Stable isotope ratios and forensic analysis of microorganisms

In the aftermath of the anthrax letters of 2001, researchers have been exploring various analytical signatures for the purpose of characterizing the production environment of microorganisms. One such signature is stable isotope ratios, which in heterotrophs, are a function of nutrient and water sources. Here we discuss the use of stable isotope ratios in microbial forensics, using as a database the carbon, nitrogen, oxygen, and hydrogen stable isotope ratios of 247 separate cultures of Bacillus subtilis 6051 spores produced on a total of 32 different culture media. In the context of using stable isotope ratios as a signature for sample matching, we present an analysis of variations between individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times. Additionally, we correlate the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen for growth medium nutrients or water with those of spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures.     

An assessment of sample processing methods for stable isotope analyses of marine food webs

Carbon and nitrogen stable isotope ratios are commonly used in the study of marine food webs. However, different sample processing methods can influence the measurement of these stable isotope ratios. The purpose of this study is to define an adequate methodology to be used in the construction of whole food webs. It is demonstrated that acidification of the samples results in a decrease in carbon stable isotope values for sedimentary organic matter, suspended particulate organic matter, plankton and invertebrates with carbonated structures. The response was variable for nitrogen isotope ratios. Based on our results we recommend sample acidification for carbon analysis in these compartments where effects of this treatment were observed. We observed a decrease in δ¹³C values after washing with distilled water, so we do not recommend washing with water after acidification. For nitrogen analysis, acidification should be avoided. The various dehydration treatments studied caused significant differences only in nitrogen isotope ratios.     

Testing the long-term stability of marine isoscapes in shelf seas using jellyfish tissues

Maps of the spatial distribution of stable isotope ratios across wide geographic areas (isoscapes) are increasingly used to study mechanisms of nutrient flux, movements of animals, and to improve trophic information derived from stable isotope analyses. Isoscapes are usually constructed from reference samples collected from known geographic positions, a time consuming and costly process. In this study, we test the temporal stability of isoscapes of carbon and nitrogen isotopes across the North Sea over a ten-year period. Using jellyfish tissues as reference organisms, we show that hydrodynamic and biogeochemical processes controlling the distribution of carbon and nitrogen isotope values, and thus that the underlying isoscapes, are temporally stable. Remarkably, broad geographic variations in stable carbon and nitrogen isotope distributions across the North Sea are consistent with previously published variations seen in archaeological cod bones from the ninth to seventeenth centuries, despite dramatic changes in land use in the past 1,000 years. Stable isotope-based studies of trophic interactions or movements in animals with lifespans of a year or more in shelf ecosystems can consequently be referenced to previously published isoscape models, and do not require construction of temporally explicit isotope baseline corrections. Scyphomedusan jellyfish are excellent reference organisms for ecological stable isotope analyses in pelagic ecosystems, due to their widespread distributions, well-defined life histories, and fast tissue growth.     

Otolith oxygen and carbon stable isotopes in wild and laboratory-reared plaice (Pleuronectes platessa)

The relationship between water temperature, growth rate, and otolith isotopic ratios was measured for juvenile plaice (Pleuronectes platessa) reared at two temperatures (11 and 17°C) and two feeding regimes (1 and 3 prey items·ml^?1). The otolith isotope ratios in individual fish ranged from ?2 to ?4 for carbon isotope ratios (δ¹³C) and from 0.2 to 1.9 for oxygen isotope ratios (δ¹⁸O). The otolith oxygen isotope ratios were significantly affected by water temperature, but not by feeding level, and there were no significant synergistic effects. The fractionation of oxygen isotopes during otolith growth was independent of individual growth rate. Carbon isotope ratios were not significantly affected by food ration or water temperature, but were related to fish growth rate. The carbon isotope ratios were negatively correlated with fish length in the colder water treatments, and tended to increase with fish length in the warm water treatments. The laboratory-determined relationship between otolith oxygen isotope ratio and water temperature was applied to individuals of five species (plaice, cod, whiting, haddock, gurnard) collected in a single trawl sample. The otolith derived temperatures often overestimated measured water temperatures. The difference between real and estimated water temperatures varied between species, and the closest fit was for field-caught plaice.     

