Purification, recovery, and laser-driven fluorination of silicon from dissolved and particulate silica for the measurement of natural stable isotope abundances.

A procedure for the purification, recovery, and determination of isotopic abundances of silicon from biogenic and lithogenic particulate matter and dissolved silicic acid is reported. Purification involves the reaction of acid molybdate with dissolved silicon in natural waters or that produced by the dissolution of particulate silica by hydrofluoric acid. The resulting silicomolybdic acid is then quantitatively precipitated by reaction with triethylamine hydrochloride. The silicon is recovered as silicon dioxide through stepwise combustion of the dried precipitate. Fluorination of the product for isotopic analysis is accomplished by laser heating under pure fluorine generated by the decomposition of a fluorine-based salt. The resulting silicon tetrafluoride is separated from hydrogen fluoride and other fluorination byproducts cryogenically using a variable-temperature cold trap. Yields for silicon recovery are 99.9% for precipitation and greater than 95% for the purification/fluorination procedure. Reproducibility of the isotopic composition for pure quartz granules processed through the procedure is ±0.1‰ for δ(30)Si.

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.

Oxygen-18 Content of Atmospheric Oxygen Does Not Affect the Oxygen Isotope Relationship between Environmental Water and Cellulose in a Submerged Aquatic Plant, Egeria densa Planch.

We determined that the oxygen isotopic composition of cellulose synthesized by a submerged plant, Egeria densa Planch., is related to the isotopic composition of environmental water by a linear function, delta(18)O cellulose = 0.48 delta(18)O water + 24.1% per thousand. The observation of a slope of less than 1 indicates that a portion of cellulose oxygen is derived from an isotopically constant source other than water. We tested whether this source might be molecular oxygen by growing plants in the presence of high concentrations of (18)O in the form of O(2) bubbled into the bottom of an aquarium. Cellulose synthesized during this experiment did not have significantly different oxygen isotope ratios than that synthesized by control plants exposed to O(2) of normal (18)O abundance. We propose that oxygen in organic matter recycled from senescent portions of the plant is incorporated into cellulose. Our findings indicate that paleoclimatic models linking the oxygen isotope composition of environmental water to cellulose from fossil plants will have to be modified to account for contributions of oxygen from this or other sources besides water.

Stable Carbon Isotope Fractionation by Methanosarcina barkeri during Methanogenesis from Acetate, Methanol, or Carbon Dioxide-Hydrogen.

Methanosarcina barkeri was cultured on methanol, H(2)-CO(2), and acetate, and the C/C ratios of the substrates and the methane produced from them were determined. The discrimination against C in methane relative to substrate decreased in the order methanol > CO(2) > acetate. The isotopic fractionation for methane derived from acetate was only one-third of that observed with methanol as the substrate. The data presented indicate that the last enzyme of methanogenesis, methylreductase, is not the primary site of isotopic discrimination during methanogenesis from methanol or CO(2). These results also support biogeochemical interpretations that gas produced in environments in which acetate is the primary methane precursor will have higher C/C ratios than those from environments where other substrates predominate.

Oxygen Isotope Exchange between Metabolites and Water during Biochemical Reactions Leading to Cellulose Synthesis.

Cellulose was produced heterotrophically from different carbon substrates by carrot tissue cultures and Acetobacter xylinum (a cellulose-producing bacterium) and by castor bean seeds germinated in the dark, in each case in the presence of water having known concentration of oxygen-18 ((18)O). We used the relationship between the amount of (18)O in the water and in the cellulose that was synthesized to determine the number and (18)O content of the substrate oxygens that exchanged with water during the reactions leading to cellulose synthesis. Our observations support the hypothesis that oxygen isotope ratios of plant cellulose are determined by isotopic exchange occurring during hydration of carbonyl groups of the intermediates of cellulose synthesis.

Oxygen and Hydrogen Isotope Ratios of Water from Photosynthetic Tissues of CAM and C(3) Plants.

