Seafloor Incubation Experiment with Deep-Sea Hydrothermal Vent Fluid Reveals Effect of Pressure and Lag Time on Autotrophic Microbial Communities

Abstract

Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed a remotely operated vehicle (ROV)-powered incubator instrument to carry out and compare results from in situ and shipboard RNA stable isotope probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labeled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes, suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations, suggesting that depressurization induced thermal stress in the metabolically active microbes in these incubations. Together, the results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under nearly native conditions in the ocean.IMPORTANCE Diverse microbial communities drive biogeochemical cycles in Earth's ocean, yet studying these organisms and processes is often limited by technological capabilities, especially in the deep ocean. In this study, we used a novel marine microbial incubator instrument capable of in situ experimentation to investigate microbial primary producers at deep-sea hydrothermal vents. We carried out identical stable isotope probing experiments coupled to RNA sequencing both on the seafloor and on the ship to examine thermophilic, microbial autotrophs in venting fluids from an active submarine volcano. Our results indicate that microbial communities were significantly impacted by the effects of depressurization and sample processing delays, with shipboard microbial communities being more stressed than seafloor incubations. Differences in metabolism were also apparent and are likely linked to the chemistry of the fluid at the beginning of the experiment. Microbial experimentation in the natural habitat provides new insights into understanding microbial activities in the ocean.

Keywords: RNA-SIP; autotrophy; deep sea; hydrothermal vent; instrumentation; metagenomics; metatranscriptomics.

FIG 1

Incubator setup for the in situ RNA stable isotope probing (RNA-SIP) experiments. Each of the four incubation chambers was heated to a chosen set point temperature. Fluid was pulled into the insulated incubation chamber from the manifold of the hydrothermal fluid and particle sampler (HFPS) through a custom titanium shutoff valve, pulling hydrogen gas and buffering acid into the chamber as it filled. (A) After the incubation period, the fluid was pulled from the incubation chamber through a 0.22-μm filter with the passive addition of an RNA preservative. (B) A cutaway view of the incubation chamber shows the incubation bag over the heating element, with the RTD used to monitor the chamber temperature near the end of the bag. (C and D) The fully assembled incubator module (as deployed in 2015) (C) slides into the HFPS sample rack (D). Fluid transfer is accomplished with the HFPS sample pump and selection valve. PFA, perfluoroalkoxy.

FIG 2

16S rRNA abundance in density gradient fractions of shipboard (A) and incubator (B) RNA-SIP experiments at 12 h. The buoyant density (grams per milliliter) of each fraction is depicted on the x axis, and the amount of 16S rRNA determined by RT-qPCR is on the y axis. The amount of 16S rRNA is displayed as the ratio of the maximum quantity in order to compare results between RNA-SIP experiments.

FIG 3

Taxonomic classification of 16S rRNA reads (A) and functionally (KO) annotated non-rRNA transcripts (B) from RNA-SIP metatranscriptomes.

FIG 4

Heat map of mean coverage across the RNA-SIP experiments of metagenome-assembled genomes (MAGs) taxonomically identified as thermophilic Epsilonbacteraeota, specifically either the genus Nitratifractor or the family Nautiliaceae, as described previously (7). Fractions from each of the four RNA-SIP experiments have been collapsed, and mean coverage is summed. The scale depicts the range of mean coverages across MAGs. MAGs were clustered based on the similarity of coverage within the RNA-SIP experiments.

FIG 5

(A) Heat map showing the 233 KO annotated genes that were differentially expressed across fractions (adjusted P value of <0.01). (B) Volcano plot of the fold change in abundance versus the adjusted P value. Genes that were significantly upregulated (adjusted P value of <0.01) in the incubator versus shipboard fractions are in red, and downregulated genes are in blue.

FIG 6

Normalized abundances of key genes and transcripts for oxygen, nitrogen, methane, hydrogen, and sulfur metabolisms within the 2015 marker 33 metagenome (MetaG) and metatranscriptome (MetaT) and the shipboard and incubator RNA-SIP experiments. Fractions from each of the four RNA-SIP experiments have been collapsed to reflect the normalized abundance of each gene in the entire experiment. Normalized abundances of metatranscriptomes were transformed to the same scale as the marker 33 metagenome. Black stars indicate a significant difference in transcript abundances (adjusted P value of <0.01) between shipboard and incubator RNA-SIP experiments. See Table S5 in the supplemental material for the specific subunits identified as significant.

FIG 7

Normalized abundances of genes and transcripts annotated for cell stress, including genes for protein chaperones, heat shock proteins, and proteases, within the 2015 marker 33 metagenome and metatranscriptome and the shipboard and incubator RNA-SIP experiments. Fractions from each of the four RNA-SIP experiments have been collapsed to reflect the normalized abundance of each gene in the entire experiment. Normalized abundances of metatranscriptomes were transformed to the same scale as the marker 33 metagenome. Black stars indicate a significant difference in transcript abundances (adjusted P value of <0.01) between shipboard and incubator RNA-SIP experiments. See Table S5 in the supplemental material for the specific subunits identified as significant.