B41A-0022
A pilot-scale study of domestic wastewater treatment by different type constructed wetland systems and its potential green house gases emissions
Constructed wetland (CV) systems are reliable, flexible in design, and can be built, operated, and maintained at lower costs compared to conventional methods of chemical treatment. There for CW systems are widely used for controlling water-body eutrophication as an ease-operation and cost-effective ecological technology in developing countriens. However, growing attention has been directed to its greenhouse side-effect and global-warming potential in recent years. In this study, three typical constructed wetlands: Vertical Flow (VF), Free Water Surface (FWS) and Subsurface Flow (SF), and combined VF-SF-FWS constructed wetlands were used not only to compare the nutrients removal performance for treatment of low C/N ratio loading domestic wastewater, but also to investigate and compare their CH4 and N2O greenhouse gas emissions characteristics. The results indicated that the four types of CWs had high removal efficiencies for organic matter and SS. Moreover, the combined wetland also showed a comparatively good performance for nitrogen and phosphorus removal, which was 93.6%, 92.8%, 81.3% and 84.5% for BOD5, SS, TN and TP, respectively. It was found that the combined CW and VF CW has a comparative lower global warming potential. The FWS CW had the highest tendency to emit CH4 and led to a higher global warming potential among four types of CWs, which was about the same as 425mgCO2/m2/h.
B41A-0023
An Assessment of Nutritional Sources of Organisms in a Temperate Coastal Marine System
The contribution of seagrass primary production to nutrient flow in temperate aquatic systems is widely debated. The purpose of this work was to verify that eelgrass ( Zostera marina) comprises a measurable fraction of the diet of consumers in recently restored eelgrass meadows. This investigation endeavored to quantify the nutritional contributions from primary producers to the aquatic food web of restored eelgrass beds in South Bay, Virginia, by monitoring the transfer of primary producer fatty acids to primary consumers and higher level consumers. Dominant primary producers, consumers, and higher trophic level fish were collected from eelgrass meadows and adjacent barren sediment habitats dominated by ephemeral floating macroalgae, mainly Ulva lactuca and Gracilaria spp. Bulk carbon, nitrogen, and sulfur isotope analysis of organisms at these sites indicated different sources of primary production, supporting the likelihood of distinct dietary compositions. Fatty acid profiles and relative concentrations of individual fatty acids in macrophytes differed among the species, allowing for source differentiation. The fatty acid composition of the tissues of macrophytes was compared to that of local consumers in an evaluation of the ultimate sources of dietary fatty acids. Particular attention was focused on the biomarker fatty acids found in eelgrass as well as other primary producers. The dietary importance of eelgrass to consumers in barren sediment sites is suggested to be negligible, resulting in fatty acid profiles differing from organisms captured at the two sites dominated by the macrophyte and seagrass primary producers.
B41A-0025
Measuring Greenhouse Gas fluxes and Carbon and Nitrogen Cycling In Situ in an Estuarine Lake
A system allowing the in situ quantification of the nitrogen cycle and various carbonaceous species in the air, water and sediment has been developed and deployed in the field to monitor the effects of sediment disturbance on the nitrogen and carbon cycles. Fluxes of nitrous oxide, methane and carbon dioxide were monitored continuously in situ at nearshore sites within a nutrient rich estuarine lake for periods in winter 2006, summer 2007 and winter 2007. Changes in these gas phase fluxes were compared with variations in aqueous and sedimentary carbonaceous and nitrogenous concentrations (OrgN, OrgC, NH3 and NO3- + NO2-) and examined in terms of carbon and nitrogen biogeochemical cycling. Links between greenhouse gas production, carbon and nitrogen cycling, water temperature, light availability, pH and dissolved oxygen concentration were also studied. The effect of sediment disturbance was also investigated with significant increases in the concentrations of aqueous NH3 and NO3- + NO2- evident. Nearshore sediments were found to act as both a source and a sink of atmospheric nitrous oxide.