Water samples from photosynthetic tissues of C(3) and Crassulacean acid metabolism (CAM) plants that grew together in the field were extracted and the stable oxygen and hydrogen isotope ratios determined. During the day, (18)O/(16)O and deuterium/hydrogen (D/H) ratios of water from CAM plants were lower than those observed in water from C(3) plants. The patterns of diurnal variation (or lack thereof) in isotope ratios of plant water are consistent with the gross anatomical and physiological characteristics of the plants studied here. Our observations support the previously advanced hypothesis that high D/H ratios in cellulose nitrate prepared from CAM plants relative to those for C(3) plants are not caused by greater deuterium enrichment in the water in CAM plants, but rather by isotopic fractionations associated with different biochemical reactions in the two types of plants.

Photosynthesis of F(1) Hybrids between C(4) and C(3)-C(4) Species of Flaveria.

Photosynthetic characteristics were studied in several F(1) hybrids between C(4) and C(3)-C(4) species of Flaveria. Stable carbon isotope ratios, O(2) inhibition of apparent photosynthesis, and phosphoenolpyruvate carboxylase activities in the hybrids were similar to the means for the parents. Values of CO(2) compensation concentrations were nearer to those of the C(4) parent and apparent photosynthesis was below that of both parents, being only 60 and 74% of that of the lowest (C(3)-C(4)) parent in two experiments. Reductions of CO(2) compensation concentration and O(2) inhibition of apparent photosynthesis as well as increases in carbon isotope ratios and phosphoenolpyruvate carboxylase activities compared to values in C(3)-C(4) species suggest transfer of a limited degree of C(4) photosynthesis to the F(1) hybrids. However, the lower apparent photosynthesis of the hybrids suggests that transfer of C(4) characteristics to non-C(4) species is detrimental unless characteristics associated with C(4) photosynthesis are fully developed. There was a highly significant negative correlation (r = -0.90) between CO(2) compensation concentration and the logarithm of phosphoenolpyruvate carboxylase activity in the parents and hybrids, suggesting involvement of this enzyme in controlling the CO(2) compensation concentration. Although bundle-sheath cells were more developed in leaves of hybrids than in C(3)-C(4) parents, they appeared to contain lower quantities of organelles than those of the C(4) parent. Reduced quantities of organelles in bundle-sheath cells could indicate incomplete compartmentation of partial pathways of the C(4) cycle in the hybrids. This may mean that the reduction of CO(2) compensation and O(2) inhibition of apparent photosynthesis relative to the C(3)-C(4) parents is less dependent on fully developed Kranz anatomy than is increased apparent photosynthesis.

Stable nitrogen and carbon isotope ratios in bone collagen as indices of prehistoric dietary dependence on marine and terrestrial resources in southern California.

The ratios of 15N to 14N and 13C to 12C tend to be higher in marine than in terrestrial organisms. The concentrations of these isotopes in human bone collagen consequently can be used to make inferences about the contribution of marine and terrestrial resources to prehistoric diets. The utility of studying 15N/14N and 13C/12C ratios in conjunction with each other is illustrated by our analysis of 40 human burials from archaeological sites in the Santa Barbara Channel area of southern California. The mean delta 13C and delta 15N values (in per mil) of collagen from these skeletons decrease progressively from the Channel Islands (delta 13C = -14.0, delta 15N = +16.3) to the mainland coast (delta 13C = -14.5, delta 15N = +14.9) to the interior (delta 13C = -17.2, delta 15N = +10.9). These data suggest that Indians living on the Channel Islands during the late prehistoric period were heavily dependent on marine resources. The inhabitants of the mainland interior, in contrast, had a diet composed largely of terrestrial foods. From their isotope ratios, it appears that the Indians who lived on the mainland coast consumed a mixed diet containing substantial quantities of both marine and terrestrial resources. Differences in 15N/14N and 13C/12C ratios of individuals from mainland sites dating from the early and late prehistoric periods show that the marine component of the diet increased substantially through time. These isotopic data are consistent with pathological, faunal, and artifactual evidence of increased marine resource exploitation during the late prehistoric period.