Isotope ratios of cellulose from plants having different photosynthetic pathways

Hydrogen and carbon isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from C₃, C₄, and Crassulacean acid metabolism (CAM) plants were determined for plants growing within a small area in Val Verde County, Texas. Plants having CAM had distinctly higher deuterium/hydrogen (D/H) ratios than plants having C₃ and C₄ metabolism. When hydrogen isotope ratios are plotted against carbon isotope ratios, each photosynthetic mode separates into a distinct cluster of points. C₄ plants had many D/H ratios similar to those of C₃ plants, so that hydrogen isotope ratios cannot be used to distinguish between these two photosynthetic modes. Portulaca mundula, which may have a modified photosynthetic mode between C₄ and CAM, had a hydrogen isotope ratio between those of the C₄ and CAM plants. When oxygen isotope ratios are plotted against carbon isotope ratios, no distinct clustering of the C₄ and CAM plants occurs. Thus, oxygen isotope ratios are not useful in distinguishing between these metabolic modes. A plot of hydrogen isotope ratios versus oxygen isotope ratios for this sample set shows considerable overlap between oxygen isotope ratios of the different photosynthetic modes without a concomitant overlap in the hydrogen isotope ratios of CAM and the other two photosynthetic modes. This observation is consistent with the hypothesis that higher D/H ratios in CAM plants relative to C₃ and C₄ plants are due to isotopic fractionations occurring during biochemical reactions.     

Growth and sporulation of Entomophthora virulenta on semidefined media in liquid culture

Entomophthora virulenta was tested for growth and sporulation under various nutritional conditions in shake cultures containing combinations of dextrose and protein hydrolysates. Darkness, a temperature of 25°C, and a pH near 6.5 induced the formation of the greatest number of zygospores when the carbon and nitrogen sources were held constant. The influence of the total concentration of nutrients and of the ratio of the carbon and nitrogen sources depended on the nitrogen source used: The highest amount of spores under these conditions was produced with yeast extract as nitrogen source, a carbon/nitrogen source ratio of 2, and a 15% total nutrient concentration. The influence of the preinoculum and inoculum on sporulation is discussed.     

稳定同位素技术在植物水分利用研究中的应用

近20a稳定同位素技术在植物生态学研究中的应用得到了长足发展,使得对植物与水分关系也有了更深一步的了解。介绍稳定同位素性碳、氢、氧同位素在研究植物水分关系中的应用及进展,以期能为国内植物水分利用研究提供参考。由于植物根系从土壤中吸收水分时并不发生同位素分馏,对木质部水分同位素分析有助于对植物利用水分来源,生态系统中植物对水分的竞争和利用策略的研究,更好地了解生态系统结构与功能。稳定碳同位素作为植物水分利用效率的一个间接指标,在不同水分梯度环境中,及植物不同代谢产物与水分关系中有着广泛的应用。同位素在土壤-植被-大气连续体水分中的应用,有助于了解生态系统的水分平衡。随着稳定同位素方法的使用,植物与水分关系的研究将取得更大的进展。     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria.

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inverse gradients in leaf wax δD and δ13C values along grass blades of Miscanthus sinensis: Implications for leaf wax reproduction and plant physiology

Compound specific hydrogen and carbon isotopic ratios of higher plant leaf waxes have been extensively used in paleoclimate and paleoenvironmental reconstructions. However, studies so far have focused on the comparison of leaf wax isotopic differences in bulk leaf samples between different plant species. We sampled three different varieties of tall grasses (Miscanthus sinensis) in six segments from base to tip and determined hydrogen and carbon isotopic ratios of leaf waxes, as well as hydrogen and oxygen isotopic ratios of leaf water samples. We found an increasing, base-to-tip hydrogen isotopic gradient along the grass blades that can probably be attributed to active leaf wax regeneration over the growth season. Carbon isotopic ratios, on the other hand, show opposite trends to hydrogen isotopic ratios along the grass blades, which may reflect different photosynthetic efficiencies at different blade locales.     

Equations for Lipid Normalization of Carbon Stable Isotope Ratios in Aquatic Bird Eggs

Stable isotope ratios are biogeochemical tracers that can be used to determine the source of nutrients and contaminants in avian eggs. However, the interpretation of stable carbon ratios in lipid-rich eggs is complicated because ¹³C is depleted in lipids. Variation in ¹³C abundance can therefore be obscured by variation in percent lipids. Past attempts to establish an algebraic equation to correct carbon isotope ratios for lipid content in eggs have been unsuccessful, possibly because they relied partly on data from coastal or migratory species that may obtain egg lipids from different habitats than egg protein. We measured carbon, nitrogen and sulphur stable isotope ratios in 175 eggs from eight species of aquatic birds. Carbon, nitrogen and sulphur isotopes were enriched in lipid-extracted egg samples compared with non extracted egg samples. A logarithmic equation using the C∶N ratio and carbon isotope ratio from the non extracted egg tissue calculated 90% of the lipid-extracted carbon isotope ratios within ±0.5‰. Calculating separate equations for eggs laid by species in different habitats (pelagic, offshore and terrestrial-influenced) improved the fit. A logarithmic equation, rather than a linear equation as often used for muscle, was necessary to accurately correct for lipid content because the relatively high lipid content of eggs compared with muscle meant that a linear relationship did not accurately approximate the relationship between percent lipids and the C∶N ratio. Because lipid extraction alters sulphur and nitrogen isotope ratios (and cannot be corrected algebraically), we suggest that isotopic measurement on bulk tissue followed by algebraic lipid normalization of carbon stable isotope ratio is often a good solution for homogenated eggs, at least when it is not possible to complete separate chemical analyses for each isotope.     