B41A-0026
Microbiology and Biogeochemical Study of Underground Research Tunnel for the Geological Disposal of Nuclear Waste
The Underground Research Tunnel (URT) located in Korea Atomic Energy Research Institute (KAERI), Daejeon, South Korea was recently constructed as an experimental site to study radionuclide transport, biogeochemistry, radionuclide-mineral interactions for the geological disposal of high level nuclear waste. Groundwater sampled from URT was used to examine microbial diversity and to enrich metal reducing bacteria for studying microbe- metal interactions. Genomic analysis indicated that the groundwater contained diverse microorganisms such as metal reducers, metal oxidizers, anaerobic denitrifying bacteria, and bacteria for reductive dechlorination. Metal- reducing bacteria enriched from the groundwater was used to study metal reduction and biomineralization. The metal-reducing bacteria enriched with acetate or lactate as the electron donors showed the bacteria reduced Fe(III)-citrate, Fe(III) oxyhydroxides, Mn(IV) oxide, and Cr(VI) as the electron acceptors. Preliminary study indicated that the enriched bacteria were able to use glucose, lactate, acetate, and hydrogen as electron donors while reducing Fe(III)-citrate or Fe(III) oxyhydroxide as the electron acceptor. The bacteria exhibited diverse mineral precipitation capabilities including the formation of magnetite, siderite, and rhodochrosite. The results indicated that Fe(III)- and metal-reducing communities are present in URT at the KAERI.
B41A-0027
Atmospheric oxygen levels, anaerobic methane oxidation, and the coupling of the global COS cycles by sulfate reduction
Changes in the partitioning between the reduced and oxidized reservoirs of carbon and sulfur are the dominant control on atmospheric oxygen levels, and the partitioning itself depends to a large degree on microbial redox processes remineralizing organic matter (OM). However, the controls of organic matter preservation in marine sediments are one of the most complex and controversial issues in contemporary biochemistry. Knowledge how the transition from one electron acceptor to another affects OM remineralization rates is scant even for the transition from aerobic to anaerobic respiration. Much less is known about the transition from anaerobic respiration to fermentation. Although the individual pathways of methane generation are known, our understanding of the complex interactions between different bacterial groups remains limited, resulting in considerable difficulties to resolve these questions in microcosm experiments. Here we show that a dramatic drop in seawater sulfate concentrations during the Early Cretaceous (Wortmann & Chernyavsky, Nature 2007) resulted in a global breakdown of microbial sulfate reduction in the marine subsurface biosphere. This event resulted in a positive excursion of the global δ13C-value, suggesting that organic matter remineralization rates dropped by more than 50%. This implies that
B41A-0028
Microbe-Sediment Interactions Controlling Growth of Modern Marine Stromatolites: Highborne Cay, Bahamas
Modern stromatolites forming in seawater of normal marine salinity along the margins of Exuma Sound, Bahamas, provide an ideal opportunity to document interactions between microbial mat communities and the environment that lead to stromatolite growth. The well-laminated and easily accessible stromatolite build-ups at Highborne Cay (76o49' W, 24o43' N) form in the back-reef of a 2.5km algal-ridge fringing reef complex along the eastern shoreline (facing Exuma Sound) of the island. The stromatolites form as ridges and columnar heads in the shallow subtidal zone. The mat communities on the surface of the stromatolites include both prokaryotic and eukaryotic organisms. The prokaryotic mats are dominated by gliding filamentous cyanobacteria and are found chiefly on the low-lying, shallow stromatolite ridges. In contrast, the mixed eukaryotic-prokaryotic mat communities are predominantly observed on columnar head stromatolites and are composed of diatoms overlying filamentous cyanobacteria. Tube diatom mats are dominant in winter and spring, whereas stalked diatoms are dominant in summer and fall. The mixed eukaryotic-prokaryotic communities accrete sediment more rapidly than the purely prokaryotic mats, with diatoms trapping sediment, which is subsequently bound by the upward migration of the underlying filamentous cyanobacteria. A major control on the distribution of microbial communities on Highborne Cay stromatolites is the migration of sand waves, both intertidal and subtidal, across the stromatolite reef. These migrating sand waves bury and expose the reef episodically for periods of days, weeks, and months. Variations in the duration of these sedimentation events reflect changes in physical energy along the reef, which when coupled with seasonality, control microbial growth and resultant stromatolite formation.
