The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C 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 (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_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 (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_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.     

Stimulation of crystallin synthesis in embryonic brain cells in culture

Summary Expression of -crystallin, a lens-specific protein, in 6-day-old chick embryonic brain cells was examined in situ and in vitro. The presence of minute amounts of -crystallin and its mRNA (-mRNA) in brain cells in situ was demonstrated by immunoblot and Northern blot analysis. In spreading cultures of the brain cells, -crystallin and -mRNA showed a significant increase from their in situ level. Immunohistological staining (peroxidase antiperoxidase) with monospecific anti-serum against -crystallin revealed that -producers were both epithelial cells and dendritic cells. Neither lentoidogenesis nor -crystallin expression was observed. Stimulation of -crystallin synthesis in cultured brain cells differed when compared with transdifferentiating cultures of neural retina cells. In the latter, -crystallin synthesis occurred concomitantly with differentiation of morphologically distinct lens cells containing -crystallin.     

Microanalysis of C and O isotopes of azooxanthellate and zooxanthellate corals by ion microprobe

We have determined the ¹⁸O and ¹³C values of azooxanthellate (Lophelia pertusa) and zooxanthellate (Porites lutea) corals at a micrometer scale using an ion microprobe (SIMS—secondary ion mass spectrometry). In P. lutea, centers of calcification are small (10 to 15 m) and difficult to locate during measurements. In L. pertusa, they are large (50 m) and arranged in lines of centers of calcification. Our results show that centers of calcification in L. pertusa have a restricted range of variation in ¹⁸O [-2.8±0.3 (PDB)], and a larger range in ¹³C [14.3 to 10.9 (PDB)]. Surrounding skeletal fibers exhibit large isotopic variation both for C and O (up to 12), and ¹³C and ¹⁸O are positively correlated. The C and O isotopic compositions of the center of calcification deviate from this linear trend at the lightest ¹⁸O values of the surrounding fibers. Ion microprobe results on P. lutea demonstrate also a large range of variation for the ¹⁸O values (up to 10). No correlation is found with C isotopes that exhibit, in comparison with L. pertusa, a small range of variation (2). This variation of ¹⁸O at a micrometer scale is probably the result of two processes: (1) an isotopic equilibrium calcification with 1 pH unit variation in the calcification fluid as indicated by direct measurements of coelenteron pH in the coral Galaxea fascicularis (Al-Horani et al. 2003) and (2) a kinetic fractionation. The ¹³C apparent disequilibrium in P. lutea may be the result of mixing between metabolic CO₂ (respiration) and dissolved inorganic carbon (DIC) coming directly from seawater.     

The role of vanadium in green plants

In a series of experiments, it is demonstrated that the trace element vanadium (4·10^-7 g-at/l as NH₄VO₃) has a considerable positive influence on the synthesis of -aminolevulinic acid (-ALA) in the autotrophically growing green algaChlorella pyrenoidosa, the effect being visible by an enhanced output of the amino acid into the culture medium in presence of levulinic acid (LA). The level of intracellularly accumulated -ALA, however, is not changed in presence of the metal. The V-effect on exogenous found -ALA is suppressed, when LA is added to the nutrient medium at low pH (pH 5), although V-uptake into the algal cells is not disturbed by LA. As demonstrated in culture media with various nitrogen sources (urea, partially hydrolized urea, ammonium salts), the development of the pH during the cultivation time is important for the presentation of the V-effect on -ALA. It is suggested that vanadium acts as a catalyst in the conversion of 4,5-dioxovaleric acid to -ALA by transamination.Abbreviations -ALA -aminolevulinic acid - LA levulinic acid - DOVA 4,5-dioxovaleric acid     

Carbon isotope composition in relation to leaf gas exchange and environmental conditions in Hawaiian Metrosideros polymorpha populations

