Assessing the habitability of the clouds of Venus.

The surface of Venus has long been considered to be unsuitable for life. The temperature here averages about 427°C, which is not only far to hot for liquid water to exist, but which would also cause the near instantaneous inactivation of any enzymes, the denaturing of any proteins, and the thermal decomposition of almost all biological organic molecules. This has not, however, prevented all speculation about the possibility of life existing on Venus, with some scientists suggesting that it might be possible for life to exist within the planet's cloud layer. One proposed theoretical dweller in the clouds of Venus is the spherical hydrogen gasbag isopyenic organism, which would keep its position within the cloud layer by means of a bag-like body containing the lighter-than-air gas hydrogen, although calculations suggest that such an organism would require a minimum diameter of 4 cm to stay aloft on Venus, which seems improbable.

The detection of phosphene gas in the clouds of Venus, which on Earth would  be considered a clear indicator of microbial activity, has led to renewed consideration of the possibility of life in the Venusian clouds, possibly in the form of microbes within fluid droplets, a habitat which would protect them from dehydration, otherwise a problem for small organisms floating in an atmosphere. However, living within floating droplets would present micro-organisms with another problem, due to the limited lifespan of droplets large enough to sustain such organisms, which would tend to settle out of the cloud layer and evaporate. 

In a paper published on the arXiv database at Cornell University on 8 April 2024, Jennifer Abreu of the Department of Physics and Astronomy at Lehman College at City University of New York, Alyxander Anchordoqui of the John F. Kennedy School in Somerville, Massachusetts, Nyamekye Fosu, Michael Kwakye, Danijela Kyriakakis, and Krystal Reynoso, also of the Department of Physics and Astronomy at Lehman College at City University of New York, and Luis Anchordoqui, again of the Department of Physics and Astronomy at Lehman College, and of the Department of Physics at the Graduate Center at City University of New York, and of the Department of Astrophysics at the American Museum of Natural History, present a potential scenario for the survival of microbes within the Venusian cloud layer. 

The cloud layer completely encircles Venus, giving it a planetary albedo of about 0.8 (i.e. causing it to reflect about 80% of the light it receives back into space). The base of the cloud layer is about 47 km above the surface, at which altitude the atmosphere has a temperature of about 100°C. The top of the cloud layer reaches about 74 km above the surface at the equator, decreasing to about 65 km at the poles. This cloud layer can further be divided into three zones, an upper layer, above 56.5 km, a middle layer between 50.5 km and 56. 5 km, and a lower layer less than 50.5 km above the ground. Droplets within the cloud layers can be divided into three types, based upon their size. Type 1 droplets have a diameter of about 0.2 µm, type 2 droplets have a diameter of 1-2 mm, and type 3 droplets have a diameter of about 4 mm. Types 1 and 2 are found in all three cloud layers, whereas type 3 droplets are only found in the middle and lower layers.

Within the cloud layer, the availability of carbon dioxide, sulphuric acid compounds, and ultraviolet light would provide any microbes with sources of food and energy. Optimum conditions for survival would probably be found about 50 km above the surface, where the temperature fluctuates between about 60°C and about 100°C, and the pressure is about one atmosphere.

On Earth, long lived aerosols, acidic clouds, and atmospheric temperatures consistently above the boiling point of water are not present. However, life, in the form of Bacteria, Pollen, and Algae, has been found in the atmosphere as far as 15 km above sealevel, and Bacteria have been found growing in droplets of moisture gathered from super-cooled clouds above the Alps. It has been proposed that microbes have been able to reach such heights via evaporation, storms, eruptions, or meteor impacts, all processes likely to have been found on an early Venus, where the surface is likely to have been more conducive to life. Unlike their Earthly counterparts, the clouds of Venus are not transient, but are a constant, global phenomenon, where individual aerosol particles can be sustained for a long period of time, rather than for a maximum of a few days, potentially providing a stable home for Venusian life.

Abreu et al. suggest that life may be present in the lower haze layers of the Venusian atmosphere as desiccated spores, ready to become activated when updrafts carry them into the habitable layer within the clouds. After reaching the habitable middle and lower cloud layers, the spores would act as condensation nuclei around which water droplets would form, allowing them to germinate and become metabolically active. Metabolically active microbes could grow by division (as with Prokaryotes on Earth) within the droplets, which would themselves grow further by coagulation. Eventually the droplets would grow so large that they would gravitationally settle out of the cloud layer into the hotter layers beneath, causing their liquid content to evaporate, and the micro-organisms to desiccate and re-enter the spore state. The desiccated spores would then be small and light enough to resist further downwards movement, remaining in the haze layer until a new updraft carries them back into the clouds and the process begins again.

Hypothetical life cycle of the Venusian microorganisms. Top panel: Cloud cover on Venus is permanent and continuous, with the middle and lower cloud layers at temperatures that are suitable for life. Bottom panel: Proposed life cycle for Venusian aerial microbial life. Abreu et al. (2024).

All living organisms have to be able to replicate, and in Bacteria, used as the model for Abreu et al.'s proposed Venusian microorganisms, this is achieved by binary fission; i.e. the division of one Bacterium into two. Thus, a population of Bacteria can double in size in a single generation, and the time which it takes for a population of Bacteria to double can be taken as a generation time. For many common Bacteria the generation time is less than an hour, for example in Escherichia coli it is typically about 20 minutes under ideal conditions (which assume an aerobic, nutrient-rich environment at an optimum temperature), which the marine Bacterium Vibrio natriegens can have a generation time as low as 7-10 minutes. 

If a Bacterium has a generation time of 20 minutes, then it would go through three generations in an hour (so that one Bacterium will become sixteen) and 36 in 12 hours (so that one Bacterium will become about 70 000 000 000, assuming a suitable source of nutrients).

Abreu et al. further calculate that a droplet with a diameter of about 0.1 µm would be effectively neutrally buoyant in Venus's cloud layer, and that a droplet would need to reach about 100 µm in diameter before it began to fall consistently at a velocity of 1 m per second, within a cloud layer that can be up to 27 km thick, enabling plenty of time for reproductive fission.

Unlike Earth, Venus lacks an intrinsic magnetic field which prevents cosmic radiation from reaching the atmosphere. This means that molecules in the upper atmosphere are ionized by ultraviolet radiation, forming an ionosphere, with interactions between this ionosphere and the solar winds producing a magnetic field within the outer atmosphere, capable of slowing down particles with energies of up to a few hundred kiloelectron volts and diverted them around the planet. More energetic particles are able to penetrate this weak magnetic field. Particles with energies of greater than one giga electron volt will provoke cascade reactions in the atmosphere, with nuclei scattered by interaction with the initial particle interacting to cause secondary, tertiary, and subsequent generations of particles, losing energy with each reaction. 