B41A-0029
Linear Chain Formation by Unicellular Bacteria During Mat Growth Under Low-Energy Flows
Biofilm morphologies and material properties are known to be functions of overlying fluid flow strength. It has been hypothesized that microbial mats and stromatolites also respond morphologically to fluid flow. We show that microscopic textures of experimentally grown mats of the unicellular cyanobacterium Synechocystis sp. PCC 6803 respond sensitively to overlying flow. Cultures were grown in two chemostats in which growth medium was replaced at a rate much faster than the bacteria's instantaneous growth rate, thus selecting for individuals attached to surfaces. Small petri dishes at the bottoms of each chemostat received innocula at the beginning of the experiment. One chemostat was stirred at a low rate such that there was no measurable flow across the tops of the dishes, while the other was stirred at a rate maintaining a flow of approximately 0.7 cm/s at 1 cm over the dishes. Cultures were grown for 10 days during which thick biofilms/mats developed in the dishes. In addition to biofilms developed on the petri dish surfaces, cultures in the rapidly stirred chemostat developed thick "streamers" which projected up into and were deflected by the overlying flow. Samples were collected from films and, in the rapidly stirred chemostat, from streamers by pipetting and by pinching between two thin bamboo sticks. Samples were examined by fluorescence microscopy with a 40x objective. Without fluid flow, cells were only loosely associated and showed little or no spatial organization. Under the modest flow set up in the rapidly stirred chemostat, cells in both biofilms and streamers formed long linear chains arranged in sheets or tight bundles. These results suggest that hydraulic factors may be significant in shaping mat textures at the scale of 10-100 μm by modifying the spatial associations of groups of cells. If preserved in microcrystalline quartz or carbonate, the chains formed in these experiments could be mistaken for filamentous bacteria. Care must be taken when inferring microfossil morphology in moderately preserved mat samples, particularly in sediments deposited under low-energy currents.
B41A-0030
Isotopic evidence for the source and fate of P in the Everglades wetlands
High phosphorus (P) influx into wetland ecosystems in the Florida Everglades continues to be a problem. The increased P loading has been linked to changes in flora and fauna and the degradation of water quality in the wetlands. The number and species of animals have dramatically declined due to the agricultural and urban development since 1900. The plant community has also shifted from P-limited sawgrass (Cladium) to P-adapted cattail (Typha) in areas impacted by agricultural runoff. Although the effects of P loading on ecosystem have been recognized, little is known about how those changes affect the biogeochemical processes regulating P availability and cycling in freshwater ecosystems. The P-O bond in phosphate is resistant to hydrolysis in inorganic systems. However, the P-O bond can be easily broken in enzyme-mediated biochemical reactions, resulting in rapid oxygen isotope exchange with surrounding water within organisms. Thus, oxygen isotopic composition of phosphate should indicate the environment and processes of its formation. Oxygen isotopes in phosphate may provide a useful tool for tracing the source and recycling of phosphorus in aquatic systems. Here, I present the results of an oxygen isotopic study of phosphate in a constructed wetland (Storm water Treatment Area STA-1W) in northern Everglades as well as in a relatively pristine wetland in the Everglades National Park (ENP). Oxygen isotopic compositions of dissolved inorganic phosphate (DIP) in water and of total phosphate in sediment were determined using a High Temperature Conversion Elemental Analyzer (TC/EA) interfaced to a Finnigan MAT Delta Plus XP stable isotope ratio mass spectrometer (IRMS) at NHMFL. The data show: 1) there is no clear relationship between the d18O of DIP and P concentration in the water; 2) the d18O value of DIP is correlated with hydrological data (what kind?); 3) d18O value of DIP is not in equilibrium with water. The DIP samples collected in July are closer to isotopic equilibrium with environmental water than in April, indicating a faster biochemical cycling in the summer than in the spring. Our data also show that the d18O values of total phosphate in sediment cores from both STA1W and ENP display a very similar pattern, likely reflecting the interaction of remineralization and influence of anthropogenic P input (e.g. fertilizer).