Summary Carbon isotope composition, photosynthetic gas exchange, and nitrogen content were measured in leaves of three varieties of Metrosideros polymorpha growing in sites presenting a variety of precipitation, temperature and edaphic regimes. The eight populations studied could be divided into two groups on the basis of their mean foliar ¹³C values, one group consisting of three populations with mean ¹³C values ca.-26 and another group with ¹³C values ca.-28. Less negative ¹³C values appeared to be associated with reduced physiological availability of soil moisture resulting from hypoxic conditions at a poorly drained high elevation bog site and from low precipitation at a welldrained, low elevation leeward site. Gas exchange measurements indicated that foliar ¹³C and intrinsic wateruse efficiency were positively correlated. Maximum photosynthetic rates were nearly constant while maximum stomatal conductance varied substantially in individuals with foliar ¹³C ranging from-29 to-24. In contrast with the patterns of ¹³C observed, leaf nitrogen content appeared to be genetically determined and independent of site characteristics. Photosynthetic nitrogenuse efficiency was nearly constant over the range of ¹³C observed, suggesting that a compromise between intrinsic water- and N-use efficiency did not occur. In one population variations in foliar ¹³C and gas exchange with leaf cohort age, caused the ratio of intercellular to atmospheric partial pressure of CO₂ predicted from gas exchange and that calculated from ¹³C to be in close agreement only in the two youngest cohorts of fully expanded leaves. The results indicated that with suitable precautions concerning measurement protocol, foliar ¹³C and gas exchange measurements were reliable indicators of potential resource use efficiency by M. polymorpha along environmental gradients.     

Estimates of nitrogen fixation by trees on an aridity gradient in Namibia

Summary Nitrogen (N₂) fixation was estimated along an aridity gradient in Namibia from the natural abundance of ¹⁵N (¹⁵N value) in 11 woody species of the Mimosacease which were compared with the ¹⁵N values in 11 woody non-Mimosaceae. Averaging all species and habitats the calculated contribution of N₂ fixation (N _f ) to leaf nitrogen (N) concentration of Mimosaceae averaged about 30%, with large variation between and within species. While in Acacia albida N _f was only 2%, it was 49% in Acacia hereroensis and Dichrostachys cinerea, and reached 71% in Acacia melifera. In the majority of species N _f was 10–30%. There was a marked variation in background ¹⁵N values along the aridity gradient, with the highest ¹⁵N values in the lowland savanna. The difference between ¹⁵N values of Mimosaceae and non-Mimosaceae, which is assumed to result mainly from N₂ fixation, was also largest in the lowland savanna. Variations in ¹⁵N of Mimosaceae did not affect N concentrations, but higher ¹⁵N-values of Mimosaeae are associated with lower carbon isotope ratios (¹³C value). N₂ fixation was associated with reduced intrinsic water use efficiency. The opposite trends were found in non-Mimosaceae, in which N-concentration increased with ¹⁵N, but ¹³C was unaffected. The large variation among species and sites is discussed.This paper is prepared in memory of J. Visser, who took part in the collection of species, but died in 1990     

Natural abundance of 15N in tropical plants with emphasis on tree legumes

Natural abundance of ¹⁵N ( ¹⁵N) of leaves harvested from tropical plants in Brazil and Thailand was analyzed. The ¹⁵N values of non-N₂-fixing trees in Brazil were +4.5±1.9, which is lower than those of soil nitrogen (+8.0±2.2). In contrast, mimosa and kudzu had very low ¹⁵N values (–1.4+0.5). The ¹⁵N values of Panicum maximum and leguminous trees, except Leucaena leucocephala, were similar to those of non-N₂-fixing trees, suggesting that the contribution of fixed N in these plants is negligible. The ¹⁵N values of non-N₂-fixing trees in Thailand were +4.9±2.0. Leucaena leucocephala, Sesbania grandiflora, Casuarina spp. and Cycas spp. had low ¹⁵N values, close to the value of atmospheric N₂ (0), pointing to a major contribution of N₂ fixation in these plants. Cassia spp. and Tamarindus indica had high ¹⁵N values, which confirms that these species are non-nodulating legumes. The ¹⁵N values of Acacia spp. and Gliricidia sepium and other potentially nodulating tree legumes were, on average, slightly lower than those of non-N₂-fixing trees, indicating a small contribution of N₂ fixation in these legumes.     

New organisms—novel genetics

This report describes a method for quantifying -endotoxin contents in spray-dried preparations ofBacillus thuringiensis strain GC-91. -Endotoxin is solubilized in the pro-toxin form and separated from other soluble compounds by ion exchange chromatography. The method does not discriminate between the different -endotoxins produced by strain GC-91. It is suitable for quality control, since -endotoxin concentrations found in different preparations correlate inversely with the LC 50 in bioassays on artificial diet against freshly hatched larvae ofSpodoptera littoralis. A typical batch of spray-dried material contains 1.22%±0.08% -endotoxin. The method is most accurate with preparations containing 0.5 to 2.0% toxin, for which triplicate estimations give standard errors close to±0.2%.     

