Alexis S. Templeton

Diverse Mn(II)-Oxidizing Bacteria Isolated from Submarine Basalts at Loihi Seamount

Abstract Metal-oxidizing bacteria may play a key role in the submarine weathering of volcanic rocks and the formation of ferromanganese crusts. Putative fossil microbes encrusted in Mn oxide phases are commonly observed on volcanic glasses recovered from the deep ocean; however, no known Mn(II)-oxidizing bacteria have been directly identified or cultured from natural weathered basalts. To isolate epilithic Mn(II) oxidizing bacteria, we collected young, oxidized pillow basalts from the cold, outer portions of Loihi Seamount, and from nearby exposures of pillow basalts at South Point and Kealakekua Bay, HI. SEM imaging, EDS spectra and X-ray absorption spectroscopy data show that microbial biofilms and associated Mn oxides were abundant on the basalt surfaces. Using a series of seawater-based media that range from highly oligotrophic to organic-rich, we have obtained 26 mesophilic, heterotrophic Mn(II)-oxidizing isolates dominated by α- and γ-Proteobacteria, such as Sulfitobacter, Methylarcula and Pseudoalteromonas spp. Additional isolates include Microbulbifer, Alteromonas, Marinobacter, and Halomonas spp. None of the isolates, nor their closest relatives, were previously recognized as Mn(II) oxidizing bacteria. The physiological function of Mn(II) oxidation is clearly spread amongst many phylogenetically diverse organisms colonizing basalt surfaces. Our findings support a biological catalysis of Mn(II) oxidation during basalt-weathering, and suggest heterotrophic Mn(II) oxidizing bacteria may be ubiquitously associated with submarine glasses within epilithic and endolithic biofilms.

Subsurface Microbial Diversity in Deep-Granitic-Fracture Water in Colorado

ABSTRACT A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This “Henderson candidate division” dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and Firmicutes. Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.

Fluid flow during active oblique convergence: A Southern Alps model from mechanical and geochemical observations

On the basis of combined mechanical modeling and isotopic observations, we propose a Southern Alps paradigm for application to fluid-flow regimes of active oblique collision zones subject to orographic precipitation. The model is derived from four spatial correlations among patterns in the distribution of mechanical forces that drive fluid flow, structural permeability, and isotopic signature. (1) A strong meteoric overprint of a deep isotopic signature in the inboard adjacent to the plate boundary is associated with oblique reverse faulting. In these rocks, exhumed rapidly from depth, the isotopic signature is dominated by rock advection and limited vertical movement of fluids. (2) The region along the main divide characterized by net expansion, rotation, and steep failure planes is associated with an isotopic signature of anomalously deep fluids in shallow rocks. (3) A high-strain zone within the root of the deforming orogen produces fluids during strain-induced metamorphism. (4) Basinal and meteoric fluids interact at shallow levels in the outboard region, which undergoes net contraction and rotation in an oblique thrust belt. We suggest that these four correlations represent a predictable pattern that is characteristic of all oblique orographic orogens. Rock advection is the dominant process influencing the isotopic signature adjacent to the plate boundary, where strain and erosion rates are high. Water advection exerts the dominant influence on isotopic signature in the region of net expansion near the main divide, where steep structures tap the source of deep fluids in the underlying high-strain detachment and crustal root.

Pb(II) distributions at biofilm–metal oxide interfaces

The distribution of aqueous Pb(II) sorbed at the interface between Burkholderia cepacia biofilms and hematite (α-Fe2O3) or corundum (α-Al2O3) surfaces has been probed by using an application of the long-period x-ray standing wave technique. Attached bacteria and adsorbed organic matter may interfere with sorption processes on metal oxide surfaces by changing the characteristics of the electrical double layer at the solid–solution interface, blocking surface sites, or providing a variety of new sites for metal binding. In this work, Pb Lα fluorescence yield profiles for samples equilibrated with 10−7 to 10−3.8 M Pb(II) were measured and modeled to determine quantitatively the partitioning of Pb(II) at the biofilm–metal oxide interface. Our data show that the reactive sites on the metal oxide surfaces were not passivated by the formation of a monolayer biofilm. Instead, high-energy surface sites on the metal oxides form the dominant sink for Pb(II) at submicromolar concentrations, following the trend α-Fe2O3 (0001) > α-Al2O3 (11̄02) > α-Al2O3 (0001), despite the greater site density within the overlying biofilms. At [Pb] > 10−6 M, significant Pb uptake by the biofilms was observed.