B41A-0031
Lipid and Phylogenetic Analysis of a Gypsum-hosted Endoevaporitic Microbial Community
Gypsum evaporites host diverse, productive and volumetrically significant microbial communities and are relevant modern-day analogs to both Precambrian sabkha deposits and, potentially, Martian evaporites. Extensive evaporites form in subaqueous environments of high salinity ponds (>150 permil) maintained by the Exportadora de Sal, S. A. (ESSA) in Guerrero Negro, B.C.S., Mexico. A gypsarenite (reworked clastic gypsum) crust found along the southeast margin of ESSA's Pond 9 was collected in February 2004 and each vibrantly colored layer in the top centimeter was sampled. Extant microbial communities from each layer were characterized using complementary culture-independent molecular techniques, lipid biomarker analysis, and compound specific isotopic analysis. Coupling molecular analysis with lipid biomarker analysis revealed that oxygenic photosynthetic organisms dominate the surface layers (top 3 mm). Polar lipids from the surface layers consisted predominantly of glycolipids, which are characteristic of algae, cyanobacteria and green anoxygenic photosynthetic bacteria. Consistent with prior analyses of gypsum evaporites, 16S rRNA gene clone libraries indicate that cyanobacterial populations belong primarily to the genus Cyanothece. The bacterial community below the surface layers is more diverse and dominated by anaerobic organisms. Phototrophic purple sulfur bacteria, sulfate-reducing bacteria (SRB), and Bacteroidetes were particularly abundant. The relative abundances of SRB increased with depth; Desulfobacteraceae clones were distributed throughout the crust, but not at the surface, while Desulfovibrionaceae clones were found predominantly in the deepest layers. These molecular results are consistent with fatty acid biomarker analysis. δ13C values of major lipid classes in the crust and sediment range from 14 to 36‰, which is considerably lower than corresponding values for benthic Microcoleus-dominated cyanobacterial mats found at lower salinities at ESSA (65-100‰). A mass balance calculation yields δ13C values of approximately 20‰ for the fatty acid fraction in surface layers, only slightly depleted relative to the surface 2 mm of the Microcoleus mat (δ13C ~ -17 ‰). The fatty acid fractions in lower anoxic layers are significantly depleted relative to surface crust, with δ13C values approaching -26 permil. In contrast, the Microcoleus mats do not exhibit a substantial trend with depth. The isotopic trend with depth in the crusts may reflect isotopic discrimination associated with carbon fixation by non-oxygenic phototrophs and/or chemautotrophs, or changes in the network of carbon flows within the ecosystem.
B41A-0032
Oxidative and reductive transformations of arsenic by photosynthetic microbial communities from hot springs on Pahoa Island, Mono Lake, California
The shoreline of Pahoa Island in hypersaline Mono Lake in California is characterized by numerous volcanogenic hot springs that display a wide range of temperatures between 30 and 85 degrees C. A variety of distinctive photosynthetic microbial mats are evident in these hot springs and their spatial distribution appears to be a function of water temperature. The suboxic hydrothermal waters of these seeps typically contain ~100 uM dissolved arsenic, which is rapidly oxidized from arsenite [As(III)] to arsenate [As(V)] as the springs flow over these microbial communities. We conducted experiments with anaerobic cultures of red or green photosynthetic bacteria from these hot springs, which we amended with radio-labeled 73As(III) or 73As(V) and incubated at 42 degrees C to measure arsenite oxidation and arsenate reduction activity. In order to assess the potential for As(III) to serve as an electron donor during anoxygenic photosynthesis, As(III) oxidation incubations were conducted under both light and dark conditions. Both light and dark incubations of these thermophiles rapidly oxidized amendments of 100 uM As(III) within 7 hours of incubation, however no significant difference was observed in the rate of As(III) oxidation for light compared to dark samples. Arsenate reduction was also observed in both light and dark anaerobic cultures after 48 hours incubation. In all cases, As oxidation or reduction activity was eliminated by autoclaving. These results suggest that biological As(III) oxidation by these bacteria is primarily a mechanism of detoxification or chemoautotrophy, however the potential significance of As(III) as a photosynthetic electron acceptor will be discussed.