Variation in stable isotope signatures of seston and a zooplanktivorous fish in a eutrophic Chinese lake

Temporal and spatial changes in ¹³C and ¹⁵N of seston (mainly phytoplankton) and isotopic relationship between seston and the lake anchovy (Coilia ectenes) were studied in the large eutrophic freshwater Lake Chaohu in China. Much of the spatial and temporal variation in ¹³C of lake anchovies was explained by variation in seston, indicating a strong link between pelagic primary production and higher order consumers. Because the lake is shallow, there were no significant differences in ¹³C and ¹⁵N of seston between surface and overlying waters. Spatially, the relatively high ¹³C and ¹⁵N of seston in the western part of the lake might be due to high levels of anthropogenically derived N and C introduced from the surrounding cities through sewage drainage systems. The trophic position of the lake anchovy in the food web of Lake Chaohu was estimated to be 2.9–4.1 (3.5 ± 0.4), which agrees well with the previous stomach content analysis suggesting that the lake anchovy fed both on zooplankton and small planktivorous fishes.     

Stable carbon and oxygen isotopic compositions of recent charophyte oosporangia and water from Malham Tarn,U.K.: palaeontological implications

Charophyte oosporangia and water samples from a highly calcareous lake were measured for stable carbon and oxygen isotopic composition. The time period over which the oosporangia calcify is short, thus any biochemical relationship between the water and oosporangia"s calcite represents only one time window (late Summer in Malham Tam). This important temporal restraint must also apply to interpretations of all fossil material measured. The ¹⁸O_c of the charophyte oosporangia is deduced to be in equilibrium with the ¹⁸O of the water for a given temperature. The ¹³ C_c of the charophyte oosporangia was approximately 2.5 per mil lower than the ¹³C_DIC in the water we measured. With the release Of CO₂ with phosphoric acid from the charophyte oosporangia, there was no significant difference in the ¹⁸O_c values obtained, regardless of whether or not the carbonate was separated from the organic center, however ¹³C_c values were marginally lower for carbonate plus organic center measurements. Our results indicate that fossil charophyte gyrogonites can be used to elucidate the geochemistry of the ancient water body in which they lived.     

Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn¹(N -OH, N -acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn⁴(N -OH, N -formyl)-1,4-diaminobutane. The N -acyl groups of Orn¹(N -OH, N -acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.     

Stable oxygen isotopes in<Emphasis Type="Italic"> Porites</Emphasis> corals monitor weekly temperature variations in the northern Gulf of Aqaba,Red Sea

In order to assess the ability of Porites corals to accurately record environmental variations, high-resolution (weekly/biweekly) coral ¹⁸O records were obtained from four coral colonies from the northern Gulf of Aqaba, which grew at depths of 7, 19, 29, and 42 m along one transect. Adjacent to each colony, hourly temperatures, biweekly salinities, and monthly ¹⁸O of seawater were continuously recorded over a period of 14 months (April 1999 to June 2000). Contrary to water temperature, which shows a regular and strong seasonal variation and change with depth, seawater ¹⁸O exhibits a weak seasonality and little change with depth. Positive correlations between seawater ¹⁸O and salinity were observed. The two parameters were related to each other by the equation ¹⁸O _Seawater (, VSMOW) = 0.281 × Salinity – 9.14. The high-resolution coral ¹⁸O records from this study show a regular pattern of seasonality and are able to capture fine details of the weekly average temperature records. They resolve more than 95% of the weekly average temperature range. On the other hand, attenuation and amplification of coral seasonal amplitudes were recorded in deep, slow-growing corals, which were not related to environmental effects (temperature and/or seawater ¹⁸O) or sampling resolution. We propose that these result from a combined effect of subannual variations in extension rate and variable rates of spine thickening of skeletal structures within the tissue layer. However, no smoothing or distortion of the isotopic signals was observed due to calcification within the tissue layer in shallow-water, fast-growing corals. The calculations from coral ¹⁸O calibrations against the in situ measurements show that temperature (T) is related to coral ¹⁸O ( _c ) and seawater ¹⁸O ( _w ) by the equation T (°C) = –5.38 ( _c – _w ) –1.08. Our results demonstrate that coral ¹⁸O from the northern Gulf of Aqaba is a reliable recorder of temperature variations, and that there is a minor contribution of seawater ¹⁸O to this proxy, which could be ignored.     

