Evaluation of lipid extraction methods for fatty acid quantification and compound-specific δ13C and δ2Hn analyses.

Lipids, with fatty acids (FA) as a crucial subset, have become a focal point for diverse medical, physiological, and ecological studies. However, a comprehensive assessment of the various pre-analytical FA extraction methods published in the scientific literature remains lacking. In this study, we examined the efficacy of seven well-established sample preparation methods, specifically focusing on their effectiveness in total lipid and fatty acid extraction and their impact on compound-specific stable hydrogen (δ2H) and carbon (δ13C) isotope values. We also considered the repercussions of FA removal efficacy on residual bulk tissue δ2Hn analysis, because lipids typically have low δ2H values. Our findings showed that in most cases chloroform-based extraction methods outperformed those without chloroform. While discrepancies were not as evident for smaller organisms, such as plankton, marked variations were discernible in the extraction efficiencies for muscle and liver samples, which was also manifested in the residual bulk tissue δ2Hn results. Notably, most extraction methods had little effect on specific δ13C or δ2H isotope values of FA; instead, an emphasis should be on using an extraction method that achieves optimal baseline peak separation of the chromatograms for C and H isotope measurements.

Compound-specific stable isotope analyses of fatty acids indicate feeding zones of zooplankton across the water column of a subalpine lake.

Spatial and temporal zooplankton feeding dynamics across the water column of lakes are key for understanding site-specific acquisition of diet sources. During this 6-week lake study, we examined stable carbon (δ13C) and nitrogen (δ15N) isotopes and conducted compound-specific fatty acid (FA) stable isotope analysis (CSIA) of edible seston in the epi-, meta-, and hypolimnion, and zooplankton of Lake Lunz, Austria. We predicted that CSIA of essential FA can discern the foraging grounds of zooplankton more accurately than the commonly used bulk stable isotopes. The δ13C and δ15N values of seston from different lake strata were similar, whereas a dual CSIA approach using stable carbon and hydrogen isotopes of FA (δ13CFA and δ2HFA) provided sufficient isotopic difference in essential FA to discern different lake strata-specific diet sources throughout the study period. We present a CSIA model that suggests strata-specific foraging grounds for different zooplankton groups, indicating higher preference of cladocerans for feeding on epilimnetic diet sources, while calanoid copepods retained more hypolimnetic resources. The CSIA approach thus yields strata-specific information on foraging strategies of different zooplankton taxa and provides more details on the spatial and temporal trophodynamics of planktonic food webs than commonly used bulk stable isotopes.

Mass-Transfer-Limited Biodegradation at Low Concentrations-Evidence from Reactive Transport Modeling of Isotope Profiles in a Bench-Scale Aquifer.

Organic contaminant degradation by suspended bacteria in chemostats has shown that isotope fractionation decreases dramatically when pollutant concentrations fall below the (half-saturation) Monod constant. This masked isotope fractionation implies that membrane transfer is slow relative to the enzyme turnover at µg L-1 substrate levels. Analogous evidence of mass transfer as a bottleneck for biodegradation in aquifer settings, where microbes are attached to the sediment, is lacking. A quasi-two-dimensional flow-through sediment microcosm/tank system enabled us to study the aerobic degradation of 2,6-dichlorobenzamide (BAM), while collecting sufficient samples at the outlet for compound-specific isotope analysis. By feeding an anoxic BAM solution through the center inlet port and dissolved oxygen (DO) above and below, strong transverse concentration cross-gradients of BAM and DO yielded zones of low (µg L-1) steady-state concentrations. We were able to simulate the profiles of concentrations and isotope ratios of the contaminant plume using a reactive transport model that accounted for a mass-transfer limitation into bacterial cells, where apparent isotope enrichment factors *ε decreased strongly below concentrations around 600 µg/L BAM. For the biodegradation of organic micropollutants, mass transfer into the cell emerges as a bottleneck, specifically at low (µg L-1) concentrations. Neglecting this effect when interpreting isotope ratios at field sites may lead to a significant underestimation of biodegradation.

Classification of nine malathion emulsion samples by using carbon isotope ratios and the ratio of organic solvents.