B41A-0033
Unraveling Molecular Mechanisms for the Unusual Fossil Preservation and Biomineralization Pathways in Tlayúa, the Mexican Solenhofen
The Tlayúa slurry constitutes the most important paleontological locality in the American continent, and constitutes the second most important locality in its genre worldwide. The importance of Tlayúa strives in the fact that a great diversity of marine and terrestrial fossils in perfect state of preservation have been found, with ages surpassing 115 million yrs. Paleomagnetic determinations and biostratigraphic determinations conducted in amonites and belemnites indicate that the formation of the Tlayúa slurry dates back to the late Albian. On the other hand, fish, reptiles, invertebrates, and vegetables fossil specimens have been found to date back to the Mesozoic Era. Because of this fact is unprecedented worldwide, Tlayúa is nowadays considered patrimony for the humanity. One of the most accepted hypothesis for explaining Tlayúa's formation relies on the deposition of sediments and fauna on a shallow platform of a tropical sea. A similar geographic place is located in Solenhofen, Germany, where slurries have been exploited for more than 200 yrs with a production of approximately 500 species. Remarkably, in the Tepexi del Rio region for the past 20 yrs more than 5,000 fossil specimens representing more than 200 species have been collected alone. An exceptional specimen preservation found in Tlayúa has been attributed to restricted circulation of water resulting in an anaerobic and/or hypersaline environment, coupled with the general absence of infaunal species. There were periods when the deposition site supported a rich planktontic community. Large quantities of calcareous ooze were produced, resulting in rapid burial of the organisms. The presence of diagnostic terrestrial and freshwater organisms, including arachnids, insects, lizards, and chelonians, along with typical marine fauna, suggests that Tlayúa lagoon had periodic freshwater inflow, in addition to the strong marine, lagoonal, and reefal influence. Some organisms were transported into the lagoon when the barrier was breached, probably during periods of heavy rains and hurricanes, or during high tides. Additionally, some fishes from Tlayua have been found to have affinities with recent families known to inhabit brackish and freshwater environments. Some of these fish preserve gut contents. Preliminary analysis of the intestinal content of these fishes has resulted in identification of freshwater insects and fern fragments. This work addresses for the first time the study of chemical and biological mechanisms contributing to fossil preservation and biomineralization pathways prevailing in Tlayúa using synchrotron techniques (XRF, - XRD, 3D--IR, XANES/EXAFS, STxM). We present chemical composition data collected from a fish egg's interior in search of fossilized structures. We also present data from well-preserved soft tissue collected from a fish soon to be named Michin scernai (newly identified specie, thus the name cannot be applied formally just yet). This fish is a Pachyrhizodontide, from the telesteos incertae sedis group already extinct. This particular sample was collected from the gastric cavity, precisely where female fish store the eggs before laying.
B41A-0034
Submicron-Chemical Speciation of Late Albian, Well-Preserved Fossil Samples from Tlayúa, the Mexican Solenhofen.
The Tlayúa slurry quarry constitutes the most important paleontological locality in the American continent, and constitutes the second most important locality in its genre worldwide. The importance of Tlayúa strives inderives from the fact that a great diversity of marine and terrestrial fossils in perfect state of preservation have been found, with ages surpassing 115 million yrs. Paleomagnetic and biostratigraphic determinations conducted in ammonites and belemnites indicate that the formation of the Tlayúa slurry dates back to the late Albian. One of the most accepted hypothesis for explaining Tlayúa's formation relies on the deposition of sediments and fauna on a shallow platform of a tropical sea. A similar geographic place is located in Solenhofen, Germany, where slurries have been exploited for more than 200 yrs with a production of approximately 500 species. Remarkably, in the Tepexi del Rio region alone for the past 20 yrs more than 5,000 fossil specimens representing more than 200 species have been collected alone. An The exceptional specimen preservation found in Tlayúa has been attributed to restricted circulation of water resulting in an anaerobic and/or hypersaline environment, coupled with the general absence of infaunal species. There were periods when the deposition site supported a rich planktontic community. Large quantities of calcareous ooze were produced, resulting in rapid burial of the organisms. The presence of diagnostic terrestrial and freshwater organisms, including arachnids, insects, lizards, and chelonians, along with typical marine fauna, suggests that Tlayúa lagoon had periodic freshwater inflow, in addition to the strong marine, lagoonal, and reefal influence. Some organisms were transported into the lagoon when the barrier was breached, probably during periods of heavy rains and hurricanes, or during high tides. Additionally, some fishes from Tlayua have been found to have affinities with recent families known to inhabit brackish and freshwater environments. In search for reconstructing paleoenvironments in Tlayúa, fish bone samples from a Pachyrhizodontide specimens, from the telesteos incertae sedis group already extinct, were analyzed using XRFmicro X-ray fluorescence, -X-ray diffraction RD, and -EXAFSnd XANES/EXAFS. Conducting Ca- EXAFS allowed us to resolve Ca-speciation in CaCO3 matrices. Micro Ca-EXAFSDiffraction and Ca K-edge XANES on bone material confirmed the presence of apatite, not hydroxyapatite consistent with highly-weathered environment. High concentrations of As were found in CaCO3) grains, Mn oxides grains as well as Celestine (SrSO4) grains dispersed in the egg core.