Mineralogy of Iron Microbial Mats from Loihi Seamount

Extensive mats of Fe oxyhydroxides and associated Fe-oxidizing microbial organisms form in diverse geochemical settings – freshwater seeps to deep-sea vents – where ever opposing Fe(II)-oxygen gradients prevail. The mineralogy, reactivity, and structural transformations of Fe oxyhydroxides precipitated from submarine hydrothermal fluids within microbial mats remains elusive in active and fossil systems. In response, a study of Fe microbial mat formation at the Loihi Seamount was conducted to describe the physical and chemical characteristics of Fe-phases using extended X-ray absorption fine structure spectroscopy, powder X-ray diffraction, synchrotron radiation X-ray total scattering, low-temperature magnetic measurements, and Mössbauer spectroscopy. Particle sizes of 3.5–4.6 nm were estimated from magnetism data, and coherent scattering domain (CSD) sizes as small as 1.6 nm are indicated by pair distribution function (PDF) analysis. Disorder in the nanostructured Fe-bearing phases results in limited intermediate-range structural order: less than that of standard two-line ferrihydrite (Fh), except for the Pohaku site. The short-range ordered natural Fh (FhSRO) phases were stable at 4°C in the presence of oxygen for at least 1 year and during 400°C treatment. The observed stability of the FhSRO is consistent with magnetic observations that point to non-interacting nanoparticles. PDF analyses of total scattering data provide further evidence for FhSRO particles with a poorly ordered silica coating. The presence of coated particles explains the small CSD for the mat minerals, as well as the stability of the minerals over time and against heating. The mineral properties observed here provide a starting point from which progressively older and more extensively altered Fe deposits may be examined, with the ultimate goal of improved interpretation of past biogeochemical conditions and diagenetic processes.

Stable isotopic evidence for mixing between metamorphic fluids and surface-derived waters during recent uplift of the Southern Alps, New Zealand

Recent studies have shown that the Southern Alps of New Zealand have active hydrothermal systems driven by tectonic uplift. These studies have concentrated on the rapidly exhumed rocks immediately adjacent to the Alpine Fault. We present new stable isotopic evidence that shows that fluid flow and fluid mixing processes thought to be restricted to rocks near the Alpine Fault also occurred in the low-uplift rate region of the Southern Alps orogen during the Kaikouran orogeny (10 Ma to present). Low δ18Ocalcite values of post-metamorphic veins in the eastern Southern Alps indicate that meteoric waters have penetrated to hot, midcrustal levels (350–300°C, >5 km depth) and mixed with metamorphic fluids in areas far removed from the Alpine Fault. In addition, the isotopic values of calcites precipitated in active faults in the MacKenzie basin define a trend of increasing δ18O, decreasing δ13C and decreasing temperature and form an isotopic mixing line between fault and vein calcites crosscutting metamorphic rocks and authigenic calcites precipitated in MacKenzie basin sediments. The isotope data indicate a second phase of mixing between a modified metamorphic fluid and a surficial diagenetic fluid at shallow depths. We suggest that mixed metamorphic–meteoric hydrothermal systems have developed in the east side of the Southern Alps during uplift and that active faults have played a critical role in transporting metamorphic fluids outward from the mountain front and into the adjacent intermontane basins.

Microscale imaging and identification of Fe speciation and distribution during fluid-mineral reactions under highly reducing conditions.

The oxidation state, speciation, and distribution of Fe are critical determinants of Fe reactivity in natural and engineered environments. However, it is challenging to follow dynamic changes in Fe speciation in environmental systems during progressive fluid-mineral interactions. Two common geological and aquifer materials-basalt and Fe(III) oxides-were incubated with saline fluids at 55 °C under highly reducing conditions maintained by the presence of Fe(0). We tracked changes in Fe speciation after 48 h (incipient water-rock reaction) and 10 months (extensive water-rock interaction) using synchrotron-radiation μXRF maps collected at multiple energies (ME) within the Fe K-edge. Immediate PCA analysis of the ME maps was used to optimize μXANES analyses; in turn, refitting the ME maps with end-member XANES spectra enabled us to detect and spatially resolve the entire variety of Fe-phases present in the system. After 48 h, we successfully identified and mapped the major Fe-bearing components of our samples (Fe(III) oxides, basalt, and rare olivine), as well as small quantities of incipient brucite associated with olivine. After 10 months, the Fe(III)-oxides remained stable in the presence of Fe(0), whereas significant alteration of basalt to minnesotaite and chlinochlore had occurred, providing new insights into heterogeneous Fe speciation in complex geological media under highly reducing conditions.

