Early Microbial colonisers of a short-lived volcanic island.

Micro-organisms are, unsurprisingly, typically the first organisms to colonise newly formed or exposed land surfaces, such as those exposed by a rockfall or glacial retreat, or the deposition of volcanic ash. These first colonisers typically comprise oligotrophic (able to survive on very limited nutrition) and autotrophic (able to generate their own energy through chemical reactions or photosynthesis) groups, which are able to survive in environments with very limited environments, as well as groups able to fix nitrogen and carbon obtained from the atmosphere. However, the precise nature of the earliest colonisers is not consistent, and varies from environment to environment. Thus, sediments exposed after the retreat of a glacier are typically first colonised by photosynthetic Cyanobacteria as are newly formed sand dunes, while newly laid lava-flow deposits are typically first colonised by chemolithic (able to obtain nutrients from rock) Micro-organisms.

Volcanic eruptions often create totally new land surfaces very rapidly, either by covering the existing landscape in a new layer of lava, ash, and tephra, or, less commonly, by creating totally new land-masses in the form of volcanic islands. These new volcanic islands are known as 'Surtseyan islands', in reference to the island of Surtsey, which was formed by a volcanic eruption on 14 November 1963. However, unlike Surtsey (which still exists), most such islands are very short lived, formed principally of soft volcanic ash, which is washed away by the sea within a few months, or at most one or two years. Surtseyan islands provide a completely new 'blank state' for micro-organisms to colonise, with little-or-no organic material within the new sediments, an unusually high level of heavy metals, and, frequently, intermittent exposure to toxic volcanic gasses.

Such environments are likely to be challenging to new microbial colonisers, as they are also for microbiologists hoping to study them. The extremely short-lived nature, and general instability, of most Surtseyan islands, makes it close to impossible to carry out field work upon them before they disappear. Persisting Surtseyan islands, however, present a much more interesting scenario, with the potential for biologists to study their long-term succession from a bare-volcanic environment to a developed terrestrial ecosystem. The two most recent persisting Surtseyan islands were Surtsey itself, which emerged in 1963, and Vulcão dos Capelinhos in the Azores, which emerged in 1957. Both of these were extensively studied by the biologists of the time, but in a period when the importance of Micro-organisms as environment shapers was poorly understood, an consequently little attention was paid to these organisms. The first survey of Micro-organisms on the island of Surysey did not take place until the year 2000, 37 years after the island formed. Based upon studies of other volcanic terrains and borehole evidence from the island itself (which expose evidence from early island surfaces covered up by subsequent volcanic activity) it has been suggested that the first Micro-organisms to settle on Surtsey were probably species capable of photosynthesis and lithotrophic species capable of oxidizing trace gasses, sulphur, and/or iron, and possibly some oligotrophic species capable of surviving on the very low levels of carbon and nitrogen found in volcanic sediments.

On 19 December 2014, the Hunga underwater volcano within the Kingdom of Tonga began a series of eruptions, which by 15 January had produced a new island, named Hunga Tonga Hunga Ha'apai, which connected the two older, and much smaller, islands of Hunga Tonga and Hunga Ha'apai into a single landmass. This new landmass included a cone of tephra and ash which rose to about 120 m above sealevel. Initially it was predicted that this new island would erode away within a few months, but this was not in fact the case, making the island the third such persistent Surtseyan island to have formed in the past 150 years. The new island in fact persisted until 2022, when it was destroyed by an explosive eruption of the Hunga volcano. However, the island was visited by scientists in October 2018 and again in October 2019, who collected samples of tephra from across its surface.

In a paper published in the journal mBio on 11 January 2023, Nicholas Dragone of the Department of Ecology and Evolutionary Biology at the University of Colorado Boulder, and the Cooperative Institute for Research in Environmental Science, Kerry Whittaker of the Corning School of Ocean Sciences at the Maine Maritime Academy, Olivia Lord of the Sea Education Association, Emily Burke of the School of Marine Science and Ocean Engineering at the University of New Hampshire, Helen Dufel of the Scripps Institute of Oceanography at the University of California San Diego, Emily Hite of the School of Earth Sciences and Environmental Sustainability at Northern Arizona University, Farley Miller and Gabrielle Page of the Université de Bretagne Occidentale, Dan Slayback of Science Systems & Applications, Inc., and the Biospheric Sciences Lab at NASA's Goddard Spaceflight Center, and Noah Fierer, also of the Department of Ecology and Evolutionary Biology at the University of Colorado Boulder, and the Cooperative Institute for Research in Environmental Science, present the results of a study of the Micro-organisms from these samples, which aimed to answer the questions:  'What taxa were the earliest microbial colonizers of sediments on Hunga Tonga Hunga Ha'apai?' 'From where did these microbial colonizers originate?' and 'What metabolic strategies were used by these microbes to persist in the challenging environmental conditions found on the recently formed landmass?