The terrestrial biosphere on Earth benefits from about 1033 g/cm² of protection against the harmful effects of cosmic rays, compared to about 200 g/cm² of shielding available to any Venusian organisms at the top of the cloud layer. However, this protection will have risen to about 1000 g/cm² in the middle cloud layer, comparable to conditions on Earth. Furthermore, cosmic rays are generally considered to be less harmful to Prokaryotic organisms than to multicellular organisms (an interaction with a charged particle may well kill an individual Bacterial cell - but this has no effect on any other cell, and the high rates of reproduction mean that lost Bacteria are replaced quickly, whereas a multicellular organism with many cells needs most of them to survive until the whole organism can reproduce).

Based upon these calculations, Abreu et al. calculate that micro-organisms with similar properties to those found on Earth could potentially exist within the clouds of Venus, reproducing exponentially within droplets in the clouds during the lifetime of those droplets.

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Talaromyces sedimenticola: A new species of Ascomycote Fungus from the deepest part of the Mariana Trench.

The Challenger Deep forms the deepest part of the Mariana Trench, reaching 10 971 m below sealevel. Curiously, recent studies have shown that the hadal depths of the oceans (areas more than 6 km deep) have a higher microbial carbon turnover than occurs at abyssal depths, between 4 km and 6 km below the surface. This has been supported by metagenomic studies which have shown significantly more genes coding for carbohydrate-active enzymes and peptidase are being expressed at these depths. This has led microbiologists to take an interest in the organisms living in the sediments of the deepest ocean trenches. 

In a paper published in the journal Antonie van Leeuwenhoek on 28 February 2024, Hongbo Zhou of the School of Minerals Processing and Bioengineering and the Key Laboratory of Biometallurgy at Central South University, Liting Xu and Wenxian Liu, also of the School of Minerals Processing and Bioengineering at Central South University, Kaiwen Ta of the Institute of Deep-Sea Science and Engineering of the Chinese Academy of Sciences, Xincun Wang of the State Key Laboratory of Mycology at the Institute of Microbiology of the Chinese Academy of Sciences, Jianwei Guo of the College of Agronomy and Life Sciences at Kunming University, Wenxi Luo, Zhiyuan Peng, and Qiaoni Huang, again of the School of Minerals Processing and Bioengineering at Central South University, and Yuguang Wang, once again of the School of Minerals Processing and Bioengineering and the Key Laboratory of Biometallurgy at Central South University, describe a new species of Ascomycote Fungi from the Challenger Deep.

The new species is described on the basis of two strains isolated from samples collected by the Research Vessel Tan Suo Yi Hao in September 2019, from a depth of 10 063 m below sealevel. A genetic analysis of these strains suggests that they belong to the same species, and, surprisingly, that that species  is a member of the genus Talaromyces, which mostly comprises terrestrial moulds, forming a sister taxon to a clade which includes Talaromyces trachyspermus, which is a serious commercial pest species, frequently infecting packaged fruit juices, and Talaromyces assiutensis, which is found growing within the leaves of Mangroves around the South China Sea. The new species is named Talaromyces sedimenticola, in reference to the environment where it was found.

Morphological characteristics of Talaromyces sedimenticola, (k), (l) conidiophores, (m) conidia. Zhou et al. (2024).

While morphologically similar, and genetically close, to other members of the genus Talaromyces, Talaromyces sedimenticola shows some remarkable physiological traits, which mark it out as distinctive. It could be grown at temperatures of between 4°C and 50°C, unlike most Talaromyces species, which typically grow between about 28°C and 40°C. It could also survive and grow over a far wider pH range than any other species within the genus, pH 1.5-12, whereas other Talaromyces species could only survive in the pH range 4-8, with the exception of Talaromyces aculeatus, a widespread soil-dwelling form, which can survive over the range pH 1-7. Talaromyces sedimenticola is also remarkably halotollerant, able to flourish on media with 0-14% sodium chloride (weight over volume). More curiously, Talaromyces sedimenticola was unable to metabolise sucrose, tryptone, or monobasic potassium phosphate, all of which can be utilised by other members of the genus, although it was able to utilise other common foodstuffs, such as glucose, maltose, lactose, xylose, soluble starch, glycerol, peptone, ammonium sulphate, potassium phosphate, potassium chloride, and magnesium sulphate. 

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Thainema salinarum: A new species of Cyanobacterium from Thailand.

Cyanobacteria are simple Prokaryotic photosynthesising micro-organisms found in practically all photic environments on Earth. They can be singular and free-living, but many form filamentous chains, which in turn come together to for microbial mats. Cyanobacteria are thought to have been the first photosynthetic organisms to have produced oxygen, starting from about 2.5 billion years ago, and are still important ecosystem formers today, particularly in extreme environments such as salterns (pools which are periodically flooded by marine waters which are then lost to evaporation, creating a high-salinity environment), where they lack predators or competitors. Due to their importance, Cyanobacteria have been studied by biologists for a long time, althouth their taxonomy remains somewhat confused, at least in part because many described species were originally based upon morphological descriptions, which are of, at best, limited use in Prokaryote taxonomy, as well as general shortage of studies of these organisms outside of temperate regions.

In a paper published in the journal PLoS One on 7 January 2022, Somayeh Rasouli-Dogaheh and Jiří Komárek of the Department of Botany at the University of South Bohemia, Thomrat Chatchawan of the Department of Biology at Chiang Mai University, and Tomáš Hauer, also of the Department of Botany at the University of South Bohemia, describe a new species of Cyanobacterium from a manmade solar saltern environment in Petchaburi Province, central Thailand.

The new species is named Thainema salinarum, where 'Thainema' means 'Thai-thread' and 'salinarum' refers to the environment where it was found. The colony appeared as a pale to bright blue-green mat on the salt-saturated soil of the saltern. It was originally thought to be a member of the genus Halomicronema, but genetic analysis based upon the structure of the 16S rRNA sequence showed that it is the sister species to all members curently assigned to the Family Leptolyngbyaceae, including Halomicronema (that is to say, all curent members of the Family Leptolyngbyaceae are more closely related to one-another than to Thainema salinarum, but that Thainema salinarum and all curent members of the Family Leptolyngbyaceae are more closely related to one-another than either is to anything else). To avoid any further confusion given the large number of single species groups within the Cyanobacteria, Thainema salinarum is added to the Family Leptolyngbyaceae, as an outlying species.

 

Light microscope view of Thainema salinarum. The golden granules at cross-walls and sheaths are observed. Scale bar is 10 μm. Rasouli-Dogaheh et al. (2022).

Filaments of Thainema salinarum are 1–3 μm wide, with trichomes (elongate hair-like structures on the the terminal cells) in a sheath with no branching, mostly straight and slightly constricted, or not constricted at the cross-walls. The Bacterial cells are 2–3.5 μm long, and not as wide as their length. The terminal cells typically rounded, without calyptra. 

 

Micrographs of Thainema salinarum obtained with a transmission electron microscope. (A), (B) Cells with parietal thylakoids, granules, and sheath. Scale bar is 0.5 μm. (C) Longitudinal section of filaments showing the presence of parietal thylakoids. Scale bar is 1 μm. (D) Short filament showing a round apical cell. Scale bar is 5 μm. Rasouli-Dogaheh et al. (2022).