The compound specific isotope analysis is nowadays an important and powerful tool in geochemical, environmental and forensics field. On November 2013, Aqli Foods Corporation in Japan dealt with complaints about stench from frozen foods produced. Subsequently, very high concentrations of organophosphorus pesticide as malathion, ethylbenzene and xylene were detected in recovered frozen foods. In particular case, we present the method to measure the stable carbon isotope ratio (δ13C) of nine malathion emulsion pesticides using gas chromatography/isotope ratio mass spectrometry (GC/IRMS) to identify the source. The δ13C values of malathion ranged from -30.6‰ to -29.5‰. Because malathion used in all malathion emulsions sold in Japan is imported from the same overseas company, Cheminova, Denmark. The δ13C values of ethylbenzene ranged from -28.2‰ to -20.8‰ and those of m,p-xylene from -28.7‰ to -25.2‰. The differences in the δ13C values may be because of the material itself and chemical processing. We also determined the ratio of ethylbenzene to m,p-xylene and finally categorized the nine malathion samples into five groups on the basis of this ratio and the δ13C values of ethylbenzene and m,p-xylene. The results of isotopic fractionation during volatilization (refrigerate, room temperature and incubator) was negligible small.

Comparison of Carbon Isotope Ratio Measurement of the Vanillin Methoxy Group by GC-IRMS and 13C-qNMR.

Site-specific carbon isotope ratio measurements by quantitative 13C NMR (13C-qNMR), Orbitrap-MS, and GC-IRMS offer a new dimension to conventional bulk carbon isotope ratio measurements used in food provenance, forensics, and a number of other applications. While the site-specific measurements of carbon isotope ratios in vanillin by 13C-qNMR or Orbitrap-MS are powerful new tools in food analysis, there are a limited number of studies regarding the validity of these measurement results. Here we present carbon site-specific measurements of vanillin by GC-IRMS and 13C-qNMR for methoxy carbon. Carbon isotope delta (δ13C) values obtained by these different measurement approaches demonstrate remarkable agreement; in five vanillin samples whose bulk δ13C values ranged from -31‰ to -26‰, their δ13C values of the methoxy carbon ranged from -62.4‰ to -30.6‰, yet the difference between the results of the two analytical approaches was within ±0.6‰. While the GC-IRMS approach afforded up to 9-fold lower uncertainties and required 100-fold less sample compared to the 13C-qNMR, the 13C-qNMR is able to assign δ13C values to all carbon atoms in the molecule, not just the cleavable methoxy group.

Authentication of Fennel, Star Anise, and Anise Essential Oils by Gas Chromatography (GC/MS) and Stable Isotope Ratio (GC/IRMS) Analyses.

The aromatic compound (E)-anethol is widely used in the flavor, fragrance, and medicinal industries. This compound is commonly produced through steam distillation of fennel, star anise, and anise seed. Given the cost of production, these natural and authentic essential oils are commonly adulterated with lower-cost natural materials or synthetic alternatives. The current study investigates essential oil profiles (gas chromatography/mass spectrometry) and stable isotope ratios (gas chromatography/isotope ratio mass spectrometry) of the abundant compound (E)-anethol in both authentic reference standards (n = 15) and commercially available samples (n = 30). This multifaceted analytical approach establishes techniques for ensuring the authenticity of essential oil sources of (E)-anethol and was then used to evaluate the current essential oil market sources of (E)-anethol. These findings report that adulteration of (E)-anethol-containing natural products takes various forms, and a multifaceted analytical approach is recommended for authentication. Of the commercial samples analyzed for this report, 27% were adulterated.

A small-scale neutral alumina column chromatography method for carbon isotope determination of hopanes in crude oils or rock extracts.

This paper presents a small-scale column chromatography method for separating hopanes in crude oil or rock extracts using neutral alumina as a solid phase adsorbent and a Pasteur pipette as a separation device. Three oil samples were selected to study the effect of solid phase adsorbent type and column length on the separation of hopanes. The oil samples were eluted with mixed reagents (V hexane: V petroleum ether = 8:2) and collected at intervals of 0.5 ml. Ten Fractions were collected and tested for the compounds using GC-MS. A quantitative approach was used to reveal distribution characteristics of compounds in each eluted Fraction. Experimental results showed that 100-200 um neutral alumina exhibited significant differences in the adsorptive capacity of biomarkers from oil samples and rock extracts. The elution order of the biomarkers in the chromatographic column (the length is 180 mm) was n-alkanes, steranes and hopanes. The separation of hopanes could be realized by collecting the eluted Fractions 4 and 5. Compared with the urea complexation, the purity of hopanes separated by column chromatography was higher. The concentration of n-alkanes (nC16-nC34) could be reduced from 1.99 to 4.83 mg/ml to 0.79-0.94 mg/ml, and the content of steranes can be reduced from the original 12% to 0.45%. Residual n-alkanes and steranes were not visible in the GC-MS detection. The Total Ion Chromatography (TIC) of hopanes was consistent with the distribution characteristics of the m/z191 mass chromatogram. The isolated hopanes could meet the detection requirements of isotope ratio mass spectrometry. The C29Ts/C29αß ratio of hopanes decreased gradually from 1.63 to 0.73 as the column length increased. It is speculated that the variation of the C29Ts/C29αß ratio is not only affected by maturity but also by the oil and gas migration. This method is a new attempt in the field of compound purification and can be widely used in the study of stable carbon isotopes of hopanes monomeric hydrocarbons.

