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 an 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, 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 near-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 delay, with shipboard microbial communities more stressed compared to 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.

Oceanic Crustal Fluid Single Cell Genomics Complements Metagenomic and Metatranscriptomic Surveys With Orders of Magnitude Less Sample Volume

“…A limitation to the RedoxSensor ™ Green cell sorting-based approach is the need for ship-based incubation that may result in shifts in community structure or function if important in situ conditions cannot be replicated (i.e., temperature, pressure, oxygen). Sample transport to the ship and manipulation on board likely causes some changes to the microbial community and their activity (e.g., Fortunato et al, 2021 ; Trembath-Reichert et al, 2021 ). Regardless, for the North Pond oxic crustal fluids, our comparison reveals that single-cell sorting using a redox-active dye and paired metagenomics/metatranscriptomics identify many of the same taxonomic groups and metabolic functions, even when sorted cells were only sequenced at low coverage.…”

Approaches to Unmask Functioning of the Uncultured Microbial Majority From Extreme Habitats on the Seafloor

“…Marine microbial communities hold a central role as drivers of major biogeochemical processes, impacting ecosystem functioning far beyond the oceans ( Falkowski et al, 2008 ). Research into these microbial consortia and the processes they mediate is, however, often constrained by technical capabilities, as is particularly evident in deep-sea research ( Fortunato et al, 2021 ). Thus, sampling hard accessible deep-sea environments is already a technical and logistical challenge, requiring the development of specialized underwater devices ( Liang et al, 2021 ; Paulus, 2021 ).…”

“…Gas-tight sampling devices are used to mitigate this effect and prevent outgassing ( Seewald et al, 2002 ; Butterfield et al, 2004 ; Miyazaki et al, 2017 ; Wu et al, 2018 ; Garel et al, 2019 ; Wang et al, 2020 ). A long and often variable lag time during ascent may change redox reactions, introducing artifacts in subsequent analyses despite the usage of pressure maintaining sampling devices ( Fortunato et al, 2021 ). Once on board, the samples are subjected to atmospheric pressure for ex situ filtration, likely causing cell lyses and release of RNA and DNA molecules ( Edgcomb et al, 2016 ).…”

New Opportunities and Untapped Scientific Potential in the Abyssal Ocean

“…High-pressure samplers that preserve native pressure conditions (e.g., McNichol et al, 2016;Cario et al, 2019;Garel et al, 2019;Peoples et al, 2019) are required to better understand how microbes and other life forms have adapted to high pressure, and how pressure affects physiology, community composition, and ecology. With extensive sample transit times through the water column, the risk of signal degradation and contamination increases, making in situ sensing (Chua et al, 2016) and sample processing and preservation (Fortunato et al, 2021) a high technical priority. The Alvin submersible is well positioned to deploy, manipulate, and collect such instruments, while also offering users the benefit of minimal "psychological distance" and the interpretive immediacy it entails (Trope and Liberman, 2010).…”