Hinako Takamiya (Department of Earth and Planetary Science, 1st Year Doctoral Student)

Mariko Kozuka (Project Researcher, Department of Earth and Planetary Science)

Yohei Suzuki (Associate Professor, Department of Earth and Planetary Science)

Key points of the presentation

• Deep-sea hydrothermal vents have been actively searched for primitive life forms as a promising candidate site for the birth of life. The interior of the metal sulfide chimney ^{(Note 1)} is considered important as a site that supported the birth of life and the evolution of early life.
• By developing a technique to visualize microorganisms living inside rocks, we discovered that extremely small microorganisms (about 1/10,000 of a millimeter) with remarkably small cells live inside metal sulfide chimneys.
• The results of genetic analysis indicate that microorganisms born in the early stages of life evolution are dominant inside the chimney, which is an important achievement in estimating the ecology of early life.

Summary of Presentation

A research group led by Associate Professor Yohei Suzuki of the Graduate School of Science, The University of Tokyo collected metal sulfide chimneys from deep-sea bottom hydrothermal vents in the southern Mariana Trough (Figure 1A) using the Hyper Dolphin, an unmanned submersible of the Japan Agency for Marine-Earth Science and Technology (Figures 1B-C). Microorganisms inside rocks near the seafloor surface, such as metal sulfide chimneys, have been a problem due to sample contamination caused by microbial invasion from seawater into the rocks during the time between collection on the seafloor and retrieval on board. The research group's previous work has developed an imaging technique to visualize microorganisms inside rocks on a cell-by-cell basis, and has revealed that microorganisms with a cell density of over 10 billion individuals ^{per cm3} can be found in cracks in lava erupted from submarine volcanoes ( related literature). The same technique was applied to metal sulfide chimneys, but because of the small pores in the rock (Figure 1D) and the small size of the microbial cells, the cells could not be observed. Therefore, using state-of-the-art electron microscopic analysis techniques, we observed the interior of the rock and discovered microscopic microorganisms coated with copper oxide nanoparticles (Figure 1E). Normally, when tiny microorganisms living in seawater invade the interior of rocks, their body surfaces are not covered with particles because copper oxide nanoparticles have the property of disappearing quickly upon contact with seawater. Therefore, the microscopic microorganisms discovered in this study did not invade the rock due to contamination (Fig. 1F-G), but were thought to have inhabited the interior of the chimney. Another important key to this discovery is that the cells of the microorganisms are covered with copper oxide nanoparticles (Fig. 1H), making observation possible. Life with smaller cell size is more primitive, and genetic analysis revealed that the microorganisms that dominate in the interior of rocks are a group that emerged in the early stages of life evolution. Therefore, the results of this study are expected to advance research on the birth and early evolution of life.

Figure 1: Metal sulfide chimney sample collection point (A), chimney collection (B), photograph of chimney cross section (C), view of the gap between mineral particles inside the chimney where tiny microbes were found (D), photograph of tiny microbes coated on copper oxide nanoparticles (E), cross section of a chimney (F), enlarged view of F . (G) showing that the rock is not contaminated by microorganisms entering from seawater (H), and an illustration (G) outlining the interior of the chimney.

Presentation

Deep-sea floor hydrothermal vents, along with terrestrial hot springs, have been actively studied as promising candidates as sites for the birth of life. At deep-sea floor hydrothermal vents, hydrothermal fluids rich in metals and hydrogen sulfide react with cold seawater to form metal-sulfide structures. The black hydrothermal fluid spewing from the tip of the structure is called a chimney because it resembles a chimney emitting black smoke (Fig. 1F). The interior of metal sulfide chimneys is of particular importance as a birthplace of life because of the abundance of material substances for biomolecular synthesis and metal catalysts necessary for their assembly.

Ninety-nine percent of microorganisms in nature cannot be cultured, and by not being able to culture them, it has been unclear how they live in harsh and extreme environments, since it is not known what food they need for their metabolism or how they breathe. Recent advances in genome analysis technology have decoded the genomes, the blueprints of life, of many extreme environment microorganisms. It is now possible to extract information on microbial metabolism from genomes, as well as reliable information on the evolution of life. In the latest phylogenetic tree based on genome information, it is clear that an archaeon called DPANN ^{(Note 2} ) diverged at a root close to the final universal common ancestor (Figure 2) and emerged at an early stage of life evolution. The gene sequence encoded in the DPANN genome is very different from that of previously known organisms, and although it is unclear what metabolism the organism thrives on despite the decoded genome, it is clear that it is an extremely small microorganism with a remarkably small cell size. Genome analysis of microorganisms living inside metal sulfide chimneys has also progressed, and DPANN has been detected in deep-sea floor hydrothermal vent environments, but it was unclear what type of environment, in a complex mixing field of hydrothermal and seawater, would be suitable for DPANN to live.