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Nitrosophilus alvini: A new species of hydrogen-oxidising chemolithoautotroph Bacterium isolated from a deep-sea hydrothermal vent in the East Pacific Rise.

The phylum 'Campylobacterota' is a phylogenetically and ecophysiologically diverse Bacterial group that consists of two classes, i.e., Desulfurellia (the former order Desulfurellales) and 'Campylobacteria' (the former class Epsilonproteobacteria). While this phylum is widely recognised as a group including pathogenic microorganisms, e.g. Helicobacter pylori and Campylobacter jejuni, and many studies have focused on these pathogens, an ever expanding number of non-pathogenic species have been identified which thrive as mesophiles or thermophiles in a wide range of natural environments (e.g. deep-sea hydrothermal fields, stratified ocean, terrestrial sulfidic caves, and oil fields) where they are recognized as important players in biogeochemical cycles. Cultivation and characterisation of these bacteria has also expanded our knowledge on the evolution and diversification of pathogenic relatives, biogeography, and the potential of biotechnological applications to mitigate global warming.

At deep-sea hydrothermal vents, Bacteria belonging to the phylum 'Campylobacterota' are known as the dominant community members, including sulphide chimney structures where they can comprise up to 85% of the microbial biomass. Taxonomically and metabolically diverse members of chemosynthetic 'Campylobacterota' are responsible for the primary production. Ever since thermophilic 'Campylobacterota' were first cultivated from hydrothermal vents, the number of culturable thermophilic members has increased with the refinement of cultivation conditions. Nevertheless, the described thermophilic species still account for only 14% of the total number of validly published species within 'Campylobacterota', and therefore there is still insufficient information on their genomes and intra-specific diversity. This also leaves the classification of thermophiles unresolved as almost all thermophilic families are composed of only a single genus, which were mostly retrieved from deepsea hydrothermal vents.

The family Nitratiruptoraceae is one of the thermophilic groups within 'Campylobacteria' that is frequently detected in deep-sea hydrothermal environments globally. This family consists so far of one validly described genus and species, Nitratiruptor tergarcus, isolated from the deep-sea hydrothermal chimney structure in the Mid-Okinawa Trough. The recently described species 'Nitratiruptor labii' was also isolated from the same deep-sea hydrothermal region. In addition to the importance of Nitratiruptoraceae species in biogeochemical cycles, their potential for industrial applications has been described. Isolation of novel Nitratiruptoraceae species and elucidation of their physiological and genomic characteristics are both necessary to help understanding the diversity of this group and the evolutionary relationships within 'Campylobacteria'.

16S rRNA gene sequences have been the universal molecular chronometer for microbial taxonomic affiliation for more than three decades, but this tool does not work well in classifying either closely related species or distantly related taxa. The rapid advances in sequencing technology over the past decade have resulted in an increase in the amount of whole genome data and have brought significant opportunities to introduce robust and accurate criteria to improve microbial taxonomy. One advance is the genome-based taxonomy based on the use of a large number of conserved core genes and indices such as in silico DNA-DNA hybridisation, average nucleotide identity, average amino acid identity, which refines phylogenetic analyses using genome sequence data. These classifiers enable the robust classification of novel species or genera, resulting in a more accurate microbial taxonomy. Genome-based methods could also be effective in classifying members within the phylum 'Campylobacterota'. However, the robust and accurate criteria using genome relatedness indices have not yet been fully evaluated for all species within 'Campylobacterota'. In order to further expand the knowledge of their phylogenetic relationships and to propose a more robust classification methodology, establishment of clear classification criteria is needed to be evaluated.

In a paper published in the journal PLoS One on 10 December 2020, Taiki Shiotani, Sayaka Mino, Wakana Sato, Sayo Nishikawa, and Masanori Yonezawa of the Laboratory of Microbiology at the Faculty of Fisheries Sciences at Hokkaido University, Stefan Sievert of the Department of Biology at the Woods Hole Oceanographic Institution, and Tomoo Sawabe, also of the Laboratory of Microbiology at the Faculty of Fisheries Sciences at Hokkaido University, report a new thermophilic Campylobacterium, strain EPR55-1T, and evaluate its taxonomic assignment using a comprehensive approach based on whole genome sequence of the phylum 'Campylobacterota'.

The sample of a sulphide chimney structure was collected from the Bio9 deep-sea hydrothermal vent on the East Pacific Rise (9.83˚ N 104.28˚ W, water depth 2511 m) by HOV Alvin during the AT26-23 scientific cruise aboard the R/V Atlantis in 2014. The interior part of the chimney sample was mixed anaerobically with 25 ml sterilised seawater containing 0.05% (w/v) neutralized sodium sulfide in 100 ml glass bottles (Schott Glaswerke) soon after HOV Alvin was recovered.

Cells of EPR55-1T are Gram-negative rods (1.0 μm long and 0.5 μm in wide). Cells are motile by means of flagella. Spore formation has not been observed. Strain EPR55-1T grew at temperature between 50˚C and 60˚C, with optimum growth at 60˚C. No growth was observed below 40˚C or above 65˚C. Growth occurred between pH 5.4 and 8.6, with optimum growth at pH 6.6. No growth was detected below pH 3.2 or above pH 9.8. Growth was observed sodium chloride concentrations between 2.4 and 3.2% (weight/volume), with optimum growth at 2.4%. No growth was observed at concentrations below 1.6% or above 4.0%. Temperature, pH, and sodium chloride ranges for growth of strain EPR55-1T were similar to those of 'Nitratiruptor labii' strain HRV44T.

 

Electron micrograph of negatively stained cells of strain EPR55-1T. Scale bar represents 1.0 μm. Shiotani et al. (2020).

Strain EPR55-1T was only able to use molecular hydrogen as electron donor. Nitrate (0.1%, weight/volume), nitrous oxide (33%, volume/volume), thiosulphate (0.1%, weight/volume), elemental sulfur (1%, weight/volume) and molecular oxygen (0.1%, volume/volume) were able to serve as the sole electron acceptors. The isolate could not utilise any organic compounds as energy or carbon sources. These results indicated that strain EPR55-1T was a strictly hydrogen-oxidizing thermophilic chemolithoautotroph. The isolate was able to use ammonium as its sole nitrogen source and utilisation of molecular nitrogen was not observed. Strain EPR55-1T utilized thiosulphate, sulfite (0.01 to 0.001%, weight/volume) and elemental sulphur as sulphur sources. None of the chemosynthetic 'Campylobacterota' isolated so far are reported as possessing the ability to utilise sulphite as its sulphur source. Strain EPR55-1T was sensitive to ampicillin, chloramphenicol, kanamycin, streptomycin and rifampicin.