The island of Hunga Tonga Hunga Ha’apai, Kingdom of Tonga (latitude, 20.536°S; longitude,175.382°W). The locations of the 32 surfaces where samples were collected are shown. The background image is from 19 August 2018 and is orthorectified. The inset image displays the islands of Hunga Ha’apai (west) and Hunga Tonga (east) on 11 September 2010, prior to the 2014–2015 eruption. Dragone et al. (2023).

Dragone et al. collected 32 samples from across the new landmass, at altitudes ranging from sealevel to the summit of the cone, at roughly 120 m higher. At the time of the collecting, some Plants and Animals had begun to settle on the island, although the majority of the samples were collected at sites away from such incursions.

The samples collected from unvegetated areas of the Hunga Tonga Hunga Ha'apai tuff cone show very low levels of nutrients, and organic carbon, but high levels of heavy metals and sulphur. The concentration of organic carbon in these tuff samples ranged from 0.19 to 0.50 mg per gram (with an average of 0.32 mg per gram), which was about ten times lower than the level from vegetated samples (i.e. samples collected from around plants, at the edge of the original landmass of Hunga Tonga). The tuff samples lacked any detectable nitrogen, while sulphur levels ranged from 0.1 to 19.8 mg per gram, with an average of 2.1 mg per gram, and iron levels ranged from 74 to 86 mg per gram, with an average of 80.1. Copper, vanadium, cobalt, and other metals were also present at levels far higher than typical of natural soils, but comparable to those often seen in contaminated soils from former industrial sites. 

All of the samples yielded both Bacterial and Archaean DNA, although the levels of DNA from tuff samples was typically two orders of magnitude lower than that from vegetated samples. The tuff samples were also apparently much less biodiverse, with an average of 108 variants on the 16SrRNA gene sequence (a highly conserved, but still variable, sequence found in all known Bacteria and Archaeans, as well as in the nuclei, mitochondria, and chloroplasts of Eukaryotes, which is considered extremely useful as a marker by Prokaryote taxonomists), compared to 473 in the vegetated samples. 

The Microbial community represented in the tuff samples was also distinct from the community found in the vegetated samples, dominated by members of the Bacterial phylum Chloroflexi making up 24.6% of the genetic reads, followed by Actinobacteria, 18.1% of genetic reads, Firmicutes, 15.7, and Proteobacteria, 15.5%. Present at lower concentrations were members of the Bacteroidetes, 6.2% of genetic reads, Planctomycetes, 5.4%, Acidobacteria, 3.3%, Cyanobacteria, 2.8%, Gemmatimonadetes, 1.5%, and candidate phylum WPS-2 'Eremiobacteria', 1.5%. The Archaean phylum Thaumarchaeota was also present in all samples, but in all cases at levels of less than 2% of the total number of genetic reads. 

The most commonly found Bacterial families were Acidiferrobacteraceae (Proteobacteria), Ktedonobacteraceae (Chloroflexi), and Sulfuricellaceae (Proteobacteria), all of which contain autotrophic chemolithotrophs capable of gaining energy by oxidizing sulphur or iron. However, many of the samples came from little known groups, so that 40% of the total genetic reads could not be classified to family level. This means that the majority of Bacteria which were colonising the new land surface belonged to taxa for which their ecological role is at best poorly understood, with only 52% belonging to families from which any member has ever been cultivated in the lab.

This Microbial community is quite distinct from that found in the initial stages of colonisation of new land surfaces in other terrestrial settings. Cyanobacteria, typically among the earliest settlers on new land surfaces in other environments, and thus widely considered to be indicative of such communities, were absent on Hunga Tonga Hunga Ha'apai. Dragone et al. suggest that this absence of Cyanobacteria may be linked to the presence of high concentrations of hydrogen sulphide, which is produced by most volcanic systems and known to be an inhibiting agent for Cyanobacteria. Members of the Phylum Chloroflexi dominated the Microbial community on Hunga Tonga Hunga Ha'apai. This dominance has not been seen elsewhere, but these Bacteria are known to be hydrogen sulphide-tollerant and are often found in volcanic settings with high hydrogen sulphide levels.

