B21B-0863 0800h
Skeletal Strength and Skeletogenetic Mechanisms Over Phanerozoic Time
Mineralized skeletons have a remarkable range of mechanical properties with respect to strength and durability. Measurements of skeletal mechanical properties show that taxonomic groups with relatively simple, `physiochemically-dominated' modes of mineralization possess skeletal strengths and durabilities that are among the lowest of any known mineralized skeletons. Organisms with relatively sophisticated, `biologically-dominated' modes of mineralization have mechanical properties among the highest values known for any materials. These extraordinarily strong and durable skeletal materials are found in mollusks, echinoderms, vertebrates, and arthropods, which are groups with primarily mobile ecological habits. These skeletons are frequently lightweight, non-massive skeletons with little phenotypic variation. By contrast, dominant reef framework builders and reef sediment formers, with physiochemically-dominated modes of mineralization, have non-mobile ecological habits and construct massive, phenotypically plastic skeletons, possessing extremely poor mechanical properties. Endolithic organisms that further degrade the mechanical properties of the mineralized skeletons of reef builders frequently ravage their massive skeletons. As a result, the skeletons of these groups commonly fragment, and play a central role in reef establishment and maintenance, as they are incorporated in reefal, wave-resistant carbonate buildups. Scleractinian corals have a physiochemically-dominated mode of mineralization and are the dominant modern reef framework builders. Mechanical properties of modern aragonitic scleractinian coral skeletons, tested alive, demonstrate skeletal strengths that are orders of magnitude lower than those seen in mollusks, echinoderms, vertebrates, and arthropods. Rudist bivalves, the dominant reef framework-building group of the Cretaceous, show prolific, massive, highly variable, calcific skeletal elements with structures similar to some reef-forming modern, non-mobile mollusks and the skeletons of other organisms with physiochemically-dominated modes of mineralization. Many aspects of the ecological habits of reef-framework building scleractinians and rudsits are similar, including relatively high skeletal growth rates, which produce massive skeletons and wave-resistant structures with entrapped bioclastic sediments. The principal adaptive role of mineralization in reef framework building groups appears to be the rapid production of massive, brittle, wave-resistant mineralized skeletons. The physiochemically-dominated mode of mineralization of these reef framework builders appears to have made them susceptible to secular variations in Phanerozoic seawater during `calcite' and `aragonite' seas, favoring scleractinians in aragonite seas and rudists during the Cretaceous calcite episode. By contrast, most mobile mollusks, echinoderms, vertebrates, and arthropods appear relatively unaffected by secular variations in seawater chemistry over the Phanerozoic
B21B-0864 0800h
Direct Microelectrodes Measurements at the Calcification Site of Foraminifera
In addition to their importance in the global carbon cycle and for paleoceanograohic studies, foraminifera are excellent model organisms for studying biomineralization at the cellular level. We have shown previously that vacuolated seawater, which is modified intacellularly to pH of $\sim$9, is the solution from which foraminifera precipitate their CaCO$_{3}$ (Bentov and Erez. 2003). In the present study we measured the pH and [Ca$^{+2}$] at the extra-cellular microenviront adjacent to the outer membrane of calcifying foraminiferal preparations using microelectrodes. These preparations are amoeboids and recovering specimens after decalcification that are produced from the foraminifer {\it Amphistegina lobifera }. We observed clear spatial polarization of pH between the apical and the basal margins of these organisms. High pH (9.2-9.4) was measured on the basal side, between the organism and the glass substrate where calcification occurs. On the apical side (facing the bulk seawater) a lower pH (8.3-8.6) was measured. The pH on the apical side is light dependent and may be associated with photosynthesis of the symbiotic algae. The [Ca$^{+2}$] measurements showed a similar pattern of polarization on the basal and apical sides. The basal [Ca$^{+2}$] near the calcifying site, varied cyclically between 11.9mM and 11.7 with a ca.30 min period. At the apical side the [Ca$^{+2}$] is 11.7mM slightly lower than ambient seawater of Gulf of Eilat (11.8 mM), and doesn't show cyclic variation. These observations are in good agreement with our model of calcification in foraminifera based on seawater vacuolization (Bentov and Erez. 2003). We suggest that calcification involves the following sequential steps: Seawater is vacuolated in large vacuoles which are cycled in the cell. At this stage the seawater in the vacuoles is being modified to increase its pH (up to $\sim$9) and possibly increase their [Ca$^{+2}$] as well. These vacuoles are exocytosed into the calcification site where high pH and [Ca$^{+2}$] create supersaturation for calcite. CaCO$_{3}$ precipitation proceeds with strict biological control on the shape, size and crystal orientation, all probably mediated by the organic matrix. During this process the pH and [Ca$^{+2}$] drop and these residual depleted seawater are exocytosed at the periphery of the organism where lower [Ca$^{+2}$] is observed. This mechanism may cause the cyclic alternation between Ca-enriched vacuoles coming to the site of calcification and Ca-depleted seawater as a result of CaCO$_{3}$ precipitation. We believe that the amoeboids and the recovering individuals represent faithfully the process of calcification in foraminifera. However, the actual values of the Ca and pH dynamics in intact organisms may be different (probably with higher amplitude), because our preparations may behave as an open system relative to the intact foraminifera.
B21B-0865 0800h
\(\delta \)\textsuperscript{44/40}Ca and Mg/Ca Ratios of \textit {N. Pachyderma} (sin.) in the Western Nordic Seas: Indications of Changing Carbonate Chemistry Controlled by Water Mass Effects or Foraminiferal Metabolism
The high temperature sensitivity of the \(\delta \)\textsuperscript{44/40}Ca signature in the polar to subpolar planktonic foraminifer of \textit {N. pachyderma} (sin.) (\(\delta \)\textsuperscript{44/40}Ca=0.21*SST(\textsuperscript{o}C)-0.61) found by previous workers has turned out to be solid and reproducible in the North Atlantic and Norwegian Sea. In this area, calcium isotopic compositions provide the possibility to resolve the amplitude of sea surface temperature (SST) changes as well as absolute SST estimates. Here we present a multi proxy record from a sediment core near the Denmark Strait, spanning the time interval from the LGM to the late Holocene. The comparison of \(\delta \)\textsuperscript{44/40}Ca, Mg/Ca, \(\delta \)\textsuperscript{18}O and foraminiferal transfer functions highlights the potential of \(\delta \)\textsuperscript{44/40}Ca as an additional SST-proxy, especially at the 'cold end' with its higher statistical uncertainties. However, in the western part of the Nordic Seas which is characterized by polar and Arctic waters, \(\delta \)\textsuperscript{44/40}Ca ratios of \textit {N. pachyderma} (sin.) are insensitive to temperature variations, reflecting the low temperature dependency found in most planktonic foraminifer species. This observed change in the calcium isotopic composition coincide with unusual high Mg/Ca ratios in \textit {N. pachyderma} (sin.) in polar and Arctic waters as published in previous works. Mg/Ca calculated temperatures in these water masses overestimate existing SSTs by more than 10\textsuperscript{o}C. Diagenetic effects, ice cover, dissolution or sample contamination were considered to influence the Mg/Ca ratio. Simultaneous measurements of Mg/Ca and \(\delta \)\textsuperscript{44/40}Ca in samples of \textit {N. pachyderma} (sin.) indicate that the change in the \(\delta \)\textsuperscript{44/40}Ca temperature sensitivity is directly linked to this unusual high Mg/Ca ratios. This failure of existing temperature calibrations of two independent carbonate-chemistry proxies in polar and Arctic waters suggests that either water mass chemistry or selective processes controlled by the foraminiferal metabolism may be responsible.