Shoot δ15N correlates with genotype and salt stress in barley

Given a uniform N source, the ¹⁵N of barley shoots provided a genotypic range within treatments and a separation between control and salt-stress treatments as great as did ¹³C^*. Plant ¹⁵N has been represented in the literature as a bioassay of external source ¹⁵N and used to infer soil N sources, thus precluding consideration of the plant as a major cause in determining its own 8¹⁵N. We believe this to be the first report of plant ¹⁵N as a genetic trait. No mechanistic model is needed for use of ¹⁵N as a trait in controlled studies; however, a qualitative model is suggested for further testing.Symbol ¹⁵N (or ¹³C) the difference between: (1) the ratio of heavy to light isotopes of the element in a sample and (2) that of its reference standard     

Effect of size on the energy acquired in species of the fish from a neotropical reservoir,Brazil

In this paper we analysed autotrophic sources of the carbon ( ¹³C) and the trophic position ( ¹⁵N) of Leporinus friderici in the influence area of Corumbá Reservoir, Brazil. We collected samples of muscles of fish from different sizes riparian vegetation, C₄ grasses, zooplankton, periphyton and particulate organic carbon (POC). There were significant differences for the carbon isotope proportion found in muscles of L.friderici in the different size groups analysed. The highest values of ¹³C recorded for middle sized individuals is attributed to the large contribution of C₄ plants in their diet. Small individuals sampled upstream also receive similar contribution from C₄ plants. In contrast the same size group sampled downstream from the reservoir, has a much smaller of C₄ plants. The ¹³C negative character of small individuals from downstream is due to the larger contribution of C₃ plants (except periphyton). At larger sizes we found intermediate ¹³C values. The ¹⁵N proportions we found for each size group were not significantly different, however we found decreasing mean values with increasing size. The trophic level calculated from the dietary data was higher than that found with the ¹³C concentration in the muscle, except for small individuals, when the values were equal.     

Feeding level and individual metabolic rate affect δ13C and δ15N values in carp: implications for food web studies

Stable isotope analyses are often used to calculate relative contributions of multiple food sources in an animals diet. One prerequisite for a precise calculation is the determination of the diet-tissue fractionation factor. Isotopic ratios in animals are not only affected by the composition of the diet, but also by the amount of food consumed. Previous findings regarding the latter point are controversial. As stable isotope analyses have often been used to investigate aquatic food webs, an experiment with carp (Cyprinus carpio L.) was carried out to test the influence of the feeding level and individual metabolic rate on ¹³C and ¹⁵N values of the whole body. After an initial phase, 49 carp were assigned randomly to four groups and fed the same diet at different levels for 8 weeks. For 15 fish, the energy budget was determined by indirect calorimetry. Feed and individual fish were analysed for their proximate composition, gross energy content and ¹³C and ¹⁵N values. ¹³C and ¹⁵N values differed significantly at different feeding levels. While ¹³C values of the lipids and ¹⁵N values decreased with increasing feeding rate, ¹³C values of the lipid-free matter showed a non-linear pattern. Data obtained from fish held in the respirometric system revealed a relationship between ¹³C values and the percentage retention of metabolizable energy. Our results show that reconstructing the diets of fish from the isotopic ratios when the feeding level and individual metabolic rates are unknown would introduce an error into the data used for back-calculation of up to 1 for both ¹³C and ¹⁵N values and may have substantial effects on the results of calculated diets. As other workers have pointed out, the development and application of stable isotopes to nutritional ecology studies is a field in its infancy and gives rise to erroneous, misleading results without nutritional, physiological and ecological knowledge.     

Keeling plots for hummingbirds: a method to estimate carbon isotope ratios of respired CO<Subscript>2</Subscript> in small vertebrates

The carbon isotope composition of an animals breath reveals the composition of the nutrients that it catabolizes for energy. Here we describe the use of Keeling plots, a method widely applied in ecosystem ecology, to measure the ¹³C of respired CO₂ of small vertebrates. We measured the ¹³C of Rufous Hummingbirds (Selasphorus rufus) in the laboratory and of Mourning (Zenaida macroura) and White-winged (Z. asiatica) Doves in the field. In the laboratory, when hummingbirds were fed a sucrose based C3 diet, the ¹³C of respired CO₂ was not significantly different from that of their diet (¹³C_{C3 diet}). The ¹³C of respired CO₂ for C3 fasted birds was slightly, albeit significantly, depleted in ¹³C relative to ¹³C_{C3 diet}. Six hours after birds were shifted to a sucrose based C4 diet, the isotopic composition of their breath revealed that birds were catabolizing a mixture of nutrients derived from both the C3 and the C4 diet. In the field, the ¹³C of respired CO₂ from Mourning and White-winged Doves reflected that of their diets: the CAM saguaro cactus (Carnegeia gigantea) and C3 seeds, respectively. Keeling plots are an easy, effective and inexpensive method to measure ¹³C of respired CO₂ in the lab and the field.     