Leaf-level metabolic changes in response to drought affect daytime CO2 emission and isoprenoid synthesis pathways.

In the near future, climate change will cause enhanced frequency and/or severity of droughts in terrestrial ecosystems, including tropical forests. Drought responses by tropical trees may affect their carbon use, including production of volatile organic compounds (VOCs), with implications for carbon cycling and atmospheric chemistry that are challenging to predict. It remains unclear how metabolic adjustments by mature tropical trees in response to drought will affect their carbon fluxes associated with daytime CO2 production and VOC emission. To address this gap, we used position-specific 13C-pyruvate labeling to investigate leaf CO2 and VOC fluxes from four tropical species before and during a controlled drought in the enclosed rainforest of Biosphere 2 (B2). Overall, plants that were more drought-sensitive had greater reductions in daytime CO2 production. Although daytime CO2 production was always dominated by non-mitochondrial processes, the relative contribution of CO2 from the tricarboxylic acid cycle tended to increase under drought. A notable exception was the legume tree Clitoria fairchildiana R.A. Howard, which had less anabolic CO2 production than the other species even under pre-drought conditions, perhaps due to more efficient refixation of CO2 and anaplerotic use for amino acid synthesis. The C. fairchildiana was also the only species to allocate detectable amounts of 13C label to VOCs and was a major source of VOCs in B2. In C. fairchildiana leaves, our data indicate that intermediates from the mevalonic acid (MVA) pathway are used to produce the volatile monoterpene trans-ß-ocimene, but not isoprene. This apparent crosstalk between the MVA and methylerythritol phosphate pathways for monoterpene synthesis declined with drought. Finally, although trans-ß-ocimene emissions increased under drought, it was increasingly sourced from stored intermediates and not de novo synthesis. Unique metabolic responses of legumes may play a disproportionate role in the overall changes in daytime CO2 and VOC fluxes in tropical forests experiencing drought.

Protocols for sample preparation and compound-specific stable-isotope analyses (δ2H, δ13C) of fatty acids in biological and environmental samples.

Compound-specific stable-isotope analysis (CSIA) of fatty acids is a powerful tool to better understand the trophic transfer of fatty acids and their biochemical fate in and across ecosystems, including tracing animal migration and understanding physiological processes. The non-exchangeable nature of C-H bonds in acyl chains, hydrogen (δ2H) and carbon (δ13C) stable-isotope values of fatty acids (FA) provide independent information about the origins of fatty acids. Several technical obstacles must be overcome to ensure accurate and reproducible measurements of FA-CSIA can be made. This protocol describes the sample preparation process for successful stable-isotope analyses of fatty acids obtained from environmental and biological samples. Numerous techniques for the preanalytical processing of fatty acid samples are available, and these often have minimal impact on δ values. Here, we provide an in-depth guide detailing our well-established laboratory protocols, ranging from the initial sample preparation, lipid extraction, and transmethylation to the instrumental arrangement, data collection, and analysis.•Protocol from obtaining a sample to standardized fatty acid specific δ2H and δ13C values.•Separate GC analysis procedures for C and H are recommended for optimal performance.

[Measurement and research status of stable hydrogen isotopes in tree-ring lignin methoxyl groups]. / æ ‘è½®æœ¨è´¨ç´ ç”²æ°§åŸºç¨³å®šæ°¢åŒä½ç´ æ¯”çŽ‡æµ‹å®šæ–¹æ³•ç ”ç©¶è¿›å±•.