With a nearly full length of 16S rRNA gene sequence of strain EPR55-1T as a query in BLAST search, 96.0%, 94.3%, 93.0%, and 93.0% similarity were estimated with 'Nitratiruptor labii' HRV44T, Nitratiruptor sp. SB155-2, Nitratiruptor tergarcus MI55-1T, and Hydrogenimonas thermophila EP1-55-1T, respectively, indicating that strain EPR55-1T may be a new species of Nitratiruptor or even a member of newly described genus with strain HRV44T. The phylogenetic analysis showed that strain EPR55-1T was closely related to the Nitratiruptor species.

 

Phylogenic tree based on 16S rRNA gene sequences. Phylogenetic tree of the members of thermophilic 'Campylobacterota', inferred by the neighbor-joining algorithm using 1,166 homologous sequence positions. Numbers at branches are bootstrap values (%) based on 1,000 replicates. Shiotani et al. (2020).

Hybrid genome assembly with Unicycler resulted in a single complete circular contig with a length of 1 807 889 base pairs. Of the 1833 genes predicted, 1783 were coding sequences, 41 tRNA genes, and 3 set of rRNA genes. These values were comparable to those of closely rerated Nitratiruptor isolates; 'Nitratiruptor labii' HRV44T (1 990 315 base pairs and 2050 coding sequences without a plasmid) Nitratiruptor tergarcus MI55-1T (1 894 691 base pairs and 1935 coding sequences) and Nitratiruptor sp. SB155-2 (1 877 931 base pairs and 1,857 coding sequences). The G + C content was 37.7%, which is similar to that of Nitratiruptor sp. SB155-2 (39.7%). 

 

Maximum likelihood tree of 160 members within 'Campylobacterota'. Maximum likelihood tree was constructed based on amino acid sequences of 139 SCGs using RAxML. Support of internal nodes was calculated using 100 bootstrap iterations. Bootstrap support of 100% and over 75% are denoted by solid and hollow, respectively. Shiotani et al. (2020).

The in silico DNA-DNA hybridisation values of strain EPR55-1T against 'Nitratiruptor labii' HRV44T, Nitratiruptor tergarcus MI55-1T and Hydrogenimonas thermophila EP1-55-1%T were 18.7%, 18.1% and 17.4%, respectively, well below a threshold of 70% in silico DNA-DNA hybridisation used for the definition of Bacterial species. In addition, average nucleotide identity values of strain EPR55-1T against 'Nitratiruptor labii' HRV44T, Nitratiruptor tergarcus MI55-1T and Hydrogenimonas thermophila EP1-55-1%T were 77.5%, 71.4% and 70.4%, respectively, well below the species threshold (95.0%). These results support the proposal that the isolate is a novel species within the class 'Campylobacteria'. Average amino acid identity values of the novel isolate against 'Nitratiruptor labii' HRV44T, Nitratiruptor tergarcus MI55-1T, Nitratiruptor sp. SB155-2 and Hydrogenimonas thermophila EP1-55-1%T were 69.9%, 64.1%, 63.7% and 59.4%, respectively, which fall within the threshold for genus-level differentiation (60–80%). The genome-wide average nucleotide identity and alignment fractions of strain EPR55-1T against the closely related species were 73.85% and 0.53 to 'Nitratiruptor labii' HRV44T, 71.45% and 0.38 to Nitratiruptor tergarcus MI55-1T, 71.32% and 0.38 to Nitratiruptor sp. SB155-2, respectively. These values below the genus level threshold (genome-wide average nucleotide identity value of 73.98 (mean) and 73,11 (median), alignment fraction value of 0.33 (mean) and median (0.345)) are indicative of genus-level differentiation of strain EPR55-1T with strain HRV44T, though percentage of conserved proteins values of strain EPR55-1T against the all three relative strains were over 70.1%, higher than genus threshold (above 50%).

A Venn-diagram showed the presence of a conserved core set of 1270 gene clusters that are shared by all Nitratiruptor genomes, representing more than a half of the proteins in each strain. In addition, strain EPR55-1T, HRV44T, MI55-1T and SB155-2 possessed 215, 345, 250 and 245 singletons, respectively.

 

Venn diagram of orthologous gene clusters among the genera Nitratiruptor and Nitrosophilus. This Venn diagram represents shared or unique orthologous gene clusters between EPR55-1^T, HRV44^T, Nitratiruptor tergarcus MI55-1^T and Nitratiruptor sp. SB155-2. Shiotani et al. (2020).

Average amino acid identity analysis between 160 genomes, which vary extensively within 'Campylobacterota, illustrates that there are genera, which need to be considered reclassification. Average amino acid identity, genome-wide average nucleotide identity, alignment fractions, and percentage of conserved proteins values between type strains of the family Nautiliaceae, Nautilia profundicola AmHT, Caminibacter mediatlanticus TB-2T, Cetia pacifica TB-6T, and Lebetimonas natsushimae HS1857T, were 71.6–74.2%, 76.1–78.0%, 0.52–0.81, and 76.02–84.0%, respectively, within or higher than the genus demarcation given by previous studies. The phylogenomic analyses based on both single-copy core genes and multilocus sequence analysis genes also showed these strains could be regarded as one clade. Although the genome-based taxonomy indicated these strains could be considered to the species belonging the same genus, the current intergenus 16S rRNA gene sequence identities of these strains were below 94.5% with the exception of Cetia pacifica TB-6T which showed more than 95% identity to all type strains of the genus Caminibacter.

 

Consensus neighbour-joining tree of 'Campylobacterota' based on single-copy core genes.The phylogenomic tree was constructed based on 139 SCG protein sequences retrieved from 160 genomes belonging to 'Campylobacterota'. The emended and newly proposed taxa in the phylum 'Campylobacterota' (Nitrosophilus, Caminibacter, Helicobacter I, II, E, F, G, and Arcobacter) are shown in bold. Shiotani et al. (2020).

In addition to the thermophilic taxa, some differences compared to the current classification were observed for the genus Helicobacter and the family Arcobacteraceae. Helicobacter pametensis and Helicobacter cholecystus, Helicobacter brantae showed low average amino acid identity values against other Helicobacter species (48.5–55.8%). Similarly, Helicobacter equorum and Helicobacter himalayensis, and Helicobacter anseris and Helicobacter mustelae showed average amino acid values below the genus threshold against other Helicobacter species (50.0–59.9% and 49.8–55.9%, respectively). Phylogenomic analyses also showed that these six species formed new three clades (Helicobacter E F, and G). Additionally, Helicobacter pylori, Helicobacter acinonychis, and Helicobacter cetorum, currently belonging to the Helicobacter clade, showed lower average amino acid identity values than the genus threshold against other current Helicobacter clade species (57.0–58.1%). In the both neighbour-joining and maximum likelihood phylogenomic trees on the basis on 139 single-copy core genes, the current Helicobacter clade branched to two clades (Helicobacter I and II). Same branching patterns were also observed in the neighbour-joining tree based on amino acid sequences of multilocus sequence analysis genes. In the genera of the family Arcobacteraceae, average amino acid identity values between species belonging to different genera Aliarcobacter, Poseidonibacter, Malaciobacter, Arcobacter, Halarcobacter were 61.9–78.7%, indicating they could be regarded as different species of the same genus. The large clade consisting of these genera was also identified by phylogenomic analyses based on both single-copy core genes and multilocus sequence analysis genes. However, some species showed the inter-genus 16S rRNA gene sequence identities below 94.5%, indicating differentiation at genus level. Average amino acid identity values among Campylobacter B were relatively lower (57.02–65.86%), possibly due to the low degree of relatedness between Campylobacter B species (less than 94.5% 16S rRNA gene similarities).