B21B-0866 0800h
AFM-Study on Calcite Nucleation by Cyanobacteria
Prokaryotic picoplankton plays an important role in lacustrine calcite precipitation, especially in oligotrophic lakes. The mechanisms of cyanobacteria-surface mediated precipitation reactions, however, remain poorly understood. For interpreting geochemical and isotopic information stored in sediments, it is essential to know the carbonate precipitation mechanisms and microscopical details of nucleation on the cell surface. A microscopy study was designed, based on previous results of cyanobacterial calcite precipitation. In order to investigate the nucleation of calcite on the cell surface under controlled conditions, experiments with Synechococcus (PCC 7942) were performed in a flow-through design of an atomic force microscope (AFM). The AFM was operated in `Tapping Mode' providing information on the topography and additionally on physicochemical properties by mapping the phase shift of the oscillating cantilever. For these experiments cells were immobilized on glass slides. As the basic requirement, growth experiments were performed in order to ensure that the fixed cells were alive. To our knowledge one of the first times the successive steps of a cyanobacterial cell division were observed by AFM. The growth of cell agglomerates was monitored over a period of 6 days. AFM images finally reveal the formation of a cyanobacterial biofilm on the glass surface. These observations were supported by the results obtained by epifluorescence microscopy. In a second step, calcite nucleation experiments were performed. Under carefully controlled physical and chemical conditions, nucleation of calcite was enhanced by flushing the fluid cell of the AFM with supersaturated solutions of CaCO$_{3}$ (CaCl$_{2}$ and NaHCO$_{3}$). These solutions were 7 and 10 times supersaturated. As the result of the addition of the CaCO$_3$-solution, changes of the microtopography and physicochemical properties of the cell surface were observed. In our study in-situ-AFM was shown to be powerful tool investigating mechanisms of biomineralization and nucleation under controlled natural conditions.
B21B-0867 0800h
Integrated Approach for Understanding Impurity Adsorption on Calcite: Mechanisms for Micro-scale Surface Phenomena
A longstanding goal within the field of environmental geochemistry has been the development of a fundamental understanding of the kinetics that governs the interactions of solution-borne impurities with the calcite mineral surface. Recent dissolution experiments using Mg$^{2+}$, Mn$^{2+}$, and Sr$^{2+}$ have shown distinct differences in the interaction of these three impurity ions with the calcite crystal surface. Because the dissolution of carbonate minerals in soils and sediments influences the uptake and migration of groundwater contaminants, a rigorous understanding of the basic processes that occur at the mineral-fluid interface is necessary. We have used vertical scanning interferometry (VSI) coupled with scanning probe microscopy (SPM) to examine calcite crystal dissolution in the presence of Mg$^{2+}$, Mn$^{2+}$, and Sr$^{2+}$, all known dissolution inhibitors and possible groundwater contaminants. We have studied the kinetics of impurity-crystal interactions at a pH 8.8, and in the presence or absence of dissolved inorganic carbon. Our data show that, when individually introduced into undersaturated solutions, Mg$^{2+}$ and Mn$^{2+}$ are shown to activate the calcite crystal surface, resulting in enhanced etch pit nucleation rates and step density. Conversely, Sr$^{2+}$ is shown to cause passivation of the calcite surface. The effect is intensified when solutions are saturated with respect to atmospheric CO$_2$. Results indicate that aqueous CO$_3^{2-}$ (or HCO$_3^-$) may influence how aqueous metal ionic complexes interact with the crystal surface. Furthermore, the influence is differently exhibited, and passivation or activation ultimately depends on the properties of the diffusing metal ion or metal-hydroxide complex. These properties include for example, differences in hydration enthalpy, the effective ionic radius, and electron shell configuration.
B21B-0868 0800h
Step Velocity Distributions, Step Spacing, and Stepwave Theory: Experimental Evidence and Modeling Results
Much of current attention in mineral dissolution reaction kinetics is devoted to the study of the relationship between the overall "bulk" rate, reflected in time-averaged solute fluxes observed in a well-mixed reactor, versus the rates of individual, "elementary" processes, such as kink nucleation, attachment and detachment, surface and edge diffusion, and resultant step motion. Regardless of the details of the interaction between these processes, implicit in many interpretations is the notion that the relationship between them is a deterministic one, i.e., that if a given set of physical and chemical conditions is imposed on a crystal surface, a predictable rate will result. This assumption has currency in many recent AFM studies of carbonate mineral dissolution that observe that parameters such as step velocity and interstep spacing acquire characteristic values. This assumption is also the basis for kink transport models. Despite an incomplete understanding of the complex relationships between kink density and kink detachment rate, characteristic values for these individual rates guarantee a predictable cumulative "bulk" rate. However, not all AFM observations validate this assumption. For example, etch pits forming on calcite show a large variation in the velocity of steps under otherwise constant conditions that may reflect mechanistic differences. These include steps migrating within relatively flat etch pit interiors, bunched steps that comprise bounding vicinal faces, and curved or roughened steps often formed during etch pit coalescence. Here we examine experimental evidence collected using both AFM and vertical scanning interferometry for these relationships in light of a theoretical framework that relates step velocity, surface diffusion, free energy, and other relevant parameters. Our goal is to understand how variations in surface micro-topography may contribute to intrinsic rate variability, and to use this understanding to clarify the relationship of "bulk" rates to surface area.
B21B-0869 0800h
Distribution of Mg in Calcite is Strongly Influence by Transport Conditions at the Mineral-Solution Interface
Mg is widely recognized as a paleotemperature indicator in biogenic calcite samples. However, uncertainly continues about whether factors other than temperature act as primary controls on calcite Mg contents. To investigate the roles of surface structure and transport conditions in Mg uptake, we grew Mg-bearing calcite overgrowths on seed crystals using solutions with carefully prescribed levels of supersaturation, [Mg], pH, ionic strength, and constant temperature. In some growth experiments solutions were gently agitated; growth rates were limited by diffusive transport of ions at the crystal-solution interface. In other experiments fluid flowed vigorously, and growth rates were limited by the surface processes of adsorption, dehydration, and lattice bond formation. Control of growth rate by diffusion or surface reaction was determined by previous atomic force microscopy studies at the same conditions. Electron probe measurements yielded detailed information about Mg contents and distributions between the two geometrically distinct flanks of individual hillocks. Under diffusion-limited conditions, Mg displays a strong preference for incorporation into step edges with an obtuse (+) geometry. By contrast, Mg partitions preferentially into acute (-) step-edges when growth rate is controlled by surface processes. Thus the distribution of Mg in calcite is strongly influenced by transport conditions at the growing mineral surface. Although we do not know in detail the cause of differential incorporation nor how transport conditions change the distribution of Mg so dramatically, but the answers likely involve differences in the adsorption lifetime of Mg cations on step terraces relative to step migration rates. This study demonstrates that factors other than temperature strongly influence impurity uptake in calcite. The rate-controlling mechanisms for calcite growth in different organisms are as yet unknown, but transport conditions may have a first-order control on biomineral compositions.
B21B-0870 0800h
Nanoscale controls of inorganic impurities and peptides on shape modification during calcite growth
Many organisms produce crystalline structures during controlled biomineralization that exhibit complex topological forms. These biominerals often express facets or pseudofacets that are not found on crystals grown from pure solutions in the laboratory. This modification of growth shape, whether by inorganic and organic modulators, is generally explained within the paradigm of "stereochemical recognition". According to this model, stereochemical matching of the growth modulator to the molecular structure of these new and otherwise unexpressed faces, stabilizes the formation of new faces to result in a new crystal shape. This idea, however, was developed primarily from bulk crystallization experiments and geometrical models that focused on interactions between impurities and atomic planes of the newly expressed faces. Over the last several years, we have reported nanoscale investigations of how small molecule modifiers (Mg, Sr, amino acids) interact with calcite surfaces during growth. Low concentrations of these `simple' impurities have significant shape-modifying effects. While the observed mechanisms of growth modification are highly diverse, in all cases, it is clear that the source of shape modification always arises from step-specific interactions that alter either the equilibrium properties of the crystal (step edge and bulk free energy) or the kinetics of step motion. The resulting macroscopic shape changes can be traced to these effects at steps on existing faces rather than to stereochemical matching to and thermodynamic stabilization of new faces. Molecular modeling shows that the essential reason for this is that steps provide non-planar environments in which non-planar modifiers can form contacts to both the lower terrace and the step riser. Our findings provide a mechanism-based understanding of shape modification. This is essential as biomineralization studies advance to investigate more complex studies of systems that employ long-chain polypeptides or the full proteins found in vivo. In these systems, we suspect that the inherently non-planar nature of steps also provides an environment in which multiple bonding accommodations provide a large binding energy for non-planar growth modifiers.
