The overall objective of the research presented here was to combine multiple geochemical parameters and molecular characterizations to provide a novel view of active microbial community ecology of sediments in a large-river deltaic estuary. In coastal and estuarine environments, a large portion of benthic respiration has been attributed to sulfate reduction and implicated as an important mechanism in hypoxia formation. The use of high-resolution sampling of individual sediment cores and high throughput nucleic acid extraction techniques combined with 454 FLX sequencing provided a robust understanding of the metabolically active benthic microbial community within coastal sediments. This was used to provide further understanding and show the importance of simultaneously analyzing the connectivity of sulfur and iron cycling to the structure and function of the microbial population. Although aqueous sulfide did not accumulate in the sediments of the northern Gulf of Mexico, active sulfate reduction was observed in all locations sampled. Microbial recycling and sequestration as iron sulfides prevented the release of sulfide from the sediment. Prominent differences were observed between the sample locations and with depth into the sediment column. This study emphasized the importance of combining novel molecular techniques with simultaneous traditional geochemical measurements to show the interdependence of microbiology and geochemistry. In addition, this study highlights the need to consider microbial community biogeography along with small-scale variations in geochemistry and biology that impact the overall cycling of redox elements when constructing biogeochemical models in marine sediments.

The redox cycling of iron is intimately linked to the cycling of C, S, N, P as well as the speciation, mobility, and bioavailability of various toxic contaminants in soils and sediments. Within these environments, the cycling of iron is catalytically driven by iron-oxidizing (FeOB) and iron reducing bacteria (FeRB) which mediate the formation, transformation, and dissolution of various iron-bearing minerals. Under oxic conditions, FeOB promote the formation of iron oxides on or in close proximity of their cell walls and extracellular polymeric substances, and such composite, termed biogenic iron oxides (BIOS), offers highly reactive heterogenous sites that efficiently immobilize trace metals and contaminants alike. However, under reducing conditions, FeRB mediate the reductive dissolution of BIOS and in turn lead to the remobilization of associated contaminants. Conversely, contaminants may become immobilized by secondary iron minerals that form from the metabolic activity of FeRB. Therefore, determining the factors that influence the reactivity of BIOS, as well as the formation of secondary iron minerals is of critical importance to develop a better understanding of the geochemical cycling of iron and in turn the transport of contaminants in the environment. This thesis investigated (1) the impact of simulated diagenesis (ageing for ~5 years at 4ºC) on the mineral stability and reactivity of BIOS towards reduction by Shewanella putrefaciens CN32, (2) the effects of phosphate at an environmentally relevant (10µM) and excess (3.9mM) concentration on the rates and extent of microbial reduction of synthetic 2-line ferrihydrite and BIOS, as well as the formation of secondary iron minerals, and (3) the impact of sterilization by γ-irradiation on the mineral stability and reactivity of BIOS. It was found that simulated diagenesis did not affect the mineralogical composition of BIOS but significantly lowered the reactivity of BIOS towards microbial reduction. The concentration of phosphate was found to have contrasting effects on the rates of reduction of ferrihydrite and BIOS, but in general, excess concentration of phosphate enhanced the extent of Fe(III) reduction. The formation of a specific secondary iron mineral was also found to depend on the concentration of phosphate, as well as, in the case for BIOS, the presence of intermixed cell derived organic matter. γ-irradiation did not alter the mineralogy and reactivity of BIOS towards microbial reduction, and it was concluded to be a suitable technique to sterilize BIOS.

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Acid mine drainage (AMD) is an acidic, iron-rich leachate that causes the dissolution of metals. It constitutes a worldwide problem of environmental contamination detrimental to aquatic life and water quality. AMD, however, is naturally attenuated at Davis Mine in Rowe, Massachusetts. We hypothesize that sulfate-reducing bacteria (SRB) are attenuating AMD. To elucidate the mechanisms by which SRB attenuate AMD, three research projects were conducted using a suite of molecular and geochemical techniques. First we established biological influence on the attenuation of AMD by comparing the microbial community and geochemical trends of microcosms of two contrasting areas within the site: AMD attenuating (AZ) and AMD generating (GZ) zones. The differences in geochemical trends between these zones were related to differences in microbial community membership. SRB were only detected in microcosms of the AZ, while iron oxidizers were only detected in the GZ. This study indicates that biological activity contributes to the attenuation of AMD and that SRB may have a role. To further describe the role of SRB, we determined the rates of sulfate reduction, the abundance, and membership of SRB in the second project. The sulfate reduction rate was weakly correlated with the abundance of SRB. This indicates that the SRB population may be utilizing another electron acceptor. One such electron acceptor would be iron, which was investigated in the third project. When SRB are inhibited, neither accumulation of reduced iron nor the formation of reduced iron sulfide precipitates occurred. Higher concentration of sulfide produced an increase in reduced iron and pH. Therefore, iron reduction mediated by reaction with biogenic sulfide contributes to the attenuation of AMD. This is the first report of the biological enhancement of iron reduction by acidotolerant SRB. The interdisciplinary research described in this dissertation provides evidence that SRB attenuate AMD through sulfate and iron reduction and a greater understanding of SRB in acidic environments. It also demonstrates how the biogeochemical cycling of sulfur is coupled to the iron cycle. Overall, the ubiquity and metabolic versatility of SRB offers boundless potential and exciting opportunities of study in the fields of bioremediation, geomicrobiology, and microbial ecology.

