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Microbial processes within the subseafloor can be examined during the ephemeral and uncommonly observed phenomena known as snowblower venting. Snowblowers are characterized by the large quantity of white floc that is expelled from the seafloor following mid-ocean ridge eruptions. During these eruptions, rapidly cooling lava entrains seawater and hydrothermal fluids enriched in geochemical reactants, creating a natural bioreactor that supports a subseafloor microbial “bloom.” Previous studies hypothesized that the eruption-associated floc was made by sulfide-oxidizing bacteria; however, the microbes involved were never identified. Here we present the first molecular analysis combined with microscopy of microbial communities in snowblower vents from samples collected shortly after the 2011 eruption at Axial Seamount, an active volcano on the Juan de Fuca Ridge. We obtained fluid samples and white flocculent material from active snowblower vents as well as orange flocculent material found on top of newly formed lava flows. Both flocculent types revealed diverse cell types and particulates when examined by phase contrast and scanning electron microscopy (SEM). Distinct archaeal and bacterial communities were detected in each sample type through Illumina tag sequencing of 16S rRNA genes and through sequencing of the sulfide oxidation gene, soxB. In fluids and white floc, the dominant bacteria were sulfur-oxidizing Epsilonproteobacteria and the dominant archaea were thermophilic Methanococcales. In contrast, the dominant organisms in the orange floc were Gammaproteobacteria and Thaumarchaeota Marine Group I. In all samples, bacteria greatly outnumbered archaea. The presence of anaerobic methanogens and microaerobic Epsilonproteobacteria in snowblower communities provides evidence that these blooms are seeded by subseafloor microbes, rather than from microbes in bottom seawater. These eruptive events thus provide a unique opportunity to observe subseafloor microbial communities.

A hyperthermophilic deep-sea methanogen, Methanocaldococcus strain JH146, was isolated from 26°C hydrothermal fluid at Axial Volcano to model high temperature methanogenesis in the subseafloor. Emphasis was placed on defining growth kinetics, cell yields and growth energy demand (GE) across a range of conditions. The organism uses H2 and CO2 as its sole carbon and energy sources. At various temperatures, pHs, and chlorinities, its growth rates and cell yields co-varied while GE remained uniform at 1.69 × 10(-11) J cell(-1)s(-1) ± 0.68 × 10(-11) J cell(-1)s(-1) (s.d., n = 23). An exception was at superoptimal growth temperatures where GE increased to 7.25 × 10(-11) J cell(-1)s(-1) presumably due to heat shock. GE also increased from 5.1 × 10(-12) J cell(-1)s(-1) to 7.61 × 10(-11) J cell(-1)s(-1) as NH4 (+) concentrations decreased from 9.4 mM to 0.14 mM. JH146 did not fix N2 or assimilate NO3 (-), lacked the N2-fixing (cluster II) nifH gene, and became nitrogen limited below 0.14 mM NH4Cl. Nitrogen availability may impact growth in situ since ammonia concentrations at Axial Volcano are < 18 μM. Our approach contributes to refining bioenergetic and carbon flux models for methanogens and other organisms in hydrothermal vents and other environments.

Microorganisms throughout the dark ocean use reduced sulfur compounds for chemolithoautotrophy. In many deep-sea hydrothermal vents, sulfide oxidation is quantitatively the most important chemical energy source for microbial metabolism both at and beneath the seafloor. In this study, the presence and activity of vent endemic Epsilonproteobacteria was examined in six low-temperature diffuse vents over a range of geochemical gradients from Axial Seamount, a deep-sea volcano in the Northeast Pacific. PCR primers were developed and applied to target the sulfur oxidation soxB gene of Epsilonproteobacteria. soxB genes belonging to the genera Sulfurimonas and Sulfurovum are both present and expressed at most diffuse vent sites, but not in background seawater. Although Sulfurovum-like soxB genes were detected in all fluid samples, the RNA profiles were nearly identical among the vents and suggest that Sulfurimonas-like species are the primary Epsilonproteobacteria responsible for actively oxidizing sulfur via the Sox pathway at each vent. Community patterns of subseafloor Epsilonproteobacteria 16S rRNA genes were best matched to methane concentrations in vent fluids, as well as individual vent locations, indicating that both geochemistry and geographical isolation play a role in structuring subseafloor microbial populations. The data show that in the subseafloor at Axial Seamount, Epsilonproteobacteria are expressing the soxB gene and that microbial patterns in community distribution are linked to both vent location and chemistry.

A hyperthermophilic deep-sea methanogen, Methanocaldococcus strain JH146, was isolated from 26°C hydrothermal fluid at Axial Volcano to model high temperature methanogenesis in the subseafloor. Emphasis was placed on defining growth kinetics, cell yields and growth energy demand (GE) across a range of conditions. The organism uses H2 and CO2 as its sole carbon and energy sources. At various temperatures, pHs, and chlorinities, its growth rates and cell yields co-varied while GE remained uniform at 1.69 × 10(-11) J cell(-1)s(-1) ± 0.68 × 10(-11) J cell(-1)s(-1) (s.d., n = 23). An exception was at superoptimal growth temperatures where GE increased to 7.25 × 10(-11) J cell(-1)s(-1) presumably due to heat shock. GE also increased from 5.1 × 10(-12) J cell(-1)s(-1) to 7.61 × 10(-11) J cell(-1)s(-1) as NH4 (+) concentrations decreased from 9.4 mM to 0.14 mM. JH146 did not fix N2 or assimilate NO3 (-), lacked the N2-fixing (cluster II) nifH gene, and became nitrogen limited below 0.14 mM NH4Cl. Nitrogen availability may impact growth in situ since ammonia concentrations at Axial Volcano are18 μM. Our approach contributes to refining bioenergetic and carbon flux models for methanogens and other organisms in hydrothermal vents and other environments.