Therefore, a submarine survey was conducted to elucidate the actual conditions by collecting metal sulfide chimneys from a deep-sea floor hydrothermal vent at a depth of 2787 m in the southern Mariana Trough, approximately 140 km offshore from Guam (Figure 1A). The research team developed an original method to visualize microorganisms living inside rocks, and succeeded in detecting microbial cells in cracks of lava erupted at a deep-sea floor hydrothermal vent. In this study, we applied the same technique that successfully detected microbial cells in lava fractures ( see related literature) to a metal sulfide chimney sample (Fig. 1B). Specifically, cell-level imaging of major biological elements such as carbon, nitrogen, sulfur, and phosphorus was performed using a high-spatial resolution secondary ion mass spectrometer ^{(Note 4)} on a 3 μm thick thin section prepared ^using focused ion beam processing technology ^{(Note 3)}. However, because the cells were too small to visualize, the thin sections were thinned down to 150 nm in thickness and observed using an electron microscope. As a result, we found that microorganisms as small as 100 nm in size (Fig. 1E) were densely packed in the narrow gaps between the mineral particles (Fig. 1D) that make up the chimney. To visualize small objects such as viruses, it is common to dye them with uranium, but in this study, it was found that the cells were covered with nanoparticles of copper oxide, which was the reason why they could be observed without dyeing. Furthermore, DNA analysis of the microorganisms in the chimney sample revealed that the dominant microorganisms inside the rock were primitive archaea classified as DPANN (Figure 2).

Figure 2: Phylogenetic tree of life evolution based on genomic information.

Microorganisms are known to inhabit the interior of chimneys where hydrothermal eruptions are actively taking place, as they are supplied with nutrients necessary for the survival of microorganisms. However, the interior of chimneys left on the seafloor after hydrothermal eruptions have ceased is nutrient-poor, and it is not known whether it can support life. The discovery of the ecosystem by the research team has revolutionized the conventional concept of deep-sea floor hydrothermal vent ecosystems, which are based on primary production from energy supplied by hydrothermal fluids. These results indicate that the interior of metal sulfide chimneys can be a habitat for photosynthesis-independent microorganisms even without hydrothermal venting, and that ecosystems can be formed even on the Earth before the birth of photosynthetic organisms. The team plans to analyze genome and gene expression, and to study the metabolism of primitive archaea and the pathways of organic matter synthesis inside rocks in the future.

Published Journals

Journal name	Frontiers in Microbiology
Title of paper	Copper-nanocoated microbial cells in grain boundaries inside an extinct vent chimney
Author(s)	Hinako Takamiya, Mariko Kouduka, Hitoshi Furutani, Hiroki Mukai, Kaoru Nakagawa, Takushi Yamamoto, Shingo Kato, Yu Kodama, Naotaka Tomioka, Motoo Ito , and Yohey Suzuki
DOI Number	10.3389/fmicb.2022.864205
Abstract URL	https://www.frontiersin.org/articles/10.3389/fmicb.2022.864205

Terminology

Note 1 Metal sulfide chimney

Chimney is an English word corresponding to a chimney, a structure formed when black hydrothermal water (black smoker), rich in metals and hydrogen sulfide, precipitates as a solid in concentric circles at the site where it erupts from the deep-sea floor. ↑up

Note 2 DPANN

A phylogenetic group of archaea that brings together multiple phyla; first proposed in 2013; very few species have been successfully cultured due to small cell and genome size. Because the genome lacks genes necessary for the maintenance of life activity, there are many unknowns about its ecology. ↑up

Note 3 Focused ion beam processing technology

A device that uses ion beams of gallium and other ions to create thin sections from solid samples in any desired shape. ↑up

Note 4 High spatial resolution secondary ion mass spectrometer

A device that can image the distribution of carbon, nitrogen, phosphorus, and sulfur in microbial cells by scanning a cesium ion beam in two dimensions with a diameter of 0.05 μm, which is smaller than that of microbial cells. ↑up