Craig L. Moyer

Neutrophilic Fe-oxidizing bacteria are abundant at the Loihi Seamount hydrothermal vents and play a major role in Fe oxide deposition.

ABSTRACT A number of hydrothermal vent sites exist on the summit of the Loihi Seamount, a shield volcano that is part of the Hawaiian archipelago. The vents are 1,100 to 1,325 m below the surface and range in temperature from slightly above ambient (10°C) to high temperature (167°C). The vent fluid is characterized by high concentrations of CO2 (up to 17 mM) and Fe(II) (up to 268 μM), but there is a general paucity of H2S. Most of the vents are surrounded by microbial mats that have a gelatinous texture and are heavily encrusted with rust-colored Fe oxides. Visually, the Fe oxides appeared homogeneous. However, light microscopy revealed that the oxides had different morphologies, which fell into three classes: (i) sheaths, (ii) twisted or irregular filaments, and (iii) amorphous oxides. A morphological analysis of eight different samples indicated that the amorphous oxides were overall the most abundant; however, five sites had >50% sheaths and filamentous oxides. These latter morphologies are most likely the direct result of microbial deposition. Direct cell counts revealed that all of the oxides had abundant microbial populations associated with them, from 6.9 × 107 to 5.3 × 108 cells per ml of mat material. At most sites, end point dilution series for lithotrophic Fe oxidizers were successful out to dilutions of 10−6 and 10−7. A pure culture was obtained from a 10−7 dilution tube; this strain, JV-1, was an obligate, microaerophilic Fe oxidizer that grew at 25 to 30°C. A non-cultivation-based molecular approach with terminal-restriction fragment length polymorphism also indicated the common presence of Fe-oxidizing bacteria at Loihi. Together, these results indicate that Fe-oxidizing bacteria are common at the Loihi Seamount and probably play a major role in Fe oxidation. A review of the literature suggests that microbially mediated Fe oxidation at hydrothermal vents may be important globally.

Abundance and diversity of microbial life in ocean crust

Oceanic lithosphere exposed at the sea floor undergoes seawater–rock alteration reactions involving the oxidation and hydration of glassy basalt. Basalt alteration reactions are theoretically capable of supplying sufficient energy for chemolithoautotrophic growth. Such reactions have been shown to generate microbial biomass in the laboratory, but field-based support for the existence of microbes that are supported by basalt alteration is lacking. Here, using quantitative polymerase chain reaction, in situ hybridization and microscopy, we demonstrate that prokaryotic cell abundances on seafloor-exposed basalts are 3–4 orders of magnitude greater than in overlying deep sea water. Phylogenetic analyses of basaltic lavas from the East Pacific Rise (9° N) and around Hawaii reveal that the basalt-hosted biosphere harbours high bacterial community richness and that community membership is shared between these sites. We hypothesize that alteration reactions fuel chemolithoautotrophic microorganisms, which constitute a trophic base of the basalt habitat, with important implications for deep-sea carbon cycling and chemical exchange between basalt and sea water.

A Novel Lineage of Proteobacteria Involved in Formation of Marine Fe-Oxidizing Microbial Mat Communities

Background For decades it has been recognized that neutrophilic Fe-oxidizing bacteria (FeOB) are associated with hydrothermal venting of Fe(II)-rich fluids associated with seamounts in the worlds oceans. The evidence was based almost entirely on the mineralogical remains of the microbes, which themselves had neither been brought into culture or been assigned to a specific phylogenetic clade. We have used both cultivation and cultivation-independent techniques to study Fe-rich microbial mats associated with hydrothermal venting at Loihi Seamount, a submarine volcano. Methodology/Principle Findings Using gradient enrichment techniques, two iron-oxidizing bacteria, strains PV-1 and JV-1, were isolated. Chemolithotrophic growth was observed under microaerobic conditions; Fe(II) and Fe0 were the only energy sources that supported growth. Both strains produced filamentous stalk-like structures composed of multiple nanometer sized fibrils of Fe-oxyhydroxide. These were consistent with mineralogical structures found in the iron mats. Phylogenetic analysis of the small subunit (SSU) rRNA gene demonstrated that strains PV-1 and JV-1 were identical and formed a monophyletic group deeply rooted within the Proteobacteria. The most similar sequence (85.3% similarity) from a cultivated isolate came from Methylophaga marina. Phylogenetic analysis of the RecA and GyrB protein sequences confirmed that these strains are distantly related to other members of the Proteobacteria. A cultivation-independent analysis of the SSU rRNA gene by terminal-restriction fragment (T-RF) profiling showed that this phylotype was most common in a variety of microbial mats collected at different times and locations at Loihi. Conclusions On the basis of phylogenetic and physiological data, it is proposed that isolate PV-1T ( = ATCC BAA-1019: JCM 14766) represents the type strain of a novel species in a new genus, Mariprofundus ferrooxydans gen. nov., sp. nov. Furthermore, the strain is the first cultured representative of a new candidatus class of the Proteobacteria that is widely distributed in deep-sea environments, Candidatus ζ (zeta)-Proteobacteria cl. nov.

Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism

ABSTRACT An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected ≤70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).

Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000 m off Hawaii

A novel hydrothermal field has been discovered at the base of Lōihi Seamount, Hawaii, at 5000 mbsl. Geochemical analyses demonstrate that ‘FeMO Deep’, while only 0.2 °C above ambient seawater temperature, derives from a distal, ultra-diffuse hydrothermal source. FeMO Deep is expressed as regional seafloor seepage of gelatinous iron- and silica-rich deposits, pooling between and over basalt pillows, in places over a meter thick. The system is capped by mm to cm thick hydrothermally derived iron-oxyhydroxide- and manganese-oxide-layered crusts. We use molecular analyses (16S rDNA-based) of extant communities combined with fluorescent in situ hybridizations to demonstrate that FeMO Deep deposits contain living iron-oxidizing Zetaproteobacteria related to the recently isolated strain Mariprofundus ferroxydans. Bioenergetic calculations, based on in-situ electrochemical measurements and cell counts, indicate that reactions between iron and oxygen are important in supporting chemosynthesis in the mats, which we infer forms a trophic base of the mat ecosystem. We suggest that the biogenic FeMO Deep hydrothermal deposit represents a modern analog for one class of geological iron deposits known as ‘umbers’ (for example, Troodos ophilolites, Cyprus) because of striking similarities in size, setting and internal structures.

Biodiversity and emerging biogeography of the neutrophilic iron-oxidizing Zetaproteobacteria

ABSTRACT Members of the neutrophilic iron-oxidizing candidate class Zetaproteobacteria have predominantly been found at sites of microbially mediated iron oxidation in marine environments around the Pacific Ocean. Eighty-four full-length (>1,400-bp) and 48 partial-length Zetaproteobacteria small-subunit (SSU) rRNA gene sequences from five novel clone libraries, one novel Zetaproteobacteria isolate, and the GenBank database were analyzed to assess the biodiversity of this burgeoning class of the Proteobacteria and to investigate its biogeography between three major sampling regions in the Pacific Ocean: Loihi Seamount, the Southern Mariana Trough, and the Tonga Arc. Sequences were grouped into operational taxonomic units (OTUs) on the basis of a 97% minimum similarity. Of the 28 OTUs detected, 13 were found to be endemic to one of the three main sampling regions and 2 were ubiquitous throughout the Pacific Ocean. Additionally, two deeply rooted OTUs that potentially dominate communities of iron oxidizers originating in the deep subsurface were identified. Spatial autocorrelation analysis and analysis of molecular variance (AMOVA) showed that geographic distance played a significant role in the distribution of Zetaproteobacteria biodiversity, whereas environmental parameters, such as temperature, pH, or total Fe concentration, did not have a significant effect. These results, detected using the coarse resolution of the SSU rRNA gene, indicate that the Zetaproteobacteria have a strong biogeographic signal.

Continuous sampling of hydrothermal fluids from Loihi Seamount after the 1996 event

For at least 9 years prior to July 1996, hydrothermal fluids flowed from Peles Vents on Loihi Seamount, Hawaii. In July–August 1996 a tectonic-volcanic event occurred that destroyed Peles Vents, creating a pit crater (Peles Pit) and several sites with hydrothermal venting. In October 1996 we deployed two new continuous water samplers (OsmoSamplers) at two of these hydrothermal sites and collected fluids using traditional sampling techniques to monitor the evolution of crustal and hydrothermal conditions after the event. The samplers were recovered in September 1997, and additional discrete vent fluid samples were collected. The OsmoSampler located along the south rift at Naha Vents captured a change in composition from a low-chlorinity, high-K fluid (relative to bottom seawater) to a high-chlorinity, low-K fluid. These changes are consistent with the fluid cooling during ascent and being derived from several different sources, which include high- (>330°C) and low- ( 330°C) into which magmatic volatiles were added. During the deployment, thermal and fluid fluxes decreased. At Naha the transport of heat and chemicals was decoupled. The chemical and thermal evolution of hydrothermal fluids after the event on Loihi is consistent with previous models based on events that have occurred along mid-ocean ridges. The event at Loihi clearly had an effect on the local hydrography; however, the integrated effect of chemical fluxes to global budgets from similar events is uncertain. Chemical fluxes from similar events may have a global impact, if ratios of chemical (e.g., CO2, Fe/Mn, Mg, sulfate, and K) to thermal anomalies greatly exceed, or are in the opposite direction to, fluxes from mid-ocean ridge hydrothermal systems.