Compensation point and isotopic characteristics of c(3)/c(4) intermediates and hybrids in panicum.

Leaf CO(2) compensation points and stable hydrogen, oxygen and carbon isotope ratios were determined for Panicum species including C(3)/C(4) intermediate photosynthesis plants, hybrids between C(3)/C(4) intermediates and C(3) plants, C(3) and C(4) plants in the Panicum genus as well as several other C(3) and C(4) plants. C(3) plants had the highest compensation points, followed by hybrids, C(3)/C(4) intermediates, and C(4) plants. delta(13)C values of cellulose nitrate and saponifiable lipids from C(4) plants were about 10 per thousand higher than those observed for cellulose nitrate and saponifiable lipids of C(3)/C(4) intermediates, hybrids, and C(3) plants. Oxygen isotope ratios of cellulose as well as those of leaf water were similar for all plants. There was substantial variability in the deltaD values of cellulose nitrate among the plants studied. In contrast, such variability was not observed in deltaD values of water distilled from the leaves, nor in the deltaD values of the saponifiable lipids. Variability in deltaD values of cellulose nitrate from C(3)/C(4) intermediates, hybrids, C(3), and C(4) plants is due to fractionations occurring during biochemical reactions specific to leaf carbohydrate metabolism.

Crassulacean Acid Metabolism in the Strangler Clusia rosea Jacq.

Observations of malic acid fluctuation, leaf anatomy, and stable carbon isotopic composition showed that the epiphytic strangler Clusia rosea, growing on Saint John, U.S. Virgin Islands, has crassulacean acid metabolism. This hemiepiphyte may be the only woody dicotyledonous tree species among the many thousands of flowering species in the 30 or more plant families that shows this type of metabolism. The finding has implications with respect to water balance during the process whereby Clusia rosea establishes itself as a tree, since crassulacean acid metabolism is a photosynthetic adaptation to water-stressed environments.

Physiological and isotopic aspects of photosynthesis in peperomia.

Physiological and isotopic aspects of several Peperomia species were investigated. All but one species had C(3)-like stomatal behavior, in that stomata were open during the day and closed during the night. In these species, most atmospheric CO(2) uptake occurred during the day. Concurrent with this stomatal behavior, there were Crassulacean acid metabolism-like acid fluctuations in most species. Carbon and hydrogen isotope ratios of cellulose nitrate from Peperomia reflect their physiological behavior. The delta(13)C values of cellulose nitrate from Peperomia species were similar to values observed in C(3) plants and consistent with the daytime uptake of exogeneous CO(2) via the C(3) photosynthetic pathway. The deltaD values of cellulose nitrate from Peperomia species approach those of Crassulacean acid metabolism plants. These elevated deltaD values are caused by fractionations occurring during biochemical reactions and not as a consequence of water relations.

Stable carbon isotope ratios of rock varnish organic matter: a new paleoenvironmental indicator.

Stable carbon isotope ratios of organic matter in rock varnishes of Holocene age from western North America and the Middle East show a strong association with the environment. This isotopic variability reflects the abundance of plants with different photosynthetic pathways in adjacent vegetation. Analyses of different layers of varnish on late Pleistocene desert landforms indicate that the carbon isotopic composition of varnish organic matter is a paleoenvironmental indicator.

Hydrogen, oxygen, and carbon isotope ratios of cellulose from submerged aquatic crassulacean Acid metabolism and non-crassulacean Acid metabolism plants.

Isotope ratios of cellulose and cellulose nitrate from aquatic Crassulacean acid metabolism (CAM) and non-CAM plants were determined. Cellulose oxygen istope ratios for all plants that grew together were virtually identical, whereas large differences were observed for hydrogen isotope ratios of cellulose nitrate between CAM and non-CAM plants. Carbon isotope ratios of cellulose nitrate did not differentiate CAM from non-CAM plants.