Increased snowfall weakens complementarity of summer water use by different plant functional groups

Winter snowfall is an important water source for plants during summer in semiarid regions. Snow, rain, soil water, and plant water were sampled for hydrogen and oxygen stable isotopes analyses under control and increased snowfall conditions in the temperate steppe of Inner Mongolia, China. Our study showed that the snowfall contribution to plant water uptake continued throughout the growing season and was detectable even in the late growing season. Snowfall versus rainfall accounted for 30% and 70%, respectively, of the water source for plants, on the basis of hydrogen stable isotope signature (δD) analysis, and accounted for 12% and 88%, respectively, on the basis of oxygen stable isotope signature (δ¹⁸O) analysis. Water use partitioning between topsoil and subsoil was found among species with different rooting depths. Increased snowfall weakened complementarity of plant water use during summer. Our study provides insights into the relationships between precipitation regimes and species interactions in semiarid regions.     

Oxygen and Hydrogen Isotope Fractionation during Cellulose Metabolism in Lemna gibba L

Lemna gibba L. B3 was grown under heterotrophic, photoheterotrophic, and autotrophic conditions in water having a variety of hydrogen and oxygen isotopic compositions. The slopes of the linear regression lines between the isotopic composition of water and leaf cellulose indicated that under the three growth conditions about 40, 70, and 100% of oxygens and carbon-bound hydrogens of cellulose exchanged with those of water prior to cellulose formation. Using the equations of the linear relationships, we estimated the overall fractionation factors between water and the exchanged oxygen and carbon bound-hydrogen of cellulose. At least two very different isotope effects must determine the hydrogen isotopic composition of Lemna cellulose. One reflects the photosynthetic reduction of NADP, while the second reflects exchange reactions that occur subsequent to NADP reduction. Oxygen isotopic composition of cellulose apparently is determined by a single type of exchange reaction with water. Under different growth conditions, variations in metabolic fluxes affect the hydrogen isotopic composition of cellulose by influencing the extent to which the two isotope effects mentioned above are recorded. The oxygen isotopic composition of cellulose is not affected by such changes in growth conditions.     

脂类抽提对北太平洋柔鱼肌肉碳、氮稳定同位素测定结果的影响

稳定同位素技术可深层次地分析头足类的摄食习性和栖息地等方面的重要信息.稳定同位素分析多选取动物肌肉为研究组织,然而,肌肉中的脂类含量会影响对其稳定同位素信息的准确解析.北太平洋柔鱼是重要的大洋经济性头足类,在海洋生态系统中具有重要的生态地位.本研究选取了53尾北太平洋柔鱼,通过分析脂类抽提对胴体肌肉稳定同位素比值的影响,探讨脂类物质对稳定同位素比值的干扰机制,对比不同碳稳定同位素比值校正模型的适用性,并提出适用于北太平洋柔鱼胴体肌肉δ¹³-C校正模型.结果表明： 北太平洋柔鱼肌肉的脂类抽提会引起稳定同位素测定结果的显著变化,δ¹³-C和δ¹⁵-N分别平均升高0.71‰和0.47‰,在分析组织碳稳定同位素比值时,脂类抽提在样品预处理过程中十分必要,其能消除脂类对样品稳定同位素分析的干扰,从而更加准确地分析研究对象食性与栖息地的变化.在单独进行氮稳定同位素比值分析时,则不需要进行脂类抽提.     

Duration of Hydrogen Formation by Anabaena cylindrica B629 in Atmospheres of Argon, Air, and Nitrogen

The time course of hydrogen formation by Anabaena cylindrica was followed beneath an argon atmosphere alone and also beneath atmospheres of argon, nitrogen, and air in the presence of carbon monoxide (0.2%) and acetylene (5%). Hydrogen production beneath argon alone was comparable in rate and duration (7 to 12 days) to that which occurred beneath air in the presence of carbon monoxide (0.2%) and acetylene (5%). However, much greater longevity (16 to 26 days) and improved rates of hydrogen formation were obtained when algae were incubated beneath argon and particularly nitrogen, each supplemented with carbon monoxide and acetylene. The total hydrogen produced by these cultures was up to three times as much as that released by cultures incubated beneath argon alone. Hydrogen-oxygen ratios for argon cultures either with or without carbon monoxide and acetylene were initially 1:5 but approximated 1:2 when measured over the entire incubation period. In each case oxygen production and nitrogenase activity (acetylene reduction) continued at reduced rates after hydrogen evolution had ceased. The effects of methionine sulfoximine (2 μM), ammonium ions (0.5 mM), or both on oxygen production were generally negligible, while effects on hydrogen production were variable depending on the atmosphere used; in most cases, eventual destabilization of the system occurred. A brief comparison was made of the time courses of anaerobic and aerobic hydrogen formation by the marine cyanobacterium Calothrix membranacea. It was found that shaking of cultures was beneficial for hydrogen production but not strictly necessary. It is concluded that hydrogen production by A. cylindrica in air and particularly nitrogen in the presence of carbon monoxide and acetylene offers the best potential of the atmospheres considered on the basis of four criteria: rates and longevity of hydrogen formation, practicality of the atmosphere used, and tolerance of hydrogen evolution to slight changes in composition of the atmosphere.     