Selenium speciation and partitioning within Burkholderia cepacia biofilms formed on α-Al2O3 surfaces

Abstract The distribution and speciation of Se within aerobic Burkholderia cepacia biofilms formed on α-Al2O3 (1-102) surfaces have been examined using grazing-angle X-ray spectroscopic techniques. We present quantitative information on the partitioning of 10−6 M to 10−3 M selenate and selenite between the biofilms and underlying alumina surfaces derived from long-period X-ray standing wave (XSW) data. Changes in the Se partitioning behavior over time are correlated with microbially induced reduction of Se(VI) and Se(IV) to Se(0), as observed from X-ray absorption near edge structure (XANES) spectroscopy. Selenite preferentially binds to the alumina surfaces, particularly at low [Se], and is increasingly partitioned into the biofilms at higher [Se]. When B. cepacia is metabolically active, B. cepacia rapidly reduces a fraction of the SeO32− to red elemental Se(0). In contrast, selenate is preferentially partitioned into the B. cepacia biofilms at all [Se] tested due to a lower affinity for binding to the alumina surface. Rapid reduction of SeO42− by B. cepacia to Se(IV) and Se(0) subsequently results in a vertical segregation of Se species at the B. cepacia/α-Al2O3 interface. Elemental Se(0) accumulates within the biofilm with Se(VI), whereas Se(IV) intermediates preferentially sorb to the alumina surface. B. cepacia/α-Al2O3 samples incubated with SeO42− and SeO32− when the bacteria were metabolically active result in a significant reduction in the mobility of Se vs. X-ray treated biofilms. Remobilization experiments show that a large fraction of the insoluble Se(0) produced within the biofilm is retained during exchange with Se-free solutions. In addition, Se(IV) intermediates generated during Se(VI) reduction are preferentially bound to the alumina surface and do not fully desorb. In contrast, Se(VI) is rapidly and extensively remobilized.

Assessment of in-situ bioremediation at a refinery waste-contaminated site and an aviation gasoline contaminated site

A combination of geochemical, microbiological and isotopic methods were used to evaluate in-situ bioremediation of petroleum hydrocarbons at one site contaminated with refinery waste and a second site contaminated with aviation gasoline at Alameda Point, California. At each site, geochemical and microbiological characteristics from four locations in the contaminated zone were compared to those from two uncontaminated background locations. At both sites, the geochemical indicators of in-situbiodegradation includeddepleted soil gas and groundwater oxygen, elevated groundwater alkalinity, and elevated soil gas carbon dioxide and methane in the contaminated zone relative to the background. Radiocarbon content of methane and carbon dioxide measured in soil gas at both sites indicated that they were derived from hydrocarbon contaminant degradation. Direct microscopy of soil core samples using cell wall stains and activity stains, revealed elevated microbial numbers and enhanced microbial activities in contaminated areas relative to background areas, corroborating geochemical findings. While microbial plate counts and microcosm studies using soil core samples provided laboratory evidence for the presence of some microbial activity and contaminant degradation abilities, they did not correlate well with either contaminant location, geochemical, isotopic, or direct microscopy data.

Low temperature S 0 biomineralization at a supraglacial spring system in the Canadian High Arctic

Elemental sulfur (S(0) ) is deposited each summer onto surface ice at Borup Fiord pass on Ellesmere Island, Canada, when high concentrations of aqueous H(2) S are discharged from a supraglacial spring system. 16S rRNA gene clone libraries generated from sulfur deposits were dominated by β-Proteobacteria, particularly Ralstonia sp. Sulfur-cycling micro-organisms such as Thiomicrospira sp., and ε-Proteobacteria such as Sulfuricurvales and Sulfurovumales spp. were also abundant. Concurrent cultivation experiments isolated psychrophilic, sulfide-oxidizing consortia, which produce S(0) in opposing gradients of Na(2) S and oxygen. 16S rRNA gene analyses of sulfur precipitated in gradient tubes show stable sulfur-biomineralizing consortia dominated by Marinobacter sp. in association with Shewanella, Loktanella, Rubrobacter, Flavobacterium, and Sphingomonas spp. Organisms closely related to cultivars appear in environmental 16S rRNA clone libraries; none currently known to oxidize sulfide. Once consortia were simplified to Marinobacter and Flavobacteria spp. through dilution-to-extinction and agar removal, sulfur biomineralization continued. Shewanella, Loktanella, Sphingomonas, and Devosia spp. were also isolated on heterotrophic media, but none produced S(0) alone when reintroduced to Na(2) S gradient tubes. Tubes inoculated with a Marinobacter and Shewanella spp. co-culture did show sulfur biomineralization, suggesting that Marinobacter may be the key sulfide oxidizer in laboratory experiments. Light, florescence and scanning electron microscopy of mineral aggregates produced in Marinobacter experiments revealed abundant cells, with filaments and sheaths variably mineralized with extracellular submicron sulfur grains; similar biomineralization was not observed in abiotic controls. Detailed characterization of mineral products associated with low temperature microbial sulfur-cycling may provide biosignatures relevant to future exploration of Europa and Mars.