The primary hydrogen (H) source for all organic compounds in the biosphere is from water, and then participates in biogeochemical cycles through photosynthesis and plant physiological metabolism. As a new proxy of paleoclimate and paleoenvironment, stable hydrogen isotope ratios in wood lignin methoxyl groups (δ2HLM) show great advantages in the studies of paleoclimatic change and have been used to reconstruct precipitation stable hydrogen isotope ratios and paleoclimate signals in many regions. Based on the lignin application mechanism and analysis method of δ2HLM, we evaluated the stability and effectiveness of δ2HLM-measurement method from lignin content and lignin monomer composition, and expounded the tree lignin methoxyl groups' stable isotope proxies of current research outcomes. In the middle latitudes, the tree-ring δ2HLM had great potential in recording temperature signals and precipitation stable hydrogen isotope ratios. However, the study of tree-ring δ2HLM was still in its infancy as evidenced by following reasons: 1) The study area was limited to the middle latitude of the northern hemisphere, and the study subjects were limited to conifer species; 2) To compensate for the limitation of hydrogen isotopic records of nitrocellulose, high resolution tree-ring δ2HLM would be studied; 3) The potential of tree-ring δ2HLM utilization in plant physiology and forest ecology remained to be exploited.

The influence of carbon limitation on growth of Heterosigma akashiwo: A case study in fatty acids composition.

Heterosigma akashiwo is an algal blooms species, and thus selected as the target microalgae in this work. This study attempted to investigate the influence of carbon limitation on the growth of H. akashiwo. Experiments were carried out in CO2-unlimited and CO2-limited systems (both include three nutritional groups). The stable isotope signatures and compound-specific stable three nutritional groups carbon isotopic composition of fatty acids were measured. Here we hypothesized that the carbon limitation could lead to the enriched of stable isotope ratios in the H. akashiwo. The results showed that carbon limitation made δ13C and δ13CFAs values more and more enriched in H. akashiwo. δ13C values were enriched in normal group of H. akashiwo within CO2-limited. δ15N values were enriched in nitrogen deficiency of H. akashiwo within both CO2-unlimited and CO2-limited. Furthermore, compared with the exponential phase, the enriched in δ13C was detected during the stationary phase in H. akashiwo within CO2-limited. A total of 8 major FAs were detected in H. akashiwo. Within CO2-unlimited, nitrogen deficiency promoted the synthesis of 4 FAs (14:1n-5c, 16:0, 18:0 and 18:3n-6c) in exponential phase. Within CO2-limited, nitrogen deficiency promoted the synthesis of FAs 14:0, 16:0 and 18:3n-3c, while phosphorus deficiency promoted the synthesis of all 6 FAs in exponential phase. δ13CFAs of H. akashiwo within CO2-limited showed the valley values and were significantly greater than those within CO2-unlimited. Furthermore, δ13CFAs of stationary phase were greater than those of exponential phase. δ13C14:1n-5c in nitrogen deficiency were the highest of the three nutrient groups within CO2-unlimited. This may point out that δ13CFAs could become an indicator of marine phytoplankton blooms. Overall, the present study may provide a novel approach to investigate the physiology and lipid metabolism of H. akashiwo blooms by using stable isotope ratios coupled with FAs profiles.

Stable isotope analysis of non-exchangeable hydrogen in carbohydrates derivatised with N-methyl-bis-trifluoroacetamide by gas chromatography - Chromium silver reduction/High temperature Conversion-isotope ratio mass spectrometry (GC-CrAg/HTC-IRMS).

A novel procedure for the rapid isotope analysis of the carbon-bound non-exchangeable (CBNE) hydrogen in mono and disaccharides has been developed to demonstrate the feasibility of detecting undeclared addition of exogenous sugar products in foods and beverages susceptible to economically motivated adulteration. The procedure utilizes a simple one-step reaction, with the derivatising agent N-methyl-bis-trifluoroacetamide, to substitute the exchangeable hydroxyl-hydrogens with trifluoroacetate derivatives that are sufficiently volatile to be separated and measured by a gas chromatograph coupled to an isotope ratio mass spectrometer. The conversion of the derivatised sugars into the measuring gas is achieved using a high temperature chromium-silver reactor that retains carbon, oxygen and fluorine whilst releasing hydrogen gas for stable isotope measurement. The new procedure has advantages over existing methods in terms of ease of use, analysis time and compound-specific information. Sugars from fruit juice and honey have been measured to demonstrate the feasibility of using this technique.

Geographical origin of Chinese wolfberry (goji) determined by carbon isotope analysis of specific volatile compounds.