B21B-0871 0800h
Aspartate Chain Length Controls Calcite Step Morphology
Most controlled crystallization is believed to be achieved under the direction of macromolecular protein-based templates with specific affinities for accumulating and assembling the desired growth units from solution. This reliance upon cellular proteins with specific sequences and structures suggests that the chemistry and stereochemistry of the amino acids forming the proteins are essential in conferring targeted activity in controlled biosynthesis. Calcium carbonate is a key biogenic mineral and model system to study the molecular mechanisms by which amino acids and, thus, polypeptides interact with mineral surfaces. Investigations of molecularly resolved crystal-peptide interactions are essential for a first order understanding of how biomolecules achieve regulated growth during biomineralization. Our previous studies have focused on the role of specific single and dipeptides species known to constitute a significant fraction of the proteins involved in biomineralization processes, in particular, acidic amino acids and aspartate. In this study we extend our investigation to the role of chain length of polyaspartates since they represent an integral part of the active site of these biomolecules' sequences. In particular, the interactions of aspartates (Asp$_{n}$, n=1,2,4,5,6) were investigated to measure their effects on the kinetics and thermodynamics of growth in parallel with theoretical calculations. Using in situ Atomic Force Microscopy (AFM) and carefully characterized solution compositions, our experimental measurements of growth kinetics and direct observations of the calcite hillock morphology at the nanoscale show that step directions with acute and obtuse geometries are affected differently for all aspartate derivatives. Short chain aspartates (n$>$=2) roughen acute steps more strongly than obtuse steps. This situation is exactly reversed for longer chain aspartates (n$>$2). Further, the critical Asp$_{n}$ concentration required for the roughening transition to occur decreases with increasing length of the Asp peptide. Circular Dichroism (CD) measurements of solution aspartate conformations were performed to determine the impact of conformation on stereochemical and steric interactions, and used as input in our molecular modeling studies. We examine the molecular origin for these experimental results using molecular modeling. Not unexpectedly, due to cooperative binding, the concentrations required to affect step edge morphology in general, decreased exponentially with a linear increase in aspartate chain length. This increased binding with chain length correlates with inhibition of step flow velocity measurements by AFM, and was explained using molecular modeling relating chain length to calcite steps binding energies. Our findings suggest that there is a qualitative change in aspartate-calcite steps interactions with a quantitative change in aspartate chain length, in addition to an exponential increase in the degree of aspartates interactions. This chain length dependent differential binding to opposing calcite steps may be an additional mechanism by which biomineralizing proteins can achieve calcite growth regulation.
B21B-0872 0800h
Modelling Coral Biomineralization: Mixed Kinetic/Equilibrium Trace Element Coprecipitation Models Reveal new Complexity
A mixed kinetic/equilibrium steady state model of trace-element co-precipitation in coral skeleton is presented, and tested against high spatial resolution observations of coral trace-element composition made previously by LA-ICP-MS. The model is implemented in PHREEQC, and simulates physicochemical precipitation from a small pocket of seawater which is isolated by the coral, and modified by enzyme exchange of 2 H$^{+}$ for Ca$^{2+}$. The model assumes that all aqueous trace-element species in the calcifying fluid are in full equilibrium and that selected trace element species compete kinetically for precipitation with the major aqueous species (Ca$^{2+}$ for cation substituents and CO$_{3}$$^{2-}$ for anion substituents). No equilibrium is assumed for the CaCO$_{3}$ skeleton. Carbon is supplied to the system by diffusion of CO$_{2}$ into the high-pH calcifying fluid, and trace elements are continuously replenished through the addition of fresh seawater. The rate at which the coral operates the enzyme pump, and the rate at which it replenishes the seawater component are independent variables, and the steady state trace-element composition of the skeleton/calcifying fluid are evaluated over a 2D grid of variables spanning realistic rates of pumping and seawater influx. It is assumed that variations in the trace element composition of the coral skeleton are the result of shifts in the steady-state caused by changes to these variables. The results indicate an unexpected complexity in the response of the trace elements. First order predictions suggest that increasing the rate of calcification by increasing the enzyme pumping should result in a mutual dilution of most trace element species by pumped Ca$^{2+}$ and diffused CO$_{2}$. However, for high rates of pumping and low seawater replenishment, the model predicts a change in the trace-element response of the system as high CO$_{3}$$^{2-}$ concentrations drive calcification and deplete Ca$^{2+}$. This added complexity makes rationalizing observations with models more difficult.
B21B-0873 0800h
Tidal Modulation of Sr/Ca Ratios in a Pacific Reef Coral
The skeletal composition of scleractinian corals reflects a combination of exogenous and endogenous processes, and represents a unique source of information about environmental as well as physiological processes linked to biomineralization. The challenge to paleoceanographic reconstructions based on coral skeletal chemistry is to identify and quantify the contribution of vital effects, thus enabling a more accurate interpretation of the proxy data in terms of climate. A good example is the strontium-to-calcium ratio (Sr/Ca) of coral skeleton, an important paleotemperature proxy that is also influenced by physiological processes. Here we present evidence for tidal forcing of coral Sr/Ca ratios on timescales of weeks to months that cannot be accounted for by changes in ocean temperature. Our ion microprobe measurements of Sr/Ca ratios in a Porites lutea coral from the north-central Pacific reveal high-frequency variations at dominant periods centered on ~6, ~10, and ~25 days. Comparison between coral Sr/Ca and in situ recorded SSTs reveals that the relationship between Sr/Ca and temperature on these short timescales does not follow trends observed at longer (annual) periods. Thus, an additional forcing is required to explain our observations. Our stochastic analyses demonstrate that Sr/Ca is correlated with both tidal water level variations and sea surface temperature, and the contribution of each forcing to the Sr/Ca content of the skeleton varies as a function of forcing period. We propose that water level influences Sr/Ca indirectly via tidal modulation of photosynthetically-active radiation (PAR) that drives large changes in zooxanthellate photosynthesis. The implication of this model is that coral Sr/Ca ratios are sensitive to the distance of the top of the colony from the sea surface. Thus coral colonies collected at different depths, massive colonies that grow and increase in height, and coral communities surviving sea level changes may reflect these changes in their Sr/Ca ratios. Additional studies will be required to determine if sea level forcing of coral Sr/Ca can provide a valuable new proxy for paleoclimate reconstructions
B21B-0874 0800h
Trace, Minor Elements, and Stable Isotopes in {\it Montastraea faveolata} as an Indicator of Stress
Coral cores were obtained along the fore reef from Looe Key Reef, Florida Keys, and analyzed for minor and trace elements by laser ablation ICP-MS and stable oxygen and carbon isotopes. Sample locations within the corals were chosen based on the location of annual bands as determined by x-radiographs. The LA-ICP-MS data were obtained along the corallite wall. Boron, magnesium, and phosphorous concentrations can be correlated among the corals analyzed. The highest elemental concentrations and the carbon and oxygen isotopic records in the Looe Key Montastraea faveolata were linked to times of reported bleaching. Boron, a common element in sea water, exists as two species, B(OH)$_{3}$ below a pH of 8.0 and B(OH)$_{4}$$_{-}$ above a pH of 8. Hemming and others (1998) determined that boron varied positively with $^{13}$C, both being coincident with high-density bands. They proposed that photosynthetic activity of zooxanthellae is the driving process, causing the shift in pH. During periods of stress, energy that would be used for normal coral activity (reproduction and growth) is diverted for tissue repair, food gathering, and waste removal. At extreme stress, these activities are reduced. As a result of decreased zooxanthellate activity, the chemistry at the organic-inorganic boundary may change as follows. 1. The pH rises, increasing the boron levels in the carbonate skeleton. 2. Phosphorous, expelled during normal growth activity, is retained, inhibiting the precipitation of "normal" aragonite. 3. The Mg/Ca ratio changes as calcium is being used preferentially. In the Looe Key Reef corals, boron, magnesium, and phosphorous all were elevated during times of reported bleaching. Within the same time intervals, the $\delta$$^{13}$C, which displayed values of between -2 % and -3 % in the "normal" light-density portion of the skeleton, approached a $\delta$$^{13}$C of 0 % in the stressed, high-density portion of the skeleton. Thus, the combination of high magnesium, boron, and phosphorous concentrations, coupled with the stable isotopic records of carbon and oxygen, correlate to stress events, such as bleaching in the Looe Key corals. These relations seem to confirm the model proposed by Hemming and others, and this chemistry may be useful in determining the record of stress events in other corals. Hemming, N.G., Guilderson, T.P. and Fairbanks, R.G., 1998, Seasonal variations in the boron isotopic composition of corals, a productivity signal?, Global Biogeochemical Cycles, v. 12, p.581-586.