 

Consensus neighbour-joining tree of 'Campylobacterota' based on multilocus sequence analysis genes. The phylogenetic tree was constructed based on amino acid sequences of multilocus sequence analysis genes (i.e. atpA, dnaK, glyA, gyrB, metG, pheS and tkt) retrieved from 154 members of 'Campylobacterota'. Arcobacter cloacae F26, Arcobacter ebronensis CECT 8441, Arcobacter mediterraneus F156-34, Campylobacter mucosalis DSM 21682, Helicobacter bizzozeronii CIII-1, Hydrogenmonas sp. MAG80, and Thiovulum sp. ES were excluded because of lack of at least one multilocus sequence analysis gene sequence. Shiotani et al. (2020).

Based on results of phylogenomic analyses, the average amino acid identity values between species belonging to same clades or the different clades were evaluated. The minimum average amino acid value between species belonging to same clades was 59.7% (Helicobacter muridarum ST1T vs Helicobacter saguini MIT 97-6194T), and the maximum average amino acid identity value between species belonging to different clades was 61.9% (Campylobacter geochelonis RC20T vs Campylobacter hominis ATCC BAA-381T) with the exception of Nitratiruptoraceae, Nautiliaceae, and Arcobacteraceae whose 16S rRNA gene sequence similarities were not reflected in the genome relatedness. These values indicated the average amino acid identity threshold for genus demarcation of 'Campylobacterota' was about 60–62%, though there are some exceptions.

Strain EPR55-1T was the first Nitratiruptoraceae species isolated from the East Pacific Rise. The strain shows some physiological differences from other Nitratiruptoraceae isolates, and represents the only Nitratiruptoraceae species which is able to utilise thiosulphate and sulphite as its sole electron acceptor and sulfur source, respectively. The ability to utilise sulphite has also never been reported in any other thermophilic Campylobacterial species. The strain EPR55-1T possessed lophotrichous flagella, unlike the monotrichous and amphitrichous flagella of 'Nitratiruptor labii' and Nitratiruptor tergarcus, respectively. 16S rRNA gene sequence similarities,genome-wide average nucleotide identity and alignment fraction values of the strain against closely related species suggested that strain EPR55-1T designate the strain as a novel genus with strain HRV44T. The strain EPR55-1T therefore represents a novel genus within a new genus of the family Nitratiruptoraceae.

Strain EPR55-1T is therefore described as a new species, Nitrosophilus alvini, where 'Nitrosophilus' derives from 'nitrosus', full of natron, intended to mean nitrate and nitrous oxide, and 'philos', loving, friendly to, and 'alvini' derives from the name of the HOV Alvin which collected the deep-sea hydrothermal samples harbouring this strain). 

Cells are rod-shaped, motile and stain Gram-negative. Anaerobic to microaerobic. Strictly chemolithoautotrophic. Thermophilic, adapted to the salinity of the ocean. On the basis of 16S rRNA gene and single-copy core-gene analyses, the genus Nitrosophilus belongs to the family Nitratiruptoraceae within the class 'Campylobacteria'.

The temperature range for growth is at 50– 60˚C (optimum 60˚C). The pH range for growth is pH 5.4–8.6 (optimum 6.6). Sodium Chloride concentration range for growth is 2.4–3.2% (weight/volume) (optimum 2.4%). Strain EPR55-1T is hydrogen-oxidising, facultatively anaerobic and chemolithoautotrophic with molecular hydrogen as its sole electron donor and with nitrate, nitrous oxide, thiosulfate, molecular oxygen or elemental sulphur as its sole electron acceptors. Ammonium is utilised as its sole nitrogen source. Thiosulphate, sulphite or elemental sulphur are utilised as its sole sulphur source. The complete genome size is 1 807 889 base pairs. The guanine + cytosine content of DNA is 37.7%. The type strain, EPR55-1T (otherwise known as JCM 32893T and KCTC 15925T), was isolated from a deep-sea hydrothermal vent in the East Pacific Rise.

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Microbial communities from deeply burried oceanic crust.

The upper oceanic crust is mainly composed of basaltic lava. It has been continuously created on Earth for about 3.8 billion years. Basaltic lava is erupted and solidified at mid-ocean ridges where high-temperature basalt-seawater reactions provide substantial energy for sustaining chemosynthetic life. On ridge flanks, circulation of crustal fluid is hydrothermally driven within the basaltic lava overburdened with sediments. The portion of basaltic lava beneath sediment cover is referred to as basaltic basement. Previous studies at 3.5- and 8-million-year-old ridge-flank systems demonstrated that these young crustal aquifers, respectively, harbor anaerobic thermophiles and aerobic mesophiles that contribute to hydrogen, carbon, and sulphur cycling. After rock fractures are filled with secondary minerals, intensities of fluid circulation and basalt-seawater reactions sharply decline with increasing crustal age; with most crustal oxidation occurring in the first 10 million years after crust formation.

More than 90% of Earth’s ocean lithosphere is older than 10 million years and long past its early stage of relatively high crustal oxidation rate. Despite its vast areal extent, the nature and extent of life in this old crust is previously unknown, in part because of the technological and analytical challenges of exploring the igneous rock habitat through scientific drilling. Alteration textures suggestive of biological activity have been observed in oceanic crust as old as 3500 million years. However, the role of microbial activities in creating these textures and the age of the crust at the time of texture formation remain unknown.

In a paper published in the journal Communications Biology on 2 April 2020, Yohey Suzuki, Seiya Yamashita, Mariko Kouduka, and Yutaro Ao of the Department of Earth and Planetary Science at the University of Tokyo, Hiroki Mukai, also of the Department of Earth and Planetary Science at the University of Tokyo, and of the Mantle Drilling Promotion Office at the Japan Agency for Marine-Earth Science and Technology, Satoshi Mitsunobu of the Department of Environmental Conservation at Ehime University, Hiroyuki Kagi of the Geochemical Research Center at the University of Tokyo, Steven D’Hondt of the Graduate School of Oceanography at the University of Rhode Island, Fumio Inagaki of the Kochi Institute for Core Sample Research and Mantle Drilling Promotion Office at the Japan Agency for Marine-Earth Science and Technology, and Yuki Morono, Tatsuhiko Hoshino, Naotaka Tomioka, and Motoo Ito, also of the Kochi Institute for Core Sample Research at the Japan Agency for Marine-Earth Science and Technology, investigate the occurrence of microbial communities in subseafloor basaltic lava older than 10 million years, recovered by Integrated Ocean Drilling Program Expedition 329 in the South Pacific Gyre. The presence of microbial cells in the iron-rich smectite on old subseafloor basaltic rock was revealed by nanoscale solid characterizations. Analysis of their lipid profiles and DNA sequences reveals the dominance of heterotrophic Bacteria, suggesting the presence of organic matter resources in the subseafloor basalt.

