isotope effects δ 13 C CO 2 = –24.1 ± 0.4‰, δ 13 C biom = –19.2 ± 0.4‰, and δ 13 C exo = –19.1 ± 0.4‰, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.