B21B-0875 0800h
Intercolony variability of skeletal oxygen and carbon isotope ratios of cultured corals: temperature-controlled experiments
The skeletal oxygen isotope ratio of {\it Porites} corals, together with the carbon isotope ratio, is the most frequently used proxy of past seawater temperature and composition for tropical and subtropical oceans. However, field calibration of the proxy signals is often difficult owing to the covariation of temperature and salinity, which control the oxygen isotope composition of the water, under natural conditions. We conducted tank experiments in which we grew {\it Porites} spp. colonies in thermostated seawater at five temperature settings between 21 \deg C and 29 \deg C under moderate light intensity of 250 \mu mol m$^{2}$ s$^{-1}$ with a 12:12 light:dark photoperiod. A skeletal isotope microprofiling technique applied along the major growth axis of each colony revealed that the oxygen isotope ratios of newly deposited skeleton in most colonies remained almost constant during tank incubation, thus providing an ideal situation for precise calibration of proxy signals. However, the oxygen isotope ratios displayed a surprisingly large intracolony variability (1 \permil at each temperature setting although the mean slope (0.14 \permil \deg C $^{-1}$) obtained for the temperature<ETH>skeletal oxygen isotope ratio relationship was close to previous results. The variations in the oxygen isotope ratios were appaently caused by kinetic isotope effects related to variations in the skeletal growth rate rather than to intraspecies variability or genetic differences among species. Although no significant correlation was found between skeletal carbon isotope ratios and temperature, carbon isotope ratios also correlated with linear growth rates, suggesting a kinetic isotope control at low growth rates.
B21B-0876 0800h
A Century of Surface Productivity Recorded in the Skeleton of a Deep-Sea Coral.
"Poor understanding of the nutrient dynamics in the Gulf of Alaska results from insufficient uninterrupted, long-term observations" (PICES, 2004). Failing an instrumental record, oceanographers must increasingly rely on proxies. The utilization of deep-water corals as environmental recorders has only recently been recognized: they may fill in the many gaps present in instrumental records. {\it Primnoa} spp. is a long-lived gorgonian coral common in the northeastern Pacific. The abundance levels of \delta$^{15}$N and \delta$^{13}$C in the gorgonin portion of the skeleton have been correlated to surface productivity (Sherwood et al, 2004). {\it Primnoa} spp. can consequently be used as an historical proxy of productivity. A {\it Primnoa} specimen was obtained from 58.38 N and 148.77 W in the Gulf of Alaska. The coral was dead when collected, and died sometime after bomb testing, in the mid-1950's. Its lifespan was more than 100 years. Annual banding in the skeleton is quite clear, allowing tight dating control. Isotopic abundances of \delta$^{15}$N ranged from 10.2 to 14.4, suggesting the specimen was carnivorous, feeding primarily on zooplankton. Levels of \delta$^{13}$C ranged from -17.7 to -19.4, and exhibit decadal cyclical characteristic of the decadal patterns in the Gulf of Mexico. By matching cycles in isotopic abundances with historical data, we have been able to estimate time of death. This one coral specimen therefore extends the instrumental record by approximately 70 years.
B21B-0877 0800h
Mg and 18O Variations in the Shell of the Chilean Gastropod Concholepas concholepas Reflect SST and Growth Rate variations
To validate the use of fossil mollusc shells as recorders of environmental conditions, a primary calibration study was carried out on modern shells of the Chilean gastropod Concholepas concholepas, the so-called southern hemisphere abalone which is particularly abundant in Holocene archaeological sites. Organisms were maintained in culture tanks and feed with live mytilids. The sea water temperature in the tank was recorded every half-an-hour by an automatic device. The experiment lasted several months. Periodical marking with calcein provided a precise chronological control of the shell growth. Thus, well-dated high resolution chemical profiles could be directly compared with temperatures during shell formation. Geochemical analyses of the calcite layers include Mg, Sr and 16O/18O composition. Trace elements were analysed using Laser Ablation ICP-MS and Electron Microprobe while stable isotopes were measured on a Secondary Ion Mass spectrometry (SIMS). The shell growth rate during two months of formation varied between 30 and 140 m/day which allows us to reach a temporal resolution for chemical profiles between a few hours and three days. The growth rate variations do not seem to be related to temperature fluctuations. Only Mg content was analytically reproducible and showed significant variations across the shells. The Mg high-resolution profiles display a grossly sinusoidal shape. Shells from different sites along the coasts of Chile showed mean Mg contents of 300 ppm and 500 ppm for mean temperatures of 17 and $20\deg$C, respectively. This suggests a gross correlation between Mg and temperature. However, high resolution Mg results do not show an exact fitting neither with temperature nor with growth rates. Other parameters, like shell ageing as suggested by an amplitude increase observed near the edge of one of the shells, or other complex biological factors, may influence Mg incorporation into the shell. \delta 18O values of the calcite vary between -1,5 and 2,0 \permil for a temperature range between 17 and $22\deg$C. Growth rate variations seem to be an important factor affecting the oxygen isotopic ratio within shells. When growth rate variations are limited, \delta 18O and temperature are well correlated. The study confirms that, like for all biogenic carbonates, elemental and isotopic composition of the calcite layer of this gastropod, should not be used in paleoenvironmental reconstructions without detailed calibration experiments, and must systematically include precise growth rate analyses. The growth rhythms, which vary under the double influence of environmental and biological factors, are of paramount importance in the relationship between environmental parameters and geochemical composition of the growth layers of the shells. Work supported by "CONCHAS" Project (PNEDC).
B21B-0878 0800h
Sclerochronological and Geochemical Study of {\it Protothaca thaca} (Veneridea, Bivalve) Shells of the Peru-Chile Region: Environmental vs Physiological Controls
Climatic and oceanographic variability along the South-American coasts of the Pacific Ocean, including ENSO impacts, might be recorded in fossil bivalve shells. Therefore, coupled sclerochronological and geochemical studies were undertaken on the aragonite shell of present {\it Protothaca thaca}, a fairly common Chilean-Peruvian ($8\deg$-$45\deg$S) coastal bivalve, to test it. Microscopic observations of thin sections combined with growth increment measurements allowed the identification of structural growth units which characterize the infra-daily to yearly (or longer) temporal scales. The lowest frequency unit is limited by conspicuous growth anomalies. The application of Bertalanffy growth equation and a comparison of growth increment width with SST show that growth rate is positively related to temperature. In northern Chile-southern Peru, this relationship is verified for temperature lower than $18\deg$C. When summer daily SST exceeds $18\deg$C, growth is strongly reduced, or stops, and a clear "summer check" forms in the shell, the width of which is closely related to the length of the period of SST $>$ $18\deg$C. During the 1997-98 El Ni\~{n}o event, long lasting high summer SST produced $>$ 520 $\mu$m-wide summer check. The relative width of summer check on fossil shells may thus be an indicator of paleo-ENSO events. Stable isotopes of O and C were analyzed on micro-milled aragonite samples taken along growth layers for precise correlations with the recorded daily SST. Delta O-18 values do not follow the SST record suggesting a non-equilibrium precipitation. Consistent delta C-13 profiles on different individuals display a quite good correlation with growth rate. Beside, unexpectedly, the C-13 composition shows some relationship with SST. This is interpreted as the result of a strong physiological control which also drives the growth rhythm variations. High-resolution trace elements studies, presently underway, may provide new clues to understand the complex interaction between metabolic, kinetic and environmental processes during the {\it P. thaca} shell growth. Work supported by "CONCHAS" Project (PNEDC).
B21B-0879 0800h
Effect of CaCO$_{3}$ Precipitation by the Organic Matrix of Sclerites Associated with Coccoliths and the Characterization of Proteinaceous Organic Matrices from the Alcyonarian, {\it Lobophytum Crassum}. (Octocorallia : Alcyoncea)
Many of the invertebrates possess calcium carbonate skeletal tissues. There is an organic matrix in the skeleton, which is supposed to provide a nucleating site for the CaCO$_{3}$ deposition and the determination of crystal forms (calcite and/or aragonite). It is reported that the calcified organic substances in skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio\-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal sclerites from alcyonarian coral, {\it Lobophytum crassum}, to analyze the sequences and functional properties of the proteins present. The separated sclerites from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of the sclerites weight. By recording the decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO$_{3}$ precipitate was revealed. SDS-PAGE analysis of the preparations showed four bands of proteins with apparent molecular weights of 102, 67, 48 and 37 kDa. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. A major band of about 67 kDa protein was determined the N-terminal amino acid sequence. The calcium-binding soluble proteins, which are responsible for calcium carbonate nucleation and crystal growth, were detected as radioactive bands by $^{45}$Ca autoradiography. The 102 and 67 kDa calcium-binding proteins were found to be radioactive. Periodic acid schiff staining indicated the 67 kDa protein was glycosylated. A carbonic anhydrase, which is thought to be play an important role in the process of calcification, revealed low level of activity. These findings suggest that the endoskeletal sclerites of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.