Chinese wolfberry or goji berry (Lycium barbarum) is an important traditional Chinese medicine. Its price and function has a close correlation with its geographical provenance. Illegal mislabeling motivated by commercial gains brings serious food safety problems and damages consumer confidence. In this work, a novel analytical strategy combined with chemometrics statistic tools was developed to determine the geographical origin of wolfberries from different provinces in China. Stable carbon isotopic ratios (δ13C) of wolfberry volatile compounds (i.e. limonene, tetramethylpyrazine, safranal, geranylacetone, and ß-ionone) were determined by gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS) with headspace-solid phase micro extraction (HS-SPME). Five types of SPME fiber (i.e. DVB/CAR/PDMS, CAR/PDMS, PDMS/DVB + OC, PDMS, and PA), extraction time, temperature and GC-IRMS conditions were comprehensively optimized to obtain the best adsorption of volatile compounds in wolfberry. Method integrity was assessed by comparing volatiles extracted using HS-SPME GC-IRMS with direct injection GC-IRMS and were in good agreement with each other. The geographical variations of volatile compounds using one-way analysis of variance (ANOVA) were explored for individual δ13C values in wolfberry samples from Gansu, Ningxia and Qinghai. Geographical origin of wolfberry was differentiated by linear discrimination analysis (LDA), with an accuracy of 89.16%, 87.77% and 85.87% for these three provinces, respectively. These results showed the combination of SPME and IRMS provides a rapid and valid method to determine the geographical origin of wolfberry.

The use of stable isotope ratio analysis to characterise saw palmetto (Serenoa Repens) extract.

Saw palmetto extract (SPE) has many pharmacological effects. Thus, its demand and value has increased steadily, along with the presence of counterfeit SPEs on the market. In this work bulk δ13C, δ2H, δ18O and fatty acid δ13C, δ2H analysis was performed in 20 authentic and 9 commercial SPEs, 12 meat fats and 4 pure fatty acids. Authentic SPEs are characterised by bulk values from -31.0‰ to -29.7‰ for δ13C, -176‰ to -165‰ for δ2H, 27.2‰ to 40.7‰ for δ18O, and values of capric, caprylic, lauric, myristic, palmitic and oleic acids from -37.4‰ to -30.5‰ for δ13C and -187‰ to -136‰ for δ2H. The isotopic values of all the commercial SPEs were out of these ranges and more similar to those of meat fat and pure fatty acids. Stable isotope ratio analysis can therefore be proposed as a suitable tool for detecting adulteration in SPEs.

H2 Kinetic Isotope Fractionation Superimposed by Equilibrium Isotope Fractionation During Hydrogenase Activity of D. vulgaris Strain Miyazaki.

We determined 2H stable isotope fractionation at natural abundances associated with hydrogenase activity by whole cells of Desulfovibrio vulgaris strain Miyazaki F expressing a NiFe(Se) hydrogenase. Inhibition of sulfate reduction by molybdate inhibited the overall oxidation of hydrogen but still facilitated an equilibrium isotope exchange reaction with water. The theoretical equilibrium isotope exchange δ2H-values of the chemical exchange reaction were identical to the hydrogenase reaction, as confirmed using three isotopically different waters with δ2H-values of - 62, +461, and + 1533‰. Expected kinetic isotope fractionation of hydrogen oxidation by non-inhibited cells was also superimposed by an equilibrium isotope exchange. The isotope effects were solely catalyzed biotically as hydrogen isotope signatures did not change in control experiments without cells of D. vulgaris Miyazaki.

C and H stable isotope ratio analysis using solid-phase microextraction and gas chromatography-isotope ratio mass spectrometry for vanillin authentication.

It is possible to distinguish precious vanillin from Vanilla species (planifolia or tahitensis) from much less expensive synthetic and nature-identical vanillin on the basis of the stable isotope ratios of H and C (2H/1H, 13C/12C). Analysis is usually performed using GC-IRMS (Gas Chromatography - Isotope Ratio Mass Spectrometry) after solvent extraction of vanillin from the sample. Recently, head-space solid-phase microextraction (HS-SPME) has been proposed as an alternative for determining 13C/12C. The aim of this study was to develop a method to analyse 2H/1H in vanillin using SPME-GC-IRMS for the first time, by testing different operating conditions and comparing the results with those obtained after solvent extraction. The ultimate scope was to develop a quick, robust and effective method to measure 2H/1H and 13C/12C in vanillin to assess the authenticity of labelling. Almost 50 authentic samples from vanilla pods, nature-identical (ex) and synthetic vanillin and 4 commercial food products were taken into account. All the samples were subjected to HS-SPME-GC-IRMS analysis and most of them to GC-IRMS analysis after solvent extraction of vanillin. The SPME method developed for 2H/1H analysis guarantees the absence of isotopic fractionation, repeatability and reproducibility standard deviation of below 7‰ and savings in terms of time (from 30 to 5 min) and solvent. HS-SPME GC-IRMS analysis of δ2H and δ13C can be proposed as a rapid and robust method to discriminate different types of vanillin and assess the authenticity of natural vanillin, also contained in food matrices.