Within the South Pacific Gyre, extremely low sedimentation rates lead to burial of sediments nearly depleted in organic matter. In this ultraoligotrophic environment, dissolved oxygen penetrates from the  ocean floor to the basaltic basement and sustains aerobic microbes throughout the sediment column. During Expedition 329, using the drilling vessel JOIDES Resolution, core samples were obtained from basaltic basement at Sites U1365, U1367, and U1368 with crustal ages of 104 million years, 33.5 million years, and 13.5 million years.

Biogeochemical characteristics of surface seawater and sediment overlying region of the drilled basaltic basement. (a) Map of annual chlorophyll-a concentration in surface seawater. Sedimentary profiles of cell abundance (b), dissolved oxygen (c), and total organic carbon (d). Profiles span the sediment column  from seafloor to basement. The vertical line in (b) marks the minimum quantification limit. Suzuki et al. (2020).

Mineral characterisations were conducted for core samples with fractures/veins to clarify the presence of clay minerals typically produced by low-temperature rock–water interactions (weathering). X-ray diffraction analysis revealed the presence of iron-rich smectite in 33.5-million-year-old and 104-million-year-old core samples but not in 13.5-million-year-old core samples. Thin sections were prepared from the 33.5-million-year-old and 104-million-year-old core samples with sample codes: U1367F-6R1, U1365E-8R4, and U1365E-12R2 at depths of 51, 109.6, and 121.8m below the seafloor and observed by scanning and transmission electron microscopies coupled to energy-dispersive X-ray spectroscopic  analysis. Iron-rich smectite was found at the rims of fractures and veins mainly filled with celadonite and iron oxyhydroxides in U1365E-8R4 and U1365E-12R2, respectively, whereas veins are filled with Iron-rich smectite in U1367F-6R1. Two types of compositionally distinct iron-rich smectite veins were observed in U1367F-6R1: one is similar to those found in U1365E-8R4 and U1365E-12R2 with high magnesium and potasium contents; the other is characterised by high iron content, as typically observed in iron-rich smectite from deep-sea hydrothermal mounds.

 

Chemical compositions of two types of Fe-rich smectite found in U1367F-6R1 and cell distributions revealed by staining of thin sections with SYBR-Green I. (a) Trigonal diagram of aluminium-iron-magnesium content in sheet layer of nontronite and energy-dispersive X-ray spectroscopic spectra of nontronite formed by basalt weathering in green and hydrothermal alteration in orange. Fluorescence microscopy images of SYBR Green I-stained microbial cells with nontronite formed by basalt weathering (b) and hydrothermal alteration (c). Suzuki et al. (2020).

Fluorescence microscopy observations of the thin sections reveal that SYBR Green I-stained cell-like fluorescence signals are extensive along the rims of the rock fractures/veins associated with irom-rich smectite in U1365E-8R4 and U1365E-12R2. Although iron-rich smectite with high magnesium and potassium contents in U1367F-6R1 is correlated with fluorescence signals, fluorescence signals were not detected from veins filled with iron-rich smectite with high iron content in U1367F-6R1.

 

Basalt interface with microbial colonization. Light and fluorescence microscopy images of SYBR Green I-stained microbial cells in a fracture filled with celadonite in of U1365E-8R4 (a) and in a vein filled with iron oxyhydroxides in U1365E-12R2 (b). Suzuki et al. (2020).

To confirm that these greenish signals originate from microbial cells rather than from autofluorescent materials, roughly 10 × 10-μm² sections with a thickness of about 3 μm were fabricated by focused ion beam, and element-mapping images were obtained using nanoscale secondary ion mass spectrometry. Focused ion beam nanoscale secondary ion mass spectrometry analysis of U1365E-8R4 revealed overlapping signals of cyano radicals, phosphorus ions, and sulphur ions on the dense spots stained with SYBR Green I, indicating that those greenish signals are derived from microbial cells. The microbial cells are localized in the proximity of microscale voids and enrobed within iron-rich smectite.

 

Single-cell characterizations of fracture-hosted microbial populations. Scanning electron microscopic image of a mineral-filled fracture in U1365E-8R4 (a). Confocal laser microscopy image of SYBR Green I-stained microbial cells (b). Scanning electron microscope image of a focused ion beam-derived thin section of U1365-8R4 with a square region (roughly 10 × 10 μm²) analyzed by the JAMSTEC nanoscale secondary ion mass spectrometry (c). Nanoscale secondary ion mass spectrometry images of cyano radicals (¹²C¹⁴N⁻) (d), phosphourus ions (³¹P⁻) (e), sulphur ions (³²S⁻) (f), silicon ions (²⁸Si⁻) (g), and iron oxide ions (⁵⁶Fe¹⁶O⁻) (h) with intensity colour contours. Overlays are shown from the gallium ion image of the focused ion beam section in black and white and the nanoscale secondary ion mass spectrometry images of cyano radicals (¹²C¹⁴N⁻) in blue, phosphourus ions (³¹P⁻) in green, and sulphur ions (³²S⁻) in red (i). Dashed rectangles and an arrow show regions presented in the following figures. Suzuki et al. (2020).

The same result was obtained by focused ion beam nanoscale secondary ion mass spectrometry of U1365E-12R2. Element mapping using scanning transmission electron microscopy equipped with energy dispersive spectroscopy showed that the microbial cells are spatially associated with laths of iron-rich smectite. Given this association and the large compositional difference between iron-rich smectite and the bentonite clay used for drilling mud, the microbial cells were not introduced from the drilling mud. These results indicate that the detected signatures along the mineral-filled fractures/veins are derived from indigenous microbial communities in the deep crustal biosphere beneath the oceanic and sedimentary biospheres.

 

Single-cell characterisations of fracture-hosted microbial populations. Scanning electron microscopy image of the mineral-filled fractures in U1365E-12R2 (a). Confocal laser microscopy image of SYBR Green I-stained microbial cells (b). Gallium ion image of a focused ion beam thin section of U1365-12R2 (3-μm thick and a square region of roughly 10 × 10 μm²) analysed using JAMSTEC nanoscale secondary ion mass spectrometry (c), (d) and images of cyano radicals (¹²C¹⁴N⁻) (e), sulphur ions (³²S⁻) (f), silicon ions (²⁸Si⁻) (g), and iron oxide ions (⁵⁶Fe¹⁶O⁻) (h) with intensity colour contours. scanning transmission electron microscopy X-ray elemental mapping images of iron (i), magnesium (j), and potassium (k). Pink arrows and dashed rectangles denote spots or regions described in subsequent figures. Suzuki et al. (2020).