Hidden in plain sight: discovery of sheath-forming, iron-oxidizing Zetaproteobacteria at Loihi Seamount, Hawaii, USA

Lithotrophic iron-oxidizing bacteria (FeOB) form microbial mats at focused flow or diffuse flow vents in deep-sea hydrothermal systems where Fe(II) is a dominant electron donor. These mats composed of biogenically formed Fe(III)-oxyhydroxides include twisted stalks and tubular sheaths, with sheaths typically composing a minor component of bulk mats. The micron diameter Fe(III)-oxyhydroxide-containing tubular sheaths bear a strong resemblance to sheaths formed by the freshwater FeOB, Leptothrix ochracea. We discovered that veil-like surface layers present in iron-mats at the Loihi Seamount were dominated by sheaths (40-60% of total morphotypes present) compared with deeper (> 1 cm) mat samples (0-16% sheath). By light microscopy, these sheaths appeared nearly identical to those of L. ochracea. Clone libraries of the SSU rRNA gene from this top layer were dominated by Zetaproteobacteria, and lacked phylotypes related to L. ochracea. In mats with similar morphologies, terminal-restriction fragment length polymorphism (T-RFLP) data along with quantitative PCR (Q-PCR) analyses using a Zetaproteobacteria-specific primer confirmed the presence and abundance of Zetaproteobacteria. A Zetaproteobacteria fluorescence in situ hybridization (FISH) probe hybridized to ensheathed cells (4% of total cells), while a L. ochracea-specific probe and a Betaproteobacteria probe did not. Together, these results constitute the discovery of a novel group of marine sheath-forming FeOB bearing a striking morphological similarity to L. ochracea, but belonging to an entirely different class of Proteobacteria.

Zeta-Proteobacteria Dominate the Colonization and Formation of Microbial Mats in Low-Temperature Hydrothermal Vents at Loihi Seamount, Hawaii

In situ colonization experiments were performed to study the pioneer populations of bacteria at Loihi Seamount, Hawaii. Over a ten-year sampling period, 41 microbial growth chambers (MGCs) were deployed and recovered in Peles Pit and the surrounding area after short-term (4–10 days) and long-term (∼1–6 years) incubations in the flow of hydrothermal effluent. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the small subunit rRNA gene (SSU rDNA) revealed that the short-term MGC communities exhibited a low number of represented populations when compared to the long-term MGC communities and naturally occurring microbial mats. Cluster analysis of T-RFLP fingerprints showed the short-term MGC communities all had similar richness but were separated into three distinct groups with different arrays of colonizing populations. Clone library analysis showed that cooler vents (T ave = 40°C) were primarily colonized by Mariprofundus ferrooxydans, a neutrophilic Fe-oxidizing ζ −Proteobacteria while warmer vents (T ave = 71°C) were colonized by Sulfurimonas spp. and other sulfur-cycling members of the ϵ −Proteobacteria. Vents with an intermediate temperature (T ave = 51°C) were colonized by representatives of both ζ -Proteobacteria and ϵ -Proteobacteria. Long-term MGC communities did not cluster with any of the short-term communities and exhibited higher richness, indicating a greater number of bacterial populations were able to colonize and grow in the long-term growth chambers.

Bacterial Variability within an Iron-Silica-Manganese-rich Hydrothermal Mound Located Off-axis at the Cleft Segment, Juan de Fuca Ridge

Terminal-restriction fragment length polymorphisms (T-RFLPs) and traditional clone library analysis were used to identify and assess the spatial variability of microbial communities within an active diffusely venting hydrothermal mound found 4 km off-axis at the Cleft Segment, Juan de Fuca Ridge. T-RFLP fingerprints were generated from three subsamples taken from different depths of a sediment core. The top and center subsamples were dominated by phylotypes clustering with the proposed ζ -Proteobacteria. The bottom of the core was dominated by a phylotype clustering in the Nitrospina group within the δ -Proteobacteria. Cluster analysis of T-RFLP fingerprints from the center of the core shows a similar community structure with those from iron-dominated microbial mat communities collected from NW Eifuku Seamount in the Mariana Island Arc and from Loihi Seamount, Hawaii. Our study demonstrates that off-axis diffuse hydrothermal activity generates microbial communities that can potentially affect their habitat through the differential precipitation of Fe-, Si-, and Mn-rich deposits.