B21B-0880 0800h
Stable isotope and chemical composition of pearls - Biomineralization in cultured pearl oysters in Ago Bay, Japan -
1. The oxygen isotopic composition of the pearl suggests that these pearls were produced around 23 degree centigrade, mainly in June, which is consistent with their occurrence in the field. In spite of high growth rate (higher than 0.2-1 g per year), where coral skeleton (~0.1 g per year) often presents non-equilibrium isotope partitioning, the pearls show complete or close to equilibrium condition. 2. The pearl samples showed rather scattered delta carbon 13 values (-1.04 - +0.47per mil, av. -0.16per mil) compared with delta oxygen 18 values. The delta carbon 13 values are ~-2.9per mil lower than those calculated from those in offshore water. It may be affected by low-delta carbon 13 waters due to degradation of organic matter in the Ba or by low-delta carbon 13 food. In the latter case, a simple mass balance calculation gives respiration component of 14%. 3. 12 trace elements of bulk pearl samples were classified into 4 groups: Group 1 (Co, Cr, Pb), Group 2 (Ba, Cs, U), Group 3 (Cu, Sn, V) and Group 4 (other elements such as Mn, Rb, Mo).Comparison with coral chemistry suggests that proteinous organic matter contain abundant Ba and Mn and significant amount of elements of Group.
B21B-0881 0800h
Iron oxyhydroxide mineralization by microbes in terrestrial environments
Many microorganisms produce extracellular organic structures that become mineralized and thus preserved. As in higher organism mineralization (e.g. shells and bones), organics template mineral nucleation and control growth. However, polymer-mediated crystal growth is a more general phenomenon that can occur in extracellular, geochemically open systems. We have been studying microbial polymers mineralized by iron oxyhydroxides in a variety of natural environments, including a flooded mine in Wisconsin, creeks in Virginia and California, and cold springs in Oregon. Enrichment culturing showed that these environments are all populated by neutrophilic iron-oxidizers. We used scanning and transmission electron microscopy (SEM and TEM), Fourier transform infrared spectroscopy (FTIR), and synchrotron-based scanning transmission X-ray microscopy (STXM) to investigate the mineralogy and organic polymer functional groups. Microscopic observations revealed that these samples, while often dominated by the sheaths and stalks generally attributed to Leptothrix spp. and Gallionella, actually contain mineralized structures with a wide range of morphologies. The extent of mineralization is variable, with some environments characterized by heavy encrustations that likely formed via abiotic precipitation. We focused on the lightly mineralized polymers as this allows us to more closely examine the organic-mineral interactions. STXM work shows that the organics in the flooded mine and Virginia creek have common functional groups, including carboxyls. However, the exact ratio of functional groups may be more dependent on environmental factors than species. Our previous work showed that organic polymer fibrils template unusually long akaganeite (beta FeOOH) crystals in biofilm samples from the flooded mine. Subsequent work has shown that polymers in other environments, including polymers contained in organized structures, also template iron oxyhydroxide mineralization. Thus, microbial polymer templation of iron oxide minerals appears to be a general phenomenon. We also examined polymer mineralization in culture and abiotic synthesis experiments. We combined alginate, a well-characterized microbial polymer with carboxylic functional groups, and Fe(III) in various forms and reproduced some of the simple structures found in nature. We used STXM (with C, N, O, and Fe NEXAFS) and Fe EXAFS to follow the influence of polymers during mineral formation. NEXAFS data collected to date clearly show an evolution in interactions between polymer functional groups as iron is bound and mineralization proceeds. An understanding of polymer-mediated formation of unique minerals will allow us to better establish these minerals as biosignatures.
B21B-0882 0800h
Oxygen Isotopic Disequilibrium in Bacteriogenic Soil Calcite Precipitated Near Leaking Oil and Gas Wells in Western Canada
About one third of all oil and gas wells drilled in Western Canada leak natural gas to surface. Leaking gas is oxidized, sometimes completely, by metanotrophic bacteria in soil near the wells. Depending on soil permeability and rate of gas leakage, zones of bacterial oxidation extend from 20 to $>$500 cm away from the well bores of leaking wells. Thin coatings and aggregates $<$300 micrometers across of authigenic calcite of both abiotic and bacteriogenic origin are found in the oxidation zones. Abiotic calcite forms crusts comprised of submicron to micron size sparry, euhedral crystals typical of inorganic soil calcite precipitates. Bacteriogenic calcite is closely associated with bacteria and microbial film and forms massive to porous aggregates of subhedral rounded crystals coating or cementing mineral grains. Growth of nanometer size calcite crystals on top of microbial surfaces indicates active involvement of bacteria with calcite precipitation. To constrain the conditions of calcite precipitation \delta$^{13}$C and \delta$^{18}$O of soil CO$_{2}$ and soil temperature at two leaking well sites in Saskatchewan were monitored for ca. two years. The \delta$^{18}$O of local soil moisture and groundwater were also measured. Results show that \delta$^{18}$O (PDB) of bacteriogenic calcite is from 3 to 6 permil lower than this that would precipitate in isotopic equilibrium with local soil moisture at any time during the year. Although \delta$^{13}$C of bacteriogenic calcite may be in isotopic equilibrium with local soil CO$_{2}$ at temperatures close to freezing, decrease of bacterial metabolic rates at such low temperatures would not favor bacteriogenic calcite precipitation. Therefore, \delta$^{13}$C may also reflect disequilibrium. In contrast, both \delta$^{18}$O and \delta$^{13}$C of abiotic calcite are in isotopic equilibrium with soil moisture and soil CO$_{2}$ at temperatures close to or higher than the average soil temperatures in the area. The small sizes of bacteriogenic calcite aggregates indicate that these precipitated in confined pools of water where local chemistry and pH are easily modified by the microorganisms. Therefore, oxygen and/or carbon isotopic disequilibrium of bacteriogenic calcite is likely the result of bacterial metabolism. The \delta$^{18}$O of soil calcite in paleosols has been widely used as a paleothermometer. Bacteriogenic calcite precipitation however is common in soils and not confined to zones of leaking gas oxidation. Likewise oxygen isotopic disequilibrium of bacteriogenic soil calcite may also be a common phenomenon. Therefore, paleosols should be examined for evidence of bacterial involvement in calcite precipitation prior to the use of \delta$^{18}$O of soil calcite as a paleothermometer.
B21B-0883 0800h
Searching for Biosignatures in Pyrite Framboids
Framboids are submillimeter raspberry-like structures with 10-10$^{9}$ microcrystallites, often composed of pyrite (FeS$_{2}$). The origin and mechanisms of formation of framboids are poorly understood. Framboid morphology can be obtained in abiotic simulations, but are also very common in the presence of microbial communities. Although some microbial communities are rich in framboids, the role that microbes potentially play in the formation of framboids, and the impact of microorganisms on framboid characteristics remains unclear. The purpose of this study was to use morphological and geochemical differences between abiotic pyrite framboids, and pyrite framboids formed in microbial mats to determine whether framboids can be used as a biosignature in the study of ancient sediments. By manipulating the experimental conditions we tried to understand more about the conditions conducive to pyrite framboid formation, and whether there was a microbial role in the genesis of sedimentary deposits containing framboids. We studied synthetic produced (abiotic) framboids with framboids formed within microbial mats and with framboids found in geological deposits. Size distribution, microtexture, composition, isotopic composition, and mineralogy of pyrite framboids was compared. Images were obtained using environmental scanning electron microscopy and the microtextures were analyzed using shape-analysis tools providing information about the overall size distribution of framboids, number of microcrystallites per unit volume, and size distribution of microcrystallites. Synthetic framboids were made in the laboratory, and framboids from microbial mats were collected from the Mangalia (Romania) subsurface sulfidic system. Neoproterozoic (750-600 Ma) manganese carbonate deposits containing fossilized pyrite framboids were collected from a mine (Hunan Province, China). Our results show that the framboids from microbial mats have a distinct size distribution, different number of crystallites, and different sizes of crystallites than abiotic framboids, and that the framboids from the Mangalia system show variability of S isotope composition within the system. We discuss criteria to address the biogenicity of (or life intervention upon) pyrite framboids from the geological record.