Core samples were evaluated for contamination using fluorescence microspheres (0.5 μm in diameter) that mimic microbial cells introduced from drilling fluid. Microscopic counting of microspheres in subsamples before and after cleaning steps such as washing with 3% sodium chloride solution and flaming the exterior showed that untreated exteriors of core samples contained detectable microspheres, but most post-treatment sample interiors contained no detectable microspheres. These results clarify that the contamination evaluation was properly conducted to show the level of drilling contamination for DNA analysis. 16S ribosomal RNA gene sequences were obtained from the V4 to V6 regions by tagsequencing from four core samples with no detected microspheres (U1365E-8R4, and -12R2 and U1367F-4R1 and U1368F-4R2), one microsphere-detected sample (U1368F-7R3), drilling fluid used at Site U1365, and a DNA extraction blank. To identify potentially contaminant operational taxonomic units from drilling and subsequent laboratory manipulations, the highly contaminated core from U1368F-7R3, the drilling fluid from U1365E, and the negative control were compared to the microsphere-undetected samples.

 

Chemical comparison of bentonite clay used for drilling fluid and nontronite spatially associated with microbial cells. Scanning electron microscopy-energy-dispersive X-ray spectroscopic spectra obtained from bentonite clay (a) and from nontronite (b). Suzuki et al. (2020).

Because α- and β-proteobacterial operational taxonomic units were identical among the contaminated sources such as the drilling fluid and the DNA extraction blank, the operational taxonomic units detected from the contaminated sources were removed from the microsphere-undetected core samples. In addition, operational taxonomic units obtained from the highly contaminated core (U1368F-7R3) were excluded for detailed analyses of indigenous microbial communities. According to phylogenetic affiliation based on 16S rRNA gene sequences, three types of microbial communities were identified.

 

Schematic diagram of procedures undertaken for contamination evaluation and decontamination of drilling fluid. Microsphere counting was performed for each step by epifluorescence microscopy. Suzuki et al. (2020).

Type SPG-I (relatively young crustal community: 13.5 million years old). At Site U1368, γ- and ε-proteobacterial sequences were proportionally abundant and included strains related to the genera Arcobacter, Thioreductor, Sulfurimonas, and Sulfurovum known as deep-sea sulphur- and/or hydrogen-oxidising chemolithoautotrophs and the genus Alteromonas globally distributed in deep-sea aquatic habitats with aerobic heterotrophy.

 

Effects of decontamination processes by washing and flaming. Microscopic counts of fluorescence microspheres in basaltic core samples after each round of decontamination. Rock pieces are represented in cm⁻³, and microsphere density in the surface wash solutions was calculated from the core volume subjected to salt washing. Suzuki et al. (2020).

Type SPG-II (aged crustal communities: 33.5–104 million years old). At Sites U1365 and U1367, β-proteobacterial sequences were predominant and closely related to aerobic organotrophs, such as Roseateles depolymerans isolated from pumice-bearing lake sediment. 

 

Phylogenetic distributions of the highly contaminated sample U1368F-7R3, the drilling-fluid sample from U1365E, and the negative control used for laboratory manipulations based on 16S rRNA gene sequences. Colours and legends represent differences in taxonomic classification ranging from genus to phylum. Each proteobacterial class or phylum is shown in parentheses. Suzuki et al. (2020).

Type SPG-III was only observed in U1365E-12R2 (a depth of 122 meters below sea floor), in which γ-proteobacterial sequences affiliated within the family Methylococaceae were predominantly detected. In general, these crustal microbial communities were comprised of Methylococaceae members typically found in methane-rich fluids emitted from the deep ocean floor, including cold seeps and hydrothermal vents.

 

Community composition in cold basaltic basement based on 16S ribosomal RNA gene sequences. Taxonomic profiles of basaltic rock cores from the South Pacific Gyre are shown as pie charts with major taxonomic groups ranging from genus to phylum. Community composition of a crustal fluid sample at North Pond (Site U1382; 8 million years old) is shown as a pie chart for comparison. Suzuki et al. (2020).

Microbial communities have previously been observed in rock core and fluid samples from North Pond (North Atlantic International Oceanic Drilling Program Site; 8 million years old), where oxygenated cold fluid actively circulates in sediment-covered basaltic basement. For microbiological investigations, a Circulation Obviation Retrofit Kit was installed to collect fluid samples from the basaltic basement at North Pond. However, it is possible that microbial communities in fluid samples are distinct from those attached to adjacent rock surfaces. Microbial communities in the North Pond fluid samples are mainly comprised of members of Campylobacterales and Alteromonadales. In contrast, rock core samples were not dominantly colonised by Campylobacterales members but Alteromonadales members. Dominant microbial populations obtained in our rock sample from Site U1368 (13.5 million years old) were similar to those obtained from the North Pond fluid samples. Thus, the nature of the basement fluid may be very similar in relatively young (8 million years old and 13.5 million years old) basaltic basement in both the Atlantic and Pacific Oceans. These results also indicate that the crustal biosphere can be technically evaluated from rock cores, as well as from circulating fluid.

Basement at 13.5 million years old and 33.5 million years old is mainly composed of pillow lava covered with 12- to 17-m thick sediment. The deepest sediment at both sites contains similar concentrations of dissolved oxygen and dissolved nitrate. Although the crustal structure and the dissolved oxidant chemistry are fairly similar at both sites, microbial community composition differs notably between the 13.5- and 33.5-million year old basements. The clay minerals that form in fractures/veins by low-temperature rock-water interactions provide information that may explain the difference between these communities; the presence and absence of iron-rich smectite in fractures/veins at Sites U1367 and U1368 indicate that iron-rich smectite formation was inhibited by vigorous seawater circulation at U1368.

 

Schematic illustrations of fluid flow regimes and key microbial populations. In basaltic basement, substrates are distinctively supplied from seawater and basalt rocks in 8–13.5 million year old (a) and 33.5–104 million year old (b). Blue and red arrows indicate abundant substrate supplies from seawater and basalt rocks, respectively. Suzuki et al. (2020).

Although the basaltic basement at Site U1365 comprises lava flows where fluid flow is generally between sheeted layers rather than along chilled margins of pillow lava, its microbial community composition is similar to that found at Site U1367, which is consistent with the presence of iron-rich smectite at Sites U1365 and U1367. Seafloor heat flow at U1367 and U1365 is consistent with conduction as the dominant mode of heat transport, while heat flow at U1368 falls below the expected conduction-only level, consistent with apparent heat transport by fluid circulation within the rocky crust. This difference is consistent with the basement ages of the respective sites, as fluid circulation and advective heat transport are generally much more vigorous in relatively young, warm crust (such as the 13.5-million year old crust at U1368) than in much older and consequently cooler crust (such as the 33.5-million year old and 104-million year old crust at, respectively, U1367 and U1365). We suggest that the basement habitability is controlled by heat and fluid flows, which generally decrease over time, versus the primary structure of the crust (e.g. pillow basalt or flow basalt). In addition, the formation of iron-rich smectite in the basaltic basement appears to be correlated with the kinds of microorganisms in aged oceanic crust.