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 C(3), C(4), 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 C(3) and C(4) metabolism. When hydrogen isotope ratios are plotted against carbon isotope ratios, each photosynthetic mode separates into a distinct cluster of points. C(4) plants had many D/H ratios similar to those of C(3) 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 C(4) and CAM, had a hydrogen isotope ratio between those of the C(4) and CAM plants. When oxygen isotope ratios are plotted against carbon isotope ratios, no distinct clustering of the C(4) 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 C(3) and C(4) plants are due to isotopic fractionations occurring during biochemical reactions.

Carbon, hydrogen, and oxygen isotope ratios of cellulose from plants having intermediary photosynthetic modes.

Carbon and hydrogen isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from species of greenhouse plants having different photosynthetic modes were determined. When hydrogen isotope ratios are plotted against carbon isotope ratios, four clusters of points are discernible, each representing different photosynthetic modes: C(3) plants, C(4) plants, CAM plants, and C(3) plants that can shift to CAM or show the phenomenon referred to as CAM-cycling. The combination of oxygen and carbon isotope ratios does not distinguish among the different photosynthetic modes. Analysis of the carbon and hydrogen isotope ratios of cellulose nitrate should prove useful for screening different photosynthetic modes in field specimens that grew near one another. This method will be particularly useful for detection of plants which show CAM-cycling.

Stable nitrogen isotope ratios of bone collagen reflect marine and terrestrial components of prehistoric human diet.

The delta 15N values of bone collagen from Eskimos and from Northwest Coast Indians dependent on salmon fishing are about 10 per mil more positive than those from agriculturalists in historic times. Among prehistoric humans, two groups dependent on marine food sources show bone collagen delta 15N values that are 4 to 6 per mil more positive than those from two agricultural groups. The nitrogen isotope ratios of bone collagen from prehistoric inhabitants of the Bahamas are anomalously low for reasons that relate to the biogeochemical cycle of nitrogen in coral reefs.

Isotopic Composition of Cellulose from C3, C4, and CAM Plants Growing Near One Another.

Cellulose from plants having crassulacean acid metabolism was enriched in deuterium but not in oxygen-18 in relation to cellulose from C(3) and C(4) plants growing in the same area, indicating that the deuterium enrichment is due to isotopic fractionation during biochemical reactions rather than during evapotranspiration. Hydrogen and oxygen stable isotope ratios of cellulose from the plants in this restricted area showed more variability than that observed in samples collected across an entire continent. Biological factors appear to be as important as environmental factors in determining the isotope ratios of plant cellulose.

Direct Respiration of Lipids During Heat Production in the Inflorescence of Philodendron selloum.

Respiration in the heat-generating, sterile florets of Philodendron selloum was examined by electron microscopy and carbon isotopic analysis of respired carbon dioxide. After the spathe unfolded, the florets switched from carbohydrate oxidation to lipid oxidation, which persisted during heating and for at least 2 days thereafter. The scarcity of glyoxysome-like organelles and the low catalase activity in this tissue indicate that the lipid was respired directly and not after conversion to carbohydrate by the glyoxylate shunt. Thus, lipid metabolism in this heat-generating plant tissue appears to mimic aspects of the biochemistry and physiology of heat production in some animal tissues.

Variable Photosynthetic Metabolism in Leaves and Stems of Cissus quadrangularis L.

By measuring titratable acidity, gas exchange parameters, mesophyll succulence, and (13)C/(12)C ratios, we have shown that Cissus quadrangularis L. has C(3)-like leaves and stems with Crassulacean acid metabolism (CAM). In addition, the nonsucculent leaves show the diurnal fluctuations in organic acids termed recycling despite the fact that all CO(2) uptake and stomatal opening occurs during the day. Young succulent stems have more C(3) photosynthesis than older stems, but both have characteristics of CAM. The genus Cissus will be a fruitful group to study the physiology, ecology, and evolution of C(3) and CAM since species occur that exhibit characteristics of both photosynthetic pathways.