The Microbial community from Hunga Tonga Hunga Ha'apai does show some similarities to Microbial communities observed on older volcanic deposits at other sites. For example, on basaltic deposits in Iceland, the Bacterial orders Planctomycetales (Planctomycetota), Rhizobiales (Proteobacteria), Rhodospirillales (Proteobacteria), and Sphingomonadales (Proteobacteria) were apparently ubiquitous, being found in all samples, as were members of the Phylum Chloroflexi.

Theoretically, the most likely source of Microbial colonisers on a new island landmass are is the surrounding ocean, followed by gut Bacteria from Birds deposited in their feces. Neither of these seems to be a particularly likely source for the Microbes colonising Hunga Tonga Hunga Ha'apai. Another possibility is that Microbes could have migrated to the new landmass from the two original landmasses, Hunga Tonga and Hunga Ha'apai. However, while Dragone et al. did find some similarities between the Microbial community of Hunga Tonga Hunga Ha'apai and that of Hunga Tonga, this did not appear to be the main source of the new landmass's Microbiota. 

As an alternative, Dragone et al. suggest that the Bacteria may have originated from nearby volcanic and/or hydrothermal vent systems. Unfortunately, not information was available on the Microbial communities at submarine or subaerial geothermal systems in Tonga prior to the 2014-15 eruption, but the Microbes observed on Hunga Tonga Hunga Ha'apai does reflect that often found in such environments. Gene sequences associated with the Planctomycetales, Rhizobiales, Rhodospirillales, and Sphingomonadales have all been recovered from volcanic environments in Alaska, Hawai'i, and New Zealand. Dragone et al. also note that many of the most abundant gene sequences from the Hunga Tonga Hunga Ha'apai deposits, including the uncultivated Chloroflexi sequences, are common in marine water samples from the deep euphotic zone, and in particular around hydrothermal vents, on organic-poor seafloor surfaces, and in organic-poor deep marine sediments. The most abundant 16S rRNA gene sequence in the Hunga Tonga Hunga Ha'apai material, Chloroflexi AD3, is identical to a sequence recovered from sediments from the Brothers Volcano Complex in the Tonga-Kermadec Arc. Other gene sequences from Hunga Tonga Hunga Ha'apai are similar to sequences recovered from hot springs in Yellowstone National Park, and hydrothermal vent fields in the Atlantic and Pacific.

The island of Hunga Tonga Hunga Ha'apai (and the original islands of Hunga Tonga and Hunga Ha'apai) was a subaerial projection of the much larger submarine Hunga Volcanic Caldara, which covers a today area of about 16 km². This is an extremely active volcanic complex, with submarine venting recorded since 1912, and significant eruptions recorded in 2009, 2014-25, and 2022. It is possible that Microbes living in sediments on submarine parts of the volcano were transported to the new land surface during the 2014-15 eruption. Something similar has been observed on Surtsey, where Micro-organisms from subsurface sediment pore waters have been shown to be transported to the surface via fumerole systems. An alternative is that the Microbes could have been blown to the island from exposed volcanic surfaces on nearby islands. Hunga is one of about 20 active volcanic systems within the Kingdom of Tonga, the closest of which is the submarine Fonuafo’ou Volcano, only 25 km from Hunga Tonga Hunga Ha'apai. The nearest volcano which reaches the surface is Tofua, about 100 km away, while Late’iki, another submarine volcano about 200 km to the north, underwent an explosive eruption in 2019, which could have aerosolised Micro-organisms. Previous work in New Zealand has suggested that Microbes could be dispersed over 850 km following a volcanic eruption. Given the presence of gene sequences associated with deep marine sediments in the Hunga Tonga Hunga Ha'apai tuff samples, Dragone et al. suggest that many of the Microbes on the island are likely to have arrived from a subsurface environment.

The taxonomic identities of the Hunga Tonga Hunga Ha'apai Microbes, combined with our current knowledge of the ecological roles of these groups, suggests that the earliest stages of settlement on the island were dominated by hemolithotrophic Bacteria and anoxygenic phototrophs. Dragone et al. found gene sequences associated with the metabolism of sulphur, the oxidation of carbon monoxide and hydrogen, and bacteriochlorophyll-mediated anoxygenic photosynthesis in tuff samples at levels two-to-five times as high as in the vegetated samples, while genes associated with other Bacteria functions were present at roughly equal levels in both sets of samples. 