B41A-0035
High Pressure Reduction of Selenite by Shewanella oneidensis MR-1
High-pressure biotopes comprise cold deep-sea environments, hydrothermal vents, and deep subsurface or deep-sea sediments. The latter are less studied, due to the technical difficulties to sample at great depths without contamination. Nevertheless, microbial sulfate reduction and methanogenesis have been found to be spatially distributed in deep deep-sea sediments (1), and sulfate reduction has been shown to be actually more efficient under high hydrostatic pressure (HHP) in some sediments (2). Sulfate-reducing bacteria obtained from the Japan Sea are characterized by an increased sulfide production under pressure (3,4). Unfortunately, investigations of microbial metabolic activity as a function of pressure are extremely scarce due to the experimental difficulty of such measurements at high hydrostatic pressure. We were able to measure the reduction of selenite Se(IV) by Shewanella oneidensis MR-1 as a function of pressure, to 150 MPa using two different high-pressure reactors that allow in situ X-ray spectroscopy measurements on a synchrotron source. A first series of measurements was carried out in a low-pressure Diamond Anvil Cell (DAC) of our own design (5) at ID22 beamline at ESRF (European Synchrotron Radiation Facility); a second one was performed in an autoclave (6) at the BM30B beamline at ESRF. Selenite reduction by strain MR-17 was monitored from ambient pressure to 150 MPa over 25 hours at 30 deg C by XANES spectroscopy (X-ray Analysis of Near Edge Structure). Spectra were recorded hourly in order to quantify the evolution of the oxidation state of selenium with time. Stationary-phase bacteria were inoculated at a high concentration into fresh growth medium containing 5 or 10 M of sodium selenite and 20 mM sodium lactate. Kinetic parameters of the Se (IV) reduction by Shewanella oneidensis strain MR-1 could be extracted from the data, as a function of pressure. They show 1) that the rate constant k of the reaction is decreased by a half at high pressure, and 2) that the yield of the reaction decreases linearly as a function of pressure. From the present study, we can infer the maximum pressure of the metabolism of Se by Shewanella oneidensis strain MR-1 at ca. 160 MPa. The present results indicate that even piezosensitive bacteria can harbor a significant amount of metabolic activity at pressure conditions that are relevant for subsurface geochemical cycles. Moreover, one can hypothetize the same implications in the cycles of more abundant elements, like Fe or Mn. 1 S. D'Hondt, et al., Science 306, 2216-2221 (2004). 2 J. Kallmeyer and A. Boetius, Appl Environ Microbiol 70, 1231-1233 (2004). 3 S. Bale, K. et al., Int J Syst Bacteriol 47, 515-521 (1997). 4 R. J. Parkes, et al. J Microbiol Methods 23, 235-249 (1995). 5 I. Daniel, et al. in prep (2007). 6 D. Testemale, et al. Rev Sci Instrum 76, 043905 (2005). 7 C. R. Myers and K. H. Nealson, J Bacteriol 172, 6232-6238 (1990).
B41A-0036
Controls on Fe Isotope Fractionation During Organic Complexation: the Importance of Covalent Bonding
Fe isotopes have been proposed as a tracer of changes to the redox state of the oceans (Rouxel et al., 2005), and for use as a biosignature (e.g., Johnson et al., 1999). Previous modeling work supports this, as they suggest redox fractionations are likely the main control over Fe isotopes.Fe isotopes have been proposed as a tracer of changes to the redox state of the oceans (Rouxel et al., 2005), and for use as a biosignature (e.g., Beard et al., 1999). Previous modeling work (Domagal-Goldman and Kubicki, submitted) that predicts greater equilibrium fractionations for redox reactions than for complexation reactions supports the former application. In this study, we try to ascertain the first-principles chemical drivers of fractionation of Fe isotopes. We do this by using Natural Bond Order (NBO) analyses and isotope fractionation predictions of Fe bound to various organic ligands at different Fe oxidation states and Fe:ligand ratios.NBO analysis re-assigns electrons in molecular orbitals to bond orbitals within a complex; this allows for the examination of the presence and strength of covalent bonding in a complex. By comparing the presence and strength of covalent Fe-O bonds in the studied complexes to other predicted variables such as bond lengths and predicted fractionation factors, we can assess the importance of these bonds to Fe isotope fractionation in nature. Byexamining the effect controlled variables such as Fe oxidation state and the number of Fe-ligand bonds have on the formation of covalent bonds, we will begin to understand what controls bonding for these types of complexes. Ultimately, this work is geared towards driving future research questions related to the isotopicfractionations of Fe and other transition metals.