Sulfur isotope inventories of atmospheric deposition, spruce forest floor and living Sphagnum along a NW–SE transect across Europe

At five European sites, differing in atmospheric Sinputs by a factor of 6, and differing in S isotope signatures ofthese inputs by up to 14 (CDT), we investigated thedirection and magnitude of an assimilation-related ³⁴S shiftand the relationship between atmospheric deposition and Sretention in selected ecosystem compartments. Bulk precipitationand spruce throughfall were collected between 1994 and 1996 inthe Isle of Mull (Scotland), Connemara (Ireland), Thorne Moors(England), Rybárenská slat' and Oceán (both Czech Republic) andanalyzed for sulfate concentrations and ³⁴S ratios. Eighteenreplicate samples per site of living Sphagnum collected inunforested peatlands and 18 samples of spruce forest floorcollected near each of the peatlands were also analyzed for Sconcentrations and ³⁴S ratios. Assimilation of S was associatedwith a negative ³⁴S shift. Plant tissues systematicallypreferred the light isotope ³²S, on average by 2. There wasa strong positive correlation between the non-marine portion ofthe atmospheric S input and total S concentration in forest floorand Sphagnum, respectively (R = 0.97 and R = 0.85). Elevated Sinputs lead to higher S retention in these two organic-richcompartments of the ecosystem. It follows that equal emphasismust be placed on organic S as on adsorption/desorption ofinorganic sulfate when studying acidification reversal inecosystems. The sea-shore sites had rainfall enriched in theheavy isotope ³⁴S due to an admixture of sea-spray. The inlandsites had low ³⁴S reflecting ³⁴S of sulfur emitted from localcoal-burning power stations. Sphagnum had always lower S contentsand higher ³⁴S ratios compared to forest floor. The within-siterange of ³⁴S ratios of Sphagnum and forest floor was wide (upto 12) suggesting that at least six replicate samples shouldbe taken when using ³⁴S as a tracer.     

The skeletal isotopic composition as an indicator of ecological and physiological plasticity in the coral genus<Emphasis Type="Italic"> Madracis</Emphasis>

Three species of the reef coral genus Madracis display skeletal isotopic characteristics that relate to depth, colony topography, and consequently to coral physiology. The joint interpretation of skeletal ¹³C and ¹⁸O provides information on the ecological plasticity and adaptation to depth of a coral species. Isotopic results are most easily understood in terms of kinetic effects, which reduce both ¹⁸O and ¹³C below isotopic equilibrium values, and metabolic effects, which only influence the skeletal ¹³C. Madracis mirabilis is adapted to depths shallower than 20 m, and shows the greatest range in kinetic effects and the strongest metabolic ¹³C enrichments caused by symbiont photosynthesis. Madracis formosa lives deeper than 40 m, and shows a reduced range of kinetic effects and relatively weak metabolic ¹³C enrichments. Madracis pharensis inhabits depths from 5 to >60 m, and does not attain the strength of kinetic effects of either of the other two species, apparently because it is not quite as well adapted to rapid growth at either extreme.     

Proton/electron stoichiometry of mitochondrialbc 1 complex. Influence of pH and transmembrane δpH

The effect of pH and transmembrane pH on the efficiency of the proton pump of the mitochondrialbc ₁ complex bothin situ and in the reconstituted state was studied. In both cases the H⁺/e ^– ratio for vectorial proton translocation by thebc ₁ complex respiring at the steady state, under conditions in which the transmembrane pH difference (pH) represents the only component of the proton motive force (p), was significantly lower than that measured under level flow conditions. The latter amounts, at neutral pH, to 1 (2 including the scalar H⁺ release). In the reconstituted system steady-state pH was modulated by changing the intravesicular buffer as well as the intra/extra-liposomal pH. Under these conditions the H⁺/e^– ratio varied inversely with the pH. The data presented show that pH exerts a critical control on the proton pump of thebc ₁ complex. Increasing the external pH above neutrality caused a decrease of the level flowH ⁺/e ^– ratio. This effect is explained in terms of proton/electron linkage inb cytochromes.