Stable Isotope Probing of Microbial Phospholipid Fatty Acids in Environmental Samples.

Phospholipid fatty acid (PLFA) extracted from environmental samples describe the microbial community pattern and are sensitive to monitor and quantify shifts in the microbial community. Linkage with the stable isotope technique adds a functional perspective and is frequently used to quantify carbon turnover in microbial communities and detect physiological changes. Here we present a PLFA extraction method by using an organic solvent water mixture, followed by lipid separation based on solid-phase extraction and an alkaline methylation. Finally, we provide a protocol for the carbon stable isotope measurements of the extracted fatty acid methyl esters (FAMEs) by gas chromatograph-isotope ratio mass spectrometer (GC-IRMS) and calculation of concentration and δ13CVPDB values.

Characteristics and compound-specific carbon isotope compositions of sedimentary lipids in high arsenic aquifers in the Hetao basin, Inner Mongolia.

Organic matter, as an electron donor, plays a vital role in As mobilization mediated by microorganisms during reductive dissolution of Fe/Mn oxides in shallow aquifers. However, the specific types and sources of organic matter involved in biogeochemical processes accelerating As mobilization are still controversial. Both sediment and groundwater samples were collected at different depths from aquifers of the Hetao Basin, a typical inland basin hosting high As groundwater. Sedimentary lipids and their compound-specific carbon isotope ratios were analyzed to evaluate characteristics and sources of organic matter. Results show that sedimentary As were well correlated with Fe and Mn oxides, suggesting that As exist as Fe/Mn oxide bound forms. Groundwater As far exceeded the drinking water guide value of 10 µg/L. Moreover, As concentrations in shallow groundwater were relatively higher. Lipids in clay were mainly originated from terrestrial higher plants, while that in fine sand samples were derived from terrestrial higher plants, microorganism and petroleum. Shallow fine sand samples were also characterized by evident in-situ biodegradation. Compound-specific carbon isotope compositions of sedimentary lipids showed that short-chain n-alkanes and n-alkanoic acids had more positive δ13C values compared to long-chain compounds, especially in shallow fine sand samples. δ13CTOC were also low in shallow fine sand samples. These results jointly indicate that these lipids in shallow fine sand samples acted as carbon source for indigenous microorganism and the short-chain components were particularly more vulnerable to biodegradation, which may contribute to high As concentrations in shallow groundwater. The new findings provide the first evidence that short chain length n-alkyl compounds afforded a source of potential electron donors for microbially mediated As mobilization process in the shallow aquifers.

Isotopic Fractionation of Sulfur in Carbonyl Sulfide by Carbonyl Sulfide Hydrolase of Thiobacillus thioparus THI115.

Carbonyl sulfide (COS) is one of the major sources of stratospheric sulfate aerosols, which affect the global radiation balance and ozone depletion. COS-degrading microorganisms are ubiquitous in soil and important for the global flux of COS. We examined the sulfur isotopic fractionation during the enzymatic degradation of COS by carbonyl sulfide hydrolase (COSase) from Thiobacillus thioparus THI115. The isotopic fractionation constant (34ɛ value) was -2.2±0.2‰. Under experimental conditions performed at parts per million by volume level of COS, the 34ɛ value for intact cells of T. thioparus THI115 was -3.6±0.7‰, suggesting that, based on Rees' model, the 34ɛ value mainly depended on COS transport into the cytoplasm. The 34ɛ value for intact cells of T. thioparus THI115 was similar to those for Mycobacterium spp. and Williamsia sp., which are known to involve the conserved region of nucleotide sequences encoding the clade D of ß-class carbonic anhydrase (ß-CA) including COSase. On the other hand, the 34ɛ value was distinct from those for bacteria in the genus Cupriavidus. These results provide an insight into biological COS degradation, which is indispensable for estimating the COS global budget based on the isotope because of the significant contribution of COS degradation by microorganisms harboring ß-CA family enzymes.