The high concentration of genes for sulphur metabolism in the Hunga Tonga Hunga Ha'apai tuff samples accords well with the high concentration of sulphur found in the same samples. Notably, genes associated with the metabolism of thiosulphate where far more abundant in the tuff samples than in the vegetated samples, notably thiosulphate reductase, thiosulphate sulphurtransferase, sulphur oxidation pathway, and thiosulphate dehydrogenase, were found in all samples. Thiosulphates serve as an intermediary phase in many sulphur metabolising pathways, with the effect that almost all sulphur-processing chemolithotrophic Microbes, including those from terrestrial volcanic systems and deep sea hydrothermal systems. Since the surface sediments on Hunga Tonga Hunga Ha'apai were well aerated and not likely to be lacking in oxygen, it is unlikely that the Bacteria here were engaged in the anaerobic reduction of sulphur, leading Dragone et al. to conclude that sulphur oxidation is likely to be the most important ecological strategy for these Micro-organisms.

No photosynthetic Cyanobacteria were found in any of the samples, nor were genes for oxygenic photosynthesis found. However, a number of genes associated with anoxygenic photosynthesis were found. These are known to be present in a wide range of Bacteria, including the Actinobacteria, which were a major component of the Hunga Tonga Hunga Ha'apai Microbiota, as well as more specific anoxygenic photosynthesis genes associated with the families Beijerinckiaceae (Proteobacteria) and Acetobaceraceae (Actinobacteria). This suggests that photosynthesis was occurring on Hunga Tonga Hunga Ha'apai, but that this was anoxygenic photosynthesis, in which sulphur is used as an electron doner.

Also widely present were genes associated with the oxidation of trace gasses, notably carbon monoxide and hydrogen. Many groups found within the samples, including the Ktedonobacteraceae and other members of the Chloroflexi are known to have this capability. The oxygenation of trace gasses is generally associated with Bacteria surviving in extremely resource-poor environments, such as Antarctic soils, and is also known to play an important role in more mature volcanic soils, 10-20 years after an eruption. Dragone et al. hypothesize that trace gas oxygenation may play the same role in volcanic soils as photosynthesis does in other newly colonised environments, enabling the very first Microbes to gain a foothold from which more developed communities can then develop.

Dragone et al.'s study indicates that Micro-organisms begin to settle on new volcanic islands very soon after their formation. However, unlike other new environments, the first colonisers on volcanic islands are not photosynthetic Cyanobacteria, but rather Bacteria capable of utilising the abundant sulphur resources of these environments, and oxidizing trace gasses. Furthermore, these Bacteria do not appear to have come from the neighbouring surface-marine or vegetated island environments, but rather to have originated from geothermal systems, potentially those deep beneath the sea or land surfaces. These Micro-organisms may well have reached the island as a result of volcanic activity elsewhere, which has the potential to disperse Micro-organisms over wide areas. It is quite possible that the Microbiota of the island went through a number of changes in the three years between the formation of the island, and the collection of the first samples there, and almost certain that, had the island survived, the Microbial community would have continued to evolve as the island developed from a bare rocky surface into a vegetated island. However, the complete destruction of the island by a new eruption in January 2022 made any further work impossible on that island, and scientists will have to wait for the formation of new volcanic islands elsewhere to take this field of study further foreward.

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Methanofollis propanolicus: A novel methanogenic Archaean from an oil well in Costa Rica.

Archaeans are Prokaryotic micro-organisms resembling Bacteria, but only distantly related to them, and more closely related to Eukaryotes. The Methanomicrobiales are a group of Archaeans which produce methane through reductive reactions, typically obtaining carbon from carbon dioxide, although there are some exceptions. Members of the genus Methanofollis, for example, produce methane by the reduction of alcohols, typically using acetate as a carbon-donor.

In a paper published in the journal Archives of Microbiology on 13 August 2022, Linda Dengler, Julia Meier, Felix Grünberger, and Annett Bellack of the Institute of Microbiology and Archaea Centre at the University of Regensburg, Reinhard Rachel of the Electron Microscopy Center at the University of Regensburg, and Dina Grohmann and Harald Huber, also of the Institute of Microbiology and Archaea Centre at the University of Regensburg, describe a new species of Methanofollis from an oil well in Costa Rica.

The new species was isolated from an exploratory drill well sunk in the Cahuