Microbial ecology of a shallow alkaline hydrothermal vent: Strýtan Hydrothermal Field, Eyjafördur, northern Iceland.

Strýtan Hydrothermal Field (SHF) is a submarine system located in Eyjafördur in northern Iceland composed of two main vents: Big Strýtan and Arnarnesstrýtan. The vents are shallow, ranging from 16 to 70 m water depth, and vent high pH (up to 10.2), moderate temperature (T max ∼70°C), anoxic, fresh fluids elevated in dissolved silica, with slightly elevated concentrations of hydrogen and methane. In contrast to other alkaline hydrothermal vents, SHF is unique because it is hosted in basalt and therefore the high pH is not created by serpentinization. While previous studies have assessed the geology and geochemistry of this site, the microbial diversity of SHF has not been explored in detail. Here we present a microbial diversity survey of the actively venting fluids and chimneys from Big Strýtan and Arnarnesstrýtan, using 16S rRNA gene amplicon sequencing. Community members from the vent fluids are mostly aerobic heterotrophic bacteria; however, within the chimneys oxic, low oxygen, and anoxic habitats could be distinguished, where taxa putatively capable of acetogenesis, sulfur-cycling, and hydrogen metabolism were observed. Very few archaea were observed in the samples. The inhabitants of SHF are more similar to terrestrial hot spring samples than other marine sites. It has been hypothesized that life on Earth (and elsewhere in the solar system) could have originated in an alkaline hydrothermal system, however all other studied alkaline submarine hydrothermal systems to date are fueled by serpentinization. SHF adds to our understandings of hydrothermal vents in relationship to microbial diversity, evolution, and possibly the origin of life.

Metabolic Strategies Shared by Basement Residents of the Lost City Hydrothermal Field.

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as molecular hydrogen (H2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirota) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal environments where H2 and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth's mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H2) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

Microbial Residents of the Atlantis Massif's Shallow Serpentinite Subsurface.

The Atlantis Massif rises 4,000 m above the seafloor near the Mid-Atlantic Ridge and consists of rocks uplifted from Earth's lower crust and upper mantle. Exposure of the mantle rocks to seawater leads to their alteration into serpentinites. These aqueous geochemical reactions, collectively known as the process of serpentinization, are exothermic and are associated with the release of hydrogen gas (H2), methane (CH4), and small organic molecules. The biological consequences of this flux of energy and organic compounds from the Atlantis Massif were explored by International Ocean Discovery Program (IODP) Expedition 357, which used seabed drills to collect continuous sequences of shallow (<16 m below seafloor) marine serpentinites and mafic assemblages. Here, we report the census of microbial diversity in samples of the drill cores, as measured by environmental 16S rRNA gene amplicon sequencing. The problem of contamination of subsurface samples was a primary concern during all stages of this project, starting from the initial study design, continuing to the collection of samples from the seafloor, handling the samples shipboard and in the lab, preparing the samples for DNA extraction, and analyzing the DNA sequence data. To distinguish endemic microbial taxa of serpentinite subsurface rocks from seawater residents and other potential contaminants, the distributions of individual 16S rRNA gene sequences among all samples were evaluated, taking into consideration both presence/absence and relative abundances. Our results highlight a few candidate residents of the shallow serpentinite subsurface, including uncultured representatives of the Thermoplasmata, Acidobacteria, Acidimicrobia, and ChloroflexiIMPORTANCE The International Ocean Discovery Program Expedition 357-"Serpentinization and Life"-utilized seabed drills to collect rocks from the oceanic crust. The recovered rock cores represent the shallow serpentinite subsurface of the Atlantis Massif, where reactions between uplifted mantle rocks and water, collectively known as serpentinization, produce environmental conditions that can stimulate biological activity and are thought to be analogous to environments that were prevalent on the early Earth and perhaps other planets. The methodology and results of this project have implications for life detection experiments, including sample return missions, and provide a window into the diversity of microbial communities inhabiting subseafloor serpentinites.

Identification and Removal of Contaminant Sequences From Ribosomal Gene Databases: Lessons From the Census of Deep Life.

Earth's subsurface environment is one of the largest, yet least studied, biomes on Earth, and many questions remain regarding what microorganisms are indigenous to the subsurface. Through the activity of the Census of Deep Life (CoDL) and the Deep Carbon Observatory, an open access 16S ribosomal RNA gene sequence database from diverse subsurface environments has been compiled. However, due to low quantities of biomass in the deep subsurface, the potential for incorporation of contaminants from reagents used during sample collection, processing, and/or sequencing is high. Thus, to understand the ecology of subsurface microorganisms (i.e., the distribution, richness, or survival), it is necessary to minimize, identify, and remove contaminant sequences that will skew the relative abundances of all taxa in the sample. In this meta-analysis, we identify putative contaminants associated with the CoDL dataset, recommend best practices for removing contaminants from samples, and propose a series of best practices for subsurface microbiology sampling. The most abundant putative contaminant genera observed, independent of evenness across samples, were Propionibacterium, Aquabacterium, Ralstonia, and Acinetobacter. While the top five most frequently observed genera were Pseudomonas, Propionibacterium, Acinetobacter, Ralstonia, and Sphingomonas. The majority of the most frequently observed genera (high evenness) were associated with reagent or potential human contamination. Additionally, in DNA extraction blanks, we observed potential archaeal contaminants, including methanogens, which have not been discussed in previous contamination studies. Such contaminants would directly affect the interpretation of subsurface molecular studies, as methanogenesis is an important subsurface biogeochemical process. Utilizing previously identified contaminant genera, we found that ∼27% of the total dataset were identified as contaminant sequences that likely originate from DNA extraction and DNA cleanup methods. Thus, controls must be taken at every step of the collection and processing procedure when working with low biomass environments such as, but not limited to, portions of Earth's deep subsurface. Taken together, we stress that the CoDL dataset is an incredible resource for the broader research community interested in subsurface life, and steps to remove contamination derived sequences must be taken prior to using this dataset.

Methane Dynamics in a Tropical Serpentinizing Environment: The Santa Elena Ophiolite, Costa Rica.

Uplifted ultramafic rocks represent an important vector for the transfer of carbon and reducing power from the deep subsurface into the biosphere and potentially support microbial life through serpentinization. This process has a strong influence upon the production of hydrogen and methane, which can be subsequently consumed by microbial communities. The Santa Elena Ophiolite (SEO) on the northwestern Pacific coast of Costa Rica comprises ~250 km2 of ultramafic rocks and mafic associations. The climatic conditions, consisting of strongly contrasting wet and dry seasons, make the SEO a unique hydrogeological setting, where water-rock reactions are enhanced by large storm events (up to 200 mm in a single storm). Previous work on hyperalkaline spring fluids collected within the SEO has identified the presence of microorganisms potentially involved in hydrogen, methane, and methanol oxidation (such as Hydrogenophaga, Methylobacterium, and Methylibium spp., respectively), as well as the presence of methanogenic Archaea (such as Methanobacterium). Similar organisms have also been documented at other serpentinizing sites, however their functions have not been confirmed. SEO's hyperalkaline springs have elevated methane concentrations, ranging from 145 to 900 µM, in comparison to the background concentrations (<0.3 µM). The presence and potential activity of microorganisms involved in methane cycling in serpentinization-influenced fluids from different sites within the SEO were investigated using molecular, geochemical, and modeling approaches. These results were combined to elucidate the bioenergetically favorable methane production and/or oxidation reactions in this tropical serpentinizing environment. The hyperalkaline springs at SEO contain a greater proportion of Archaea and methanogens than has been detected in any terrestrial serpentinizing system. Archaea involved in methanogenesis and anaerobic methane oxidation accounted from 40 to 90% of total archaeal sequences. Genes involved in methanogenic metabolisms were detected from the metagenome of one of the alkaline springs. Methanogenic activities are likely to be facilitated by the movement of nutrients, including dissolved inorganic carbon (DIC), from surface water and their infiltration into serpentinizing groundwater. These data provide new insight into methane cycle in tropical serpentinizing environments.

Serpentinization-Influenced Groundwater Harbors Extremely Low Diversity Microbial Communities Adapted to High pH.