B21B-0884 0800h
Magnetite in Black Sea Turtles (Chelonia agassizi)
Previous studies have reported experimental evidence for magnetoreception in marine turtles. In order to increase our knowledge about magnetoreception and biogenic mineralization, we have isolated magnetite particles from the brain of specimens of black sea turtles Chelonia agassizi. Our samples come from natural deceased organisms collected the reserve area of Colola Maruata in southern Mexico. The occurrence of magnetite particles in brain tissue of black sea turtles offers the opportunity for further studies to investigate possible function of ferrimagnetic material, its mineralogical composition, grain size, texture and its location and structural arrangement within the host tissue. After sample preparation and microscopic examination, we localized and identified the ultrafine unidimensional particles of magnetite by scanning electron microscope (SEM). Particles present grain sizes between 10.0 to 40.0Mm. Our study provides, for the first time, evidence for biogenic formation of this material in the black sea turtles. The ultrafine particles are apparently superparamagnetic. Preliminary results from rock magnetic measurements are also reported and correlated to the SEM observations. The black turtle story on the Michoacan coast is an example of formerly abundant resource which was utilized as a subsistence level by Nahuatl indigenous group for centuries, but which is collapsing because of intensive illegal commercial exploitation. The most important nesting and breeding grounds for the black sea turtle on any mainland shore are the eastern Pacific coastal areas of Maruata and Colola, in Michoacan. These beaches are characterized by important amounts of magnetic mineral (magnetites and titanomagnetites) mixed in their sediments.
B21B-0885 0800h
Deciphering Seasonal Variations of Diet and Water in Modern White-Tailed Deer by {\it In Situ} Analysis of Osteons in Cortical Bone
{\it In situ} stable carbon and oxygen isotope compositions of biogenic apatite were obtained from longitudinally-cut sections of cortical bone from femurs of modern domesticated sheep and free-range White-Tailed deer, using an IR-laser and a GC-continuous flow interface. Ablation pits averaged 200x50 microns, making it possible to analyze individual osteons. Since cortical bone is remodelled along osteons throughout a mammal's lifetime, isotopic data at this resolution provides information about seasonal variations in diet and drinking water. The O-isotope results were calibrated using laser analyses of NBS-18 and NBS-19, which produced a value of 26.39$\pm$0.46 permil (n=27) for WS-1 calcite (accepted value, 26.25 permil). C-isotope results were calibrated using a CO$_{2}$ reference gas, producing a value of 0.76$\pm$0.40permil (n=27) for WS-1, also in excellent agreement with its accepted value of 0.74 permil. Average O- and C-isotope values for a local domestic sheep (southwestern Ontario, Canada) were 12.20$\pm$0.58 and -15.70$\pm$0.35 permil (n=27), respectively. No isotopic trend occurred along or across individual osteons. This pattern is consistent with the sheep's relatively unchanging food and water sources. The free-range White-Tailed deer came from Pinery Provincial Park (PPP), southwestern Ontario. Its O- and C-isotope compositions varied systematically across individual osteons and were negatively correlated (R$^{2}$=0.56). O-isotope values ranged from 13.4 to 15.5 permil; the highest values correlated with summer and the lowest values, with winter. The O-isotope compositions of the main water source (Old Ausable River Channel) varied similarly during the deer's lifetime: winter average, -10.7$\pm$0.5 permil; summer average, -8.6$\pm$0.4 permil. The C-isotope results for the deer osteons varied from -19.7 to -15.9 permil. This variation can be explained by changes in food sources. Summer diets of deer in PPP consist mainly of leafy fractions of C3 vegetation, especially sumac, cedar, oak and pine (average leaf C-isotope value, -28.4$\pm$0.8 permil). During winter, when leafy material is unavailable and deep snow inhibits access to vegetation in general, deer strip bark from vegetation (average bark C-isotope value, -25.6$\pm$0.8 permil). Certain C4 grasses (little bluestem and sandreed grass, average C-isotope value, -12.7$\pm$0.2 permil), which are abundant in unforested dune areas of PPP, commonly stand above the snow cover, and hence are also available for consumption. Deer may also range more widely in the winter, feeding on corn stalks and husks that escaped both harvest and snow cover (average C-isotope value, -11.3$\pm$0.2 permil).
B21B-0886 0800h
Influence of iron deficiency on the growth rate and physiological state of Prorocentrum micans Ehrenberg
Previous researches had shown that iron is an important limiting element to marine primary production. However, the mechanism of how iron affects marine algae is not well understood. Prorocentrum micans Ehrenberg is an armoured marine planktonic dinoflagellate, which causes harmful red tide when blooming. In this research, we discussed the mechanism of iron deficiency affecting the growth rate and physiological state of P. micans Ehrenberg, based on the observation of the growth of P. micans Ehrenberg under iron deficiency. The results showed that the growth rate of P. micans Ehrenberg decreased under iron deficiency, as the time to reach the peak of cell numbers was delayed 3-4 days compared to the control group. Meanwhile, the maximal cell number and the concentration of chlorophyll a dropped slightly. Examination of cell morphology by transmission electron microscope showed that the arrangement of P. micans Ehrenberg chloroplast granum was disturbed under iron deficiency. The thylakoids exhibited twisted structure with larger interstices among the thylakoid layers. Chloroplast membrane system folded abnormally and fewer starch particles were synthesized and accumulated compared to the control group. In addition, many cavities appeared in mitochondria, and a few cells developed incomplete nuclear envelop. The energy spectrogram of the algal cells showed that the relative ratio of the contents of the elements in cell also changed as the degree of iron deficiency changed. The iron deficiency-induced morphological changes of P. micans Ehrenberg cell organelles may be due to the misfolding of some core proteins that originally require iron ion as folding center. The structural abnormality of the major cell organelles further led to the functional retardation or loss in photosynthesis, electron transport, and metabolism, which blocks normal growth of P. micans Ehrenberg. Taken together, the research helped to improve our understanding on the limiting effects of iron on marine algae growth and proposed a potential way to control red tides caused by algae blooming.
B21B-0887 0800h
THE ROLE OF MICROBIAL Fe (III) REDUCTION IN THE CLAY FLOCCULATION
This study was undertaken to investigate the changes in flocculation properties of ferruginous smectite particles (Nau-1), including settling velocity, aggregate size, and floc architecture as a result of microbial reduction in the smectite structure. Dissimilatory iron reducing bacterium, Shewanella oneidensis MR-1 was inoculated with lactate as the electron donor and Fe(III) in smectite as the sole electron acceptor for 3, 12, 24, and 48 hours in an anaerobic chamber. Two controls were utilized; the first was identical to the experimental treatments except that heat-killed cells were used (nonreduced control), and the second control was the same as the first except that the incubation was carried out in an aerobic environment. The extent of Fe(III) reduction for the 48-hr incubation was observed to reach up to 18 %. Neither the nonreduced control nor the aerobically inoculated sample showed Fe(III) reduction. Compared with the nonreduced control, there was a 2.7-Ym increase of mean aggregate size and a 30-times increase in average settling velocity in the bioreduced smectite suspensions as measured with a Micromeritics Sedigraph. The aerobically inoculated smectite showed a similar aggregate size distribution to that of the nonreduced control. Significant changes in physical properties of smectite suspensions induced by microbial Fe(III) reduction were directly measured using transmission electron microscope (TEM). Floc architecture of the bioreduced smectite reveals no open structures (pore areas) as observed in nonreduced control. The aspect ratio (thickness/length) of individual smectite particle increased from 0.11 for the nonreduced control to 0.18 for the bioreduced smectite suspensions. The effects of pH on the clay flocculation were minimal in this study because the value of pH remained nearly constant at pH =7.0 -V 7.3 before and after the experiments. We, therefore, suggest that the increase in net negative charge caused by microbial Fe(III) reduction significantly promoted clay flocculation by increasing the electrochemical attraction in the smectite suspensions.
B21B-0888 0800h
Dissolved Free Amino Acids in Hydrothermal Springs at Yellowstone National Park, U.S.A.