The bioavailability of iron is limited by the solubility of Fe(III) at circumneutral pH. In the High Nutrient-Low Chlorophyll (HNLC) zones of the ocean, the natural or anthropogenic addition of iron stimulates primary productivity and consumes carbon dioxide. As a result, iron fertilization has been proposed to mitigate anthropogenic carbon emissions and lower global temperatures. The natural sources of iron to the ocean are not fully constrained and include eolian depositions as well as inputs from continental shelf sediments, rivers, hydrothermal vents, and icebergs. Regardless of their source, the effectiveness of iron additions in promoting carbon fixation depends on the presence of organic ligands either natural or produced by microorganisms that stabilize or solubilize Fe(III) at neutral pH. For example, siderophores are well known to be expressed extracellularly by prokaryotes in the photic zones of the oceans to increase the bioavailability of iron. In this dissertation, the production of iron nanoparticles is demonstrated in vent fluids from the 90 North hydrothermal system. These iron nanoparticles may either catalyze the oxidation of sulfide to thiosulfate and produce a potential electron acceptor for microbial respiration or provide a source of iron that stimulates primary production at great distances from the hydrothermal vents. In addition, dissolved iron under the form of soluble organic-Fe(III) complexes is demonstrated to constitute a significant source of iron in estuarine sediments that receive large amounts of particulate iron from flocculation and precipitation at the salinity transition of this estuary. A novel competitive ligand equilibration absorptive cathodic stripping voltammetry (CLE-ACSV) technique reveals that the speciation of iron changes from largely colloidal or particulate in the upper estuary to truly dissolved organic-Fe(III) in the lower estuary. It is also demonstrated that organic-Fe(III) complexes are produced far below the sediment-water interface, suggesting that dissimilatory iron-reducing bacteria may play an important role in their production. These complexes then diffuse across the sediment-water interface and provide a significant source of iron to the continental shelf. The mechanism of reduction of iron oxides by iron-reducing bacteria is not fully understood and presents a unique physiological problem for the organism, as the terminal reductase has to transfer electrons to a solid electron acceptor. In this dissertation, it is demonstrated for the first time using random mutagenesis that the respiration of solid Fe(III) oxides by Shewanella oneidensis, a model iron-reducing prokaryote, first proceeds through a non-reductive dissolution step involving organic ligands that are released extracellularly by the cells. These soluble complexes are then reduced by the organism to produce Fe(II) and recycle the ligand for additional solubilization. Incubations with deletion mutants of the proteins involved in the respiration of Fe(III) revealed that the type-II secretion system, which translocates proteins on the outer membrane of gram-negative bacteria, is involved in the production of organic-Fe(III) complexes by secreting an endogenous iron-solubilizing ligand or a protein involved in the biosynthesis of this ligand on the outer membrane. In addition, periplasmic decaheme cytochromes produced by Shewanella appear to be involved in the mechanism of production of the endogenous organic ligand either directly or through a sensing mechanism that controls its production. In turn, two decaheme cytochromes positioned on the outer-membrane and hypothesized to be involved in the electron transfer to the mineral surface do not appear to be involved in the solubilization mechanism, suggesting either that the cells regulate the ligand production via periplasmic sensing systems or that these cytochromes are not involved in the solubilization mechanism. Altogether this research shows the production of organic-Fe(III) complexes in sediments generates a significant flux of dissolved iron to support primary production in continental shelf waters and that these complexes may be partly produced by iron-reducing bacteria. Indeed, experiments with a model organism demonstrate dissimilatory iron reducing bacteria produce endogenous organic ligands with high iron-binding constants to non-reductively solubilize iron oxides during the anaerobic respiration of iron oxides. The organic ligand is apparently recycled several times to minimize the energy cost associated with its biosynthesis. These findings demonstrate that the solubilization of iron oxides by organic ligands may be an important, yet underappreciated process in aquatic systems.

Thesis (Ph. D.)--Miami University, Dept. of Geology, 2007. / Title from second page of PDF document. Includes bibliographical references.

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