Serpentinization is a widespread geochemical process associated with aqueous alteration of ultramafic rocks that produces abundant reductants (H2 and CH4) for life to exploit, but also potentially challenging conditions, including high pH, limited availability of terminal electron acceptors, and low concentrations of inorganic carbon. As a consequence, past studies of serpentinites have reported low cellular abundances and limited microbial diversity. Establishment of the Coast Range Ophiolite Microbial Observatory (California, U.S.A.) allowed a comparison of microbial communities and physicochemical parameters directly within serpentinization-influenced subsurface aquifers. Samples collected from seven wells were subjected to a range of analyses, including solute and gas chemistry, microbial diversity by 16S rRNA gene sequencing, and metabolic potential by shotgun metagenomics, in an attempt to elucidate what factors drive microbial activities in serpentinite habitats. This study describes the first comprehensive interdisciplinary analysis of microbial communities in hyperalkaline groundwater directly accessed by boreholes into serpentinite rocks. Several environmental factors, including pH, methane, and carbon monoxide, were strongly associated with the predominant subsurface microbial communities. A single operational taxonomic unit (OTU) of Betaproteobacteria and a few OTUs of Clostridia were the almost exclusive inhabitants of fluids exhibiting the most serpentinized character. Metagenomes from these extreme samples contained abundant sequences encoding proteins associated with hydrogen metabolism, carbon monoxide oxidation, carbon fixation, and acetogenesis. Metabolic pathways encoded by Clostridia and Betaproteobacteria, in particular, are likely to play important roles in the ecosystems of serpentinizing groundwater. These data provide a basis for further biogeochemical studies of key processes in serpentinite subsurface environments.

Metagenomic identification of active methanogens and methanotrophs in serpentinite springs of the Voltri Massif, Italy.

The production of hydrogen and methane by geochemical reactions associated with the serpentinization of ultramafic rocks can potentially support subsurface microbial ecosystems independent of the photosynthetic biosphere. Methanogenic and methanotrophic microorganisms are abundant in marine hydrothermal systems heavily influenced by serpentinization, but evidence for methane-cycling archaea and bacteria in continental serpentinite springs has been limited. This report provides metagenomic and experimental evidence for active methanogenesis and methanotrophy by microbial communities in serpentinite springs of the Voltri Massif, Italy. Methanogens belonging to family Methanobacteriaceae and methanotrophic bacteria belonging to family Methylococcaceae were heavily enriched in three ultrabasic springs (pH 12). Metagenomic data also suggest the potential for hydrogen oxidation, hydrogen production, carbon fixation, fermentation, and organic acid metabolism in the ultrabasic springs. The predicted metabolic capabilities are consistent with an active subsurface ecosystem supported by energy and carbon liberated by geochemical reactions within the serpentinite rocks of the Voltri Massif.

Insights into environmental controls on microbial communities in a continental serpentinite aquifer using a microcosm-based approach.

Geochemical reactions associated with serpentinization alter the composition of dissolved organic compounds in circulating fluids and potentially liberate mantle-derived carbon and reducing power to support subsurface microbial communities. Previous studies have identified Betaproteobacteria from the order Burkholderiales and bacteria from the order Clostridiales as key components of the serpentinite-hosted microbiome, however there is limited knowledge of their metabolic capabilities or growth characteristics. In an effort to better characterize microbial communities, their metabolism, and factors limiting their activities, microcosm experiments were designed with fluids collected from several monitoring wells at the Coast Range Ophiolite Microbial Observatory (CROMO) in northern California during expeditions in March and August 2013. The incubations were initiated with a hydrogen atmosphere and a variety of carbon sources (carbon dioxide, methane, acetate, and formate), with and without the addition of nutrients and electron acceptors. Growth was monitored by direct microscopic counts; DNA yield and community composition was assessed at the end of the 3 month incubation. For the most part, results indicate that bacterial growth was favored by the addition of acetate and methane, and that the addition of nutrients and electron acceptors had no significant effect on microbial growth, suggesting no nutrient- or oxidant-limitation. However, the addition of sulfur amendments led to different community compositions. The dominant organisms at the end of the incubations were closely related to Dethiobacter sp. and to the family Comamonadaceae, which are also prominent in culture-independent gene sequencing surveys. These experiments provide one of first insights into the biogeochemical dynamics of the serpentinite subsurface environment and will facilitate experiments to trace microbial activities in serpentinizing ecosystems.

Drivers of epsilonproteobacterial community composition in sulfidic caves and springs.

Epsilonproteobacteria are widely distributed in marine, freshwater, and terrestrial environments, although most well-studied groups are from hydrothermal vents and the human intestinal tract. The environmental variables that control epsilonproteobacterial communities in sulfidic terrestrial environments, however, are poorly understood. Here, the environmental variables that influence epsilonproteobacterial community composition in geographically separated sulfidic caves and springs were determined by coarse and fine-scale approaches: denaturing gradient gel electrophoresis profiling of 23S rRNA PCR amplicons and clone library sequencing of the 16S-ITS-23S rRNA operon. Sequences retrieved from this study were not closely related to cultured representatives, indicating that existing culture collections do not adequately capture the diversity of terrestrial Epsilonproteobacteria. Comparisons of 16S-ITS-23S rRNA operon sequences from four sites revealed that some distant communities (> 8000 km) share closely related populations of Epsilonproteobacteria, while other sites have nearly clonal and phylogenetically distinct populations. Statistical evaluations of sequence data reveal that multiple environmental variables (e.g. temperature, pH, salinity, dissolved oxygen, and bicarbonate concentrations) influence Epsilonproteobacteria community composition. Locations with clonal populations tended to be from higher temperatures and intermediate dissolved oxygen concentrations. rRNA operon sequences outside of the 16S rRNA gene may be critical to recognizing environmental drivers of epsilonproteobacterial community composition.

Temporal study of Helicobacter pylori presence in coastal freshwater, estuary and marine waters.

Helicobacter pylori, a gastric pathogen, is believed to be transmitted via the fecal-oral route as well as the oral-oral route. Its presence and viability in environmental waters is not well characterized. The goals of this study were to test H. pylori presence via molecular methods in freshwater, estuarine and beach sites in Delaware over both short and long time scales and to establish whether fecal indicator bacteria, including total Enterococcus and human-specific Bacteroidetes species, are predictive of the pathogen in these waters. The presence of Helicobacter pylori was initially tested by PCR with newly designed 23S rRNA gene primers against Helicobacter spp. and confirmed by sequencing. Two coastal beach sites were repeatedly positive in 2007. Clone library analysis indicated the persistence of one operational taxonomic unit (OTU) over time at one site. Detection of H. pylori was also determined by PCR assays from DNA and RNA for the 16S rRNA gene, as well as DNA for the ureA and cagA genes. Approximately 21% of the samples were positive for H. pylori 16S rRNA gene and 80% of those were also positive for H. pylori 16S rRNA, indicating that this potential pathogen is not only present in natural waters, but also probably viable. There was no correlation between the occurrence of H. pylori and fecal indicator bacteria, suggesting that standard water quality tests are ineffective in predicting the presence of this pathogen in natural waters. These results demonstrate the widespread presence of potentially viable H. pylori in coastal marine and estuarine waters. Additionally, the repeatedly positive samples indicate either a continual contamination source or persistence of H. pylori in marine waters.

UNRAVELING THE REGULATION OF NITROGEN ASSIMILATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE): DIURNAL VARIATIONS IN TRANSCRIPT LEVELS FOR FIVE GENES INVOLVED IN NITROGEN ASSIMILATION(1).

We examined the diurnal expression of five genes encoding nitrogen-assimilating enzymes in the marine diatom Thalassiosira pseudonana (Hust.) Hasle et Heimdal following a transition from NH4 (+) - to NO3 (-) -supplemented media. The accumulation of nia transcripts (encoding nitrate reductase, NR) following the transition to NO3 (-) -supplemented media was similar to previously reported changes in NR abundance and activity. Nia mRNA levels varied diurnally, and the diurnal oscillations were abolished when cells were transferred to continuous light. Genes encoding chloroplastic (niiA) and cytosolic (nirB) nitrite reductases were identified in the genome of T. pseudonana. NiiA and nirB transcript levels increased within 2 h following the addition of NO3 (-) and varied diurnally. Patterns of diurnal variation in nia, niiA, and glnII (encoding the chloroplast-localized glutamine synthetase) mRNA abundances were similar. NirB and glnN (encoding the cytosolic-localized glutamine synthetase) mRNA levels also oscillated diurnally; however, the oscillation was out of phase with nia, niiA, and glnII. We propose that NO3 (-) is assimilated into organic molecules in both the chloroplast and cytosol of diatoms and that enzymes encoded by nirB and glnN contribute to the ecologically important dark assimilation of NO3 (-) observed in marine diatoms. As with nia, the diurnal variations in niiA, nirB, glnII, and glnN were abolished when cells were transferred to continuous light. Our results demonstrate that transcript accumulation is not circadian controlled, but, rather, changes in metabolic pools triggered by light:dark (L:D) transitions may be important in regulating the cellular mRNA levels encoding these key nitrogen assimilating enzymes.