Insights into the organic geochemistry of hydrothermal systems, as well as the dynamics of biotic processes in hot spring ecosystems, can be gained by identifying and quantifying dissolved free amino acids (DFAA). Hydrothermal systems form a unique environmental subset relative to other aqueous settings due to their higher temperatures, largely uncharacterized and exotic microbiology, wider pH range, and elevated levels of rare metals, sulfur, and dissolved gases. Previous studies of hot spring and geothermal systems (e.g. Mukhin et al., 1979; Svensson et al., 2004) indicated the presence of micromolar quantities of various amino acids, but the underlying mechanisms controlling amino acid production and disappearance/consumption have continued to remain elusive. DFAA were identified and quantified in five hot springs at Yellowstone National Park that span a range of pH (2 to 8) and temperature (75 to 93$\deg$C/boiling). Biotic uptake experiments and enantiomeric analyses on samples from one location were also performed to elucidate biotic pathways. Analyses were performed using high pressure anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), which is able to resolve amino acids as well as certain carbohydrates, oligopeptides, and a variety of related biological molecules. Preliminary data indicate that total DFAA concentrations are quite low (sub-micromolar range) and that amino acids with aliphatic and nitrogen-containing R-groups are predominant in the DFAA fraction. The types and concentrations of amino acids were variable across the sites. Obsidian Pool (pH 5.1, 77.5$\deg$C), where multiple microbiological studies have been conducted, was found to have a DFAA fraction consisting primarily of glycine with trace amounts of arginine, lysine, and histidine. In comparison, an acidic spring in the Sylvan Springs area (pH 1.9, 79.7$\deg$C) had higher total DFAA concentrations and was found to contain primarily arginine, lysine, and leucine, together with trace amounts of alanine, proline, and histidine. At least six other unknown compounds were also observed, one of them possibly at near-micromolar levels, and there was evidence for higher levels of organic compounds in general. The generally low concentrations observed in this study suggest that amino acids participate in highly dynamic biotic pathways in Yellowstone hot springs. Our observations of lower concentrations of amino acids and less diversity differ from literature results, but are consistent with suggestions of a positive correlation between acidic conditions and higher levels of DFAA (Svensson et al., 2004). References: Mukhin L.M., Bondarev V.B., Vakin E.A., Il'yukhina N.I., Kalinichenko V.I., Milekhina E.I., Safonova E.N. (1979) Doklady Akademii Nauk SSSR 244(4), 974-7. Svensson E., Skoog A., and Amend J.P. (2004) Organic Geochemistry 35, 1001-1014.
http://geopig.asu.edu
B21B-0889 0800h
Structural Disorder and Origin of Kerogen in the Apex Chert: A Comparison With Fischer-Tropsch-Type Carbon
Carbonaceous filamentous features preserved in the 3.5 Ga Apex Chert in Western Australia are currently the oldest microfossils on Earth. However, the biogenicity of these microfossils has been called into question on several counts. Two points of contention concern the structural disorder and origin of the carbon in the proposed microfossils. Although the features were originally described as composed of kerogen--an amorphous, insoluble, carbon-rich material common in oil shales--recent investigations using laser-Raman spectroscopy have concluded that this carbonaceous material is partially graphitized. Unfortunately, Raman spectroscopy may not be sensitive enough to distinguish disordered carbon species. An abiotic organic Fischer-Tropsch-type (FTT) synthesis has been proposed as the source of the carbon in the Apex Chert. To address these issues, we have studied residual carbonaceous material in the Apex Chert and two FTT carbon products using transmission electron microscopy (TEM), electron energy-loss spectroscopy (EELS), and x-ray absorption near-edge structure spectroscopy (XANES). The FTT carbon was synthesized under hydrothermal conditions at 175\deg C and 250\deg C using a montmorillonite clay catalyst by Rushdi and Simoneit (Orig. Life. Evol. Biosph. 31, 103, 2001). In TEM images, the Apex Chert residual carbon is localized at grain boundaries between quartz grains, similar to kerogen distribution in microfossils from the Gunflint Formation. High-resolution TEM images do not show the presence of graphite lattice fringes. EELS and XANES spectra of this material show the large rounded peak for single C-C bonding from 290 - 300 eV and a peak for benzene ring structures at 285 eV but no graphite peak (291 eV). In addition, XANES spectra of the residual carbon show a peak due to carbonyl (C=O) at 288.5 eV. These observations indicate that the residual carbon and the carbon in the proposed microfossils is either kerogen or an abiotic kerogen-like material. EELS and XANES spectra of FTT carbon show carbon-carbon single and double bonds and carbonyl bonding, but no graphite peak. This is generally the same as spectra of Apex Chert carbon, but the carbonyl peak for FTT carbon is much more intense. However, it is possible that decarbonation of silicified FTT material during diagenesis could reduce the amount of carbonyl such that the resulting energy-loss spectrum would be similar to spectra of Apex Chert carbon. Therefore, it is possible that the proposed microfossils in the Apex Chert could be produced by preserved FTT carbon in a hydrothermal system.
B21B-0890 0800h
Silicate-carbonate-microorganism interface studied by high resolution TEM and electron energy loss spectroscopy
The biogeochemical alteration of an Mg-Fe orthopyroxene (Tatahouine meteorite) reacted under arid conditions in a desert environment was studied by transmission electron microscopy (TEM). The focused ion beam method has been used to prepare an electron transparent cross section of the interface between a single microorganism, an orthopyroxene and nanometer-sized calcite crystals. The microorganism en route to fossilization and the nanometer-sized calcite crystals were identified using X-ray energy dispersive spectroscopy and electron energy loss spectroscopy (EELS). High resolution TEM observations reveal a close relationship between the microorganism and the surface of the orthopyroxene. Moreover, a 100 nm deep depression was observed in the orthopyroxene close to the microorganism, suggesting an enhanced dissolution mediated by the microbe. EELS measurements have also shown the existence of carbonyl or carboxyl groups associated with the calcite nano-crystals that could be related to the unusual morphology and structure of these calcite crystals.
B21B-0891 0800h
Predictive Microbiology in Hydrothermal Ecosystems
Metabolisms of high-temperature microorganisms are not revealed by molecular phylogenies, but, if known, could connect microbial and geochemical processes in hydrothermal ecosystems. Disequilibria among oxidation-reduction reactions, established by kinetic barriers to electron-transfer reactions, provide energy, and life provides the catalyst. In more-or-less closed systems, such as slowly-accumulating detrital sediments, life taps as much energy as conversion efficiency will allow, and many redox couples are driven to near-equilibrium states. In contrast, open systems like hot springs maintain persistent states of redox disequilibria that support highly diverse communities of microorganisms. In Yellowstone National Park hot springs, the magnitude of these redox disequilibria can be predicted based solely on pH, guided by past measurements of hot spring geochemistry. Geochemical diversity at Yellowstone National Park produces hydrothermal ecosystems over a pH range from less than 2 to greater than 8, with associated major and trace element concentration changes. We have assessed the supply of chemical energy in the form of redox reactions that are far from equilibrium in the Fe-S-C-O-H-N system. Field measurements of temperature, pH, dissolved oxygen, total sulfide, nitrate, nitrite, total ammonia, ferrous iron, and bicarbonate alkalinity are combined with lab analyses of sulfate, iron mineralogy, and gas composition (hydrogen, carbon dioxide, methane, carbon monoxide) in a thermodynamic analysis of the state of redox disequilibria in more than 50 hot spring habitats. Initial results (using only inorganic forms of C) yield nearly 200 reactions that are out of redox equilibrium, and which could supply energy if catalyzed. Some of these reactions, such as hydrogen oxidation, are pH independent, and the energy supply is nearly constant at about 24 kcal per mole of electrons over the entire pH range. Other reactions, which are pH dependent, show greater or lesser variations in energy supply as pH changes. As an example, the oxidation of dissolved ferrous iron to goethite varies from 26 kcal per mole of electrons (more energy-yielding than hydrogen oxidation) near pH 8, to 10 kcal per mole of electrons at pH 2. Taken together, these trends provide the first comprehensive framework for predicting which thermophilic metabolisms will prevail in which hydrothermal environments. Merging molecular microbiological methods with this type of predictive geochemical data will produce a new integrated biogeochemical approach to solving problems in microbial ecology.