Tree-ring growth and stable isotopes (13C and 15N) detect effects of wildfires on tree physiological processes in Pinus sylvestris L.

Forest fires may alter the physiological and growth processes of trees by causing stress in trees and modifying the availability of soil nutrient. We investigated if, after a high-severity fire, changes in tree-ring growth can be observed, as well as changes in the nitrogen and carbon isotope composition of tree rings of surviving trees. Two wildfires that occurred in Pinus sylvestris L. stands in Northern Italy, one at the beginning and one at the end of the vegetative season, were chosen as the focus of this study. After the fires, the surviving trees showed growth suppression with very narrow tree rings or locally absent rings. The carbon isotope ratio was more negative in tree rings formed in the 5 years following fire, indicating better water supply in a situation of less competition. The nitrogen isotope ratio followed opposite trends in the two wildfire stands. In trees cored in the stand where the fire happened at the beginning of the vegetative season, there was no change in the nitrogen isotope ratio, whereas in samples collected in the other fire site, higher nitrogen isotope ratios were observed in the tree rings formed after the fire, reflecting changes in the soil nitrogen supply. Modifications in the growth and isotope composition of the fire-stressed trees disappeared from 6 to 10 years after the fire. By studying trees before and after fire, we were able to show that fire affects not only the growth of surviving trees, but also their physiological processes.     

稳定氢氧同位素定量植物水分来源的不确定性解析

稳定氢氧同位素技术被广泛运用于生态系统、特别是干旱区生态系统中植物水分来源的研究,其理论假设为"水分被植物根系吸收并向木质部运输过程中不发生氢氧同位素分馏"。生态系统中不同水源的氢氧同位素组成普遍存在显著差异,为从水源混合体中区分出各水源的贡献率提供了前提条件。但在实际应用过程中,诸多因素导致稳定氢氧同位素技术定量植物水分来源的结果具有不确定性。综合已有研究并加以分析,举证说明植物吸收水分相对于水源同位素变化的滞后性、水源同位素的季节性变化、蒸发作用和水源之间的混合作用对水源同位素的影响等导致植物水分来源定量结果不确定性的几个因素,以期为今后稳定氢氧同位素技术在植物水分来源领域的应用提供参考。     

Biosynthesis of amylase inhibitor by Streptomycete cultures

We studied the effects of different sources of carbon, nitrogen, and dietary elements on the biosynthesis of amylase inhibitor produced by two strains from the Russian National Collection of Industrial Microorganisms: Streptomyces violaceus (code: Ac-1734) and Streptomyces lucensis (Ac-1743). We found that the biosynthesis of amylase inhibitor can be regulated by limiting carbon and oxygen concentrations, maintaining constant a C: N ratio and a stable proportion of carbohydrates (dextrins, maltose and glucose) in the medium, and by adding an extra organic source of nitrogen to the medium. The inhibitors produced by the cultures are pseudo-polysaccharides.     

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.     

Control of Origin of Sesame Oil from Various Countries by Stable Isotope Analysis and DNA Based Markers—A Pilot Study

The indication of origin of sesame seeds and sesame oil is one of the important factors influencing its price, as it is produced in many regions worldwide and certain provenances are especially sought after. We joined stable carbon and hydrogen isotope analysis with DNA based molecular marker analysis to study their combined potential for the discrimination of different origins of sesame seeds. For the stable carbon and hydrogen isotope data a positive correlation between both isotope parameters was observed, indicating a dominant combined influence of climate and water availability. This enabled discrimination between sesame samples from tropical and subtropical/moderate climatic provenances. Carbon isotope values also showed differences between oil from black and white sesame seeds from identical locations, indicating higher water use efficiency of plants producing black seeds. DNA based markers gave independent evidence for geographic variation as well as provided information on the genetic relatedness of the investigated samples. Depending on the differences in ambient environmental conditions and in the genotypic fingerprint, a combination of both analytical methods is a very powerful tool to assess the declared geographic origin. To our knowledge this is the first paper on food authenticity combining the stable isotope analysis of bio-elements with DNA based markers and their combined statistical analysis.