Observations of microbial cells in focused ion beam sections (10 μm × 10 μm × 3 μm) suggest a cell density range of 3 300 000 000 cells/cm³. In the focused ion beam sections, 15 and 2 cyanide-bearing spots derived from microbial cells are visualized in U1365E-8R4 and U1365E-12R2, which gives approximate cell numbers of 50 000 000 000 and 7 000 000 000 cells/cm³. This cell density is narrowly limited to the iron-rich smectite at the interface between basalt and alteration minerals. Within that interface, cell density is exceedingly high in comparison with cell density in the deepest sediment overlying the basaltic basements at Sites U1365 and U1367 (roughly 100 cells/cm³), and in comparison with low-temperature fluids collected from 8-million year old basalt basement at North Pond (roughly 10 000 cells/cm³). The range of cell density estimated for the iron-rich smectite of the basalt-water interface is nearly the same or higher as in organic-rich near-seafloor sediment deposited on continental margins.

To verify the cell density estimates in the two focused ion beam sections, μ-Raman spectroscopy was used to obtain a diagnostic spectrum from the microbe-smectite assemblage. The spectrum is composed of broad peaks at 1200–1600 cm⁻¹ attributed to amorphous organic matter and a slope increasing with Raman shift attributed to smectite. The fingerprint spectrum was obtained throughout the interface regions filled with iron-rich smectite with high magnesium and potassium contents in U1367F-6R1, U1365E-8R4, and U1365E-12R2, but not from that filled with iron-rich smectite with the high iron content in U1367F-6R1. The lack of the fingerprint spectrum from iron-rich smectite with high iron content may be due to its formation at a deep-sea hydrothermal mound near the mid-ocean ridge.

 

μ-Raman spectra of the assemblage composed of microbes and Ferich smectite in U1365E-8R4. Optical microscopic image of the focused ion beam section with a laser spot where a μ-Raman spectrum was obtained (a). Circles indicate points analysed by μ-Raman spectroscopy in the gallium ion image overlain with nanoscale secondary ion mass spectrometry images (b). μ-Raman spectra from yellow circles associated with microbial cells (c) and from white circles without microbial cells (d). Suzuki et al. (2020).

Smectite is a fine-grained clay mineral, with a large surface area to adsorb organic matter. As dominant microbial communities detected from 33- and 104-million year old basaltic basements are heterotrophic, it is conceivable that organic matter bound to iron-rich smectite may help to sustain the high cell density at the basalt interface. Clay fractions were separated from the core samples and their organic carbon content was quantified. The clay fractions mainly composed of iron-rich smectite contained up to 22-fold higher organic carbon than the bulk core samples, supporting the inference that mineral-bound organic matter fuels heterotrophic activities of microorganisms at the basalt interface. Fourier transform infrared-ray spectra were obtained from the clay fractions to clarify the presence of lipids, based on the aliphatic CH₃/CH₂ absorbance ratios (R₃/₂). Given that the R₃/₂ values are domain-specific: Eukarya 0.3–0.5, Bacteria 0.6–0.7 and Archaea 0.8–1.0, the R₃/₂ ranges of the clay fractions from U1365E-8R4, U1365-12R2, and U1367F-6R1 were approximately in the Bacterial range, which agrees with the dominance of Bacteria indicated by 16S rRNA gene sequences from the corresponding core samples U1365E-8R4 and U1365-12R2 and from the other core sample collected from the same site(U1367F-4R1).

 

Raman spectra of microbe-nontronite assemblages in fractures/veins of basaltic basement. Broad peaks at 1200–1600 cm⁻¹ attributed to amorphous organic matter and a slope increasing with Raman shift attributed to smectite were obtained at the basalt interface in U1365E-8R4 (a), U1365E-12R2 (b), and U1367F-6R1 (c). Weak broad peaks at 1200–1600 cm⁻¹ attributed to amorphous organic matter at magnesium-poor nontronite in U1367F-6R1 (d). Yellow dots show points where strong peaks were obtained at 1200–1600 cm⁻¹. White dots show points where weak peaks were obtained at 1200–1600 cm⁻¹. Suzuki et al. (2020).

16S rRNA gene sequences related to aerobic and anaerobic methanotrophs were prominent among the 16S rRNA gene sequences detected from the mineral-filled fractures in 104-million year old basaltic basement. Almost half of the 16S rRNA gene sequences analyzed from U1365E-12R2 were closely related to Methyloprofundus sedimenti, an aerobic methanotrophic Bacterium isolated from a deep-sea sediment sample associated with a Whale fall. Additionally, anaerobic methane-oxidising Archaea subtype 1 (ANME-1) was detected from U1365E-8R4. As methane concentrations are below the detection limit (below 1.3 μM) in all sediment samples at Site U1365, methane bound to iron-rich smectite might be a source of energy for their persistence in situ.

The results of this study greatly extend understanding of bioenergetics and habitability in Earth’s upper oceanic crust. Previous studies of bioenergetics in subseafloor basalt have generally focused on chemoautotrophic mineral oxidation, which mostly occurs in crust younger than about 10 million years. Our results indicate that cells encased in iron-rich smectite densely coat rock surfaces of much older (33.5 million years and 104 million years) basalt and are largely sustained by aerobic heterotrophy and methanotrophy. Organic matter that may sustain these communities in the upper crustal aquifer includes (i) dissolved organic matter in the seawater that flows through the fractures and veins, and (ii) organic matter abiotically synthesized during rock weathering (e.g. Lost City where amino-acid production associated with formation of iron, magnesium-rich smectite in gabbroic basement at the Lost City hydrothermal field).

These results also have important implications for understanding the abundance and global distribution of microbial cells in the upper oceanic crust. Mineral (iron and sulphur) oxidation rates are highest in crust younger than about 10 million years. The number of cells that might be supported by aerobic iron oxidation in the upper marine crust has been estimated as 2 400 000 000 000 000 000 000 000 000 cells, potentially equivalent to 10% of total cell abundance in marine sediment. Because the abundant microbes reliant on aerobic heterotrophy and methanotrophy reside in much older crust (33.5 million years and 104 million years), inclusion of these heterotrophic and methanotrophic cells may substantialy increase estimate of total cell abundance in the upper oceanic crust.

The results of this study also have implications for the possibility of life on Mars and other planetary bodies. Basaltic crust is ubiquitous on other planets, such as Mars, as well on Earth. The Martian basaltic crust formed 4 billion years ago, to be followed by formation of iron, magnesium-rich smectite via hydrothermal alteration and weathering at the surface and in the subsurface until about 3 billion years ago. On modern Mars, the surface is cold and dry under high vacuum conditions, and methane is emitted from the subsurface into the atmosphere. Recently, the presence of subsurface liquid water has been indicated, which spurred international interest in the search for extraterrestrial life. Given the subsurface presence of methane and liquid water on Mars, the communities fueled by organic matter and methane in subseafloor basalt on Earth provide a clear model for extant life and/or biosignatures from past life in the subsurface of Mars and other planets.

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