http://geopig.asu.edu
B21B-0892 0800h
Organic Acids as Hetrotrophic Energy Sources in Hydrothermal Systems
Many thermophilic microbes are heterotrophs, but little is known about the organic compounds present in hydrothermal ecosystems. More is known about what these organisms will metabolize in lab experiments than what they do metabolize in nature. In an effort to bridge this gap, we have begun to incorporate organic analyses into ongoing research on Yellowstone hydrothermal ecosystems. After filtering at least a liter of hot spring water to minimize contamination, samples were collected into sixty-milliliter serum vials containing ultra-pure phosphoric acid, sodium hydroxide, or benzalkonium chloride. Approximately 80 sites were sampled spanning temperatures from 60 to $90\degC$ and pH values from 2 to 9. Analytical data for organic acid anions (including formate, acetate, lactate, and succinate) were obtained by ion chromatography. Preliminary results indicate that concentrations of organic acids anions range from 5 to 300 ppb. These results can be used with other field and lab data (sulfate, sulfide, nitrate, ammonia, bicarbonate, pH, hydrogen) in thermodynamic calculations to evaluate the amounts of energy available in heterotrophic reactions. Preliminary results of such calculations show that sulfate reduction to sulfide coupled to succinate oxidation to bicarbonate yields about 6 kcal per mole of electrons transferred. When formate oxidation to bicarbonate or hydrogen oxidation to water is coupled to sulfate reduction there is less energy available by approximately a factor of two. A comparison with nitrate reduction to ammonia involving succinate and/or formate oxidation reveals several similarities. Using formate to reduce nitrate can yield about as much energy as nitrate reduction with hydrogen (typically 12 to 14 kcal per mole of electrons transferred), but using succinate can yield more than twice as much energy. In fact, reduction of nitrate with succinate can provide more energy than any of the inorganic nitrate reduction reactions involving sulfur, iron minerals, sulfide, carbon monoxide or methane in Yellowstone hot springs. This difference suggests that small organic compounds in hydrothermal fluids can be major sources of metabolic energy for microbes, and may explain why so many heterotrophs are found in themophilic microbial culture experiments.
B21B-0893 0800h
Ultrahigh Resolution Mass Spectrometry of Leachable Organic Matter yFrom Wood after Progressive Fungal Decay
Dissolved organic matter (DOM) leaching from forest floor coarse woody debris is an yimportant mediator in metal cycling, microbial activity, and mineral dissolution in the yassociated soils. Much research has been directed at characterizing the chemical ycomposition and molecular weight range of DOM to better assess and predict its yreactivity. Unfortunately, these chemical and structural properties of DOM are extremely ydifficult to assess and consequently a wide variety of indirect analytical techniques are ytypically employed. Direct analysis of DOM by High-field Fourier Transform Ion yCyclotron Resonance Mass Spectrometry combined with Electrospray Ionization (ESI yFT-ICR MS) has only recently been applied to a limited number of environmental yorganic matter samples. With a resolving power of greater than 200,000, this is currently ythe only analytical technique capable of fully resolving individual molecules in such ycomplex mixtures. y To simulate leachates from coniferous woody debris samples of red spruce (Picea yrubens) wood were degraded with brown rot fungi over a period of 43 weeks and yextracted with water. Water-soluble fractions of the degraded residue were analyzed yusing a 9.4 Tesla ultra-high resolution FT-ICR MS. Supporting information on the yleachate and solid residue chemistry was obtained using on-line 13C-labelled ytetramethylammonium hydroyide (TMAH) thermochemolysis GC-MS, solid-state NMR yand elemental analysis. y The FT-ICR mass spectra reveal structures at every nominal mass from approximately yy250 to 1200 Daltons as well as molecular families containing ions that differ from each yother in degree of saturation or number of functional group substitutions. There is a yprogressive increase in the number of individual compounds within a nominal mass unit, yfrom 2 compounds at 4 weeks decay to 6 compounds at 32 weeks, resulting in thousands yof discrete extractable compounds at the conclusion of decay. Degradation of wood over ytime does not change the overall molecular weight range of products, however, a shift in ylocation of the data distribution on the Kendrick plot demonstrates changes in yhomologous series with degradation. This data is currently being analyzed using the van yKrevelen diagram to gain additional information.y
B21B-0894 0800h
An Assessment of Fungal Diversity Using Oligonucleotide Fingerprinting of rRNA Genes, A Macroarray-based Technique
Environmental controls over the diversity and community composition of microbial decomposers are poorly understood. In this experiment, we examined the effects of litter quality and competitive exclusion on fungal diversity. Specifically, we expected to see greater fungal diversity in litter containing higher nitrogen concentrations in that this high quality substrate would be able to support fungal groups with a range of nutrient requirements. Additionally, to test the competitive exclusion principle that a limited number of decomposer groups can be supported by a given substrate, we excluded from the litter one phylum of decomposer fungi, the Ascomycota. An increase in diversity within the remaining fungal phyla would indicate a release from competitive exclusion. To test these hypotheses, we performed a decomposition experiment in a boreal forest near Delta Junction, Alaska. Senescent leaves of {\it Populus tremuloides} (quaking aspen) from two sites that varied in soil fertility were collected in fall 2002. Leaves from the more fertile site had nitrogen concentrations of 2.26%, and those from the less fertile site had nitrogen concentrations of 1.59%. Leaves were placed into 1-mm mesh bags and incubated in a third site from September 2002 to July 2003. The fungicide Benomyl, which eliminates Ascomycetes, was applied to a subset of the bags at the onset of decomposition. Directly following collection, active fungal DNA was isolated using a nucleotide analog probe. Fungal communities were characterized by oligonucleotide fingerprinting of rRNA genes, a macroarray-based technique. Cluster analyses of fingerprints identified fungal groups representing taxonomic levels ranging from genus to class. The number of fungal groups in each treatment was approximated using the Chao1 estimator. A total of 524 $\pm$ 10 fungal groups were estimated to occur across all treatments, with less than 60 of these groups previously identified in Genbank. We found that litter quality did not strongly affect fungal diversity, since the number of fungal groups did not differ significantly between site of litter origin (P = 0.0691). However, the overall fungal diversity in the fungicide treatment was significantly reduced compared to the control (P = 0.0358). This indicates that the remaining fungal groups did not completely radiate to fill all available niches following the elimination of competitors. Thus, we did not find strong evidence for competitive exclusion as an important control over the microbial community composition in this system. An additional analysis of the diversity of non-Ascomycetes across treatments will be conducted to further elucidate any potential role of competitive exclusion.
B21B-0895 0800h
Behavior of rare earth and trace elements in Lake Tanganyika and its three major tributaries
Water samples were collected, during the rainy and dry seasons 2003, from three major rivers and several locations of the Lake Tanganyika. They were directly filtered (0.45 m pore size) into pre-washed polyethylene bottles, and acidified at pH 2. Finnigan Element 2 high resolution (HR)-IC-MPS was used to measure trace and rare earth elements (REE) concentrations under clean laboratory conditions, and (115In) was used as an internal standard. Because of the close relationship between light rare earth element (LREE) and Fe, riverine REE of the three were used to study the process trace element scavenging by Fe oxyhydroxides in three different two sub-basins of the lake. This confirmed by the significant positive correlation between Nd and Fe. The vertical distribution of Fe and Mn oxides were also used to investigate removal and release of trace elements in the water column. The normalized lacustrine REE to their riverine counterpart showed a gradual removal of REE across the lake, which was in the order of LREE>MREE>HREE. Hence, the rivers are the sole source of the lacustrine REE abundance. Coincidence of Fe maxima with those of Ce anomalies and La indicates that trace element profiles are chiefly controlled by the coating of Fe oxyhydroxides through oxidation of Fe2+ to Fe3+ under high dissolved oxygen contents and pH and vice versa. Due to differences in hydrodynamics between the extreme ends of the lake (upwelling in the southern end during the dry season), high mixing between bottom water and surface was observed at the surface in the Southern Basin while the mixing occurred mainly between 40 m and 80 m depth in the Northern Basin. There was also a clear similarity between Ba and NO3- and PO43- profiles in the southern end of the lake, supporting the idea that deep anoxic water, rich in nutrients and trace elements, are bought the surface during this period of intensive upwelling. In conclusion, the surface water chemical compositions of Lake Tanganyika are controlled by fluvial inputs and the seasonal changes in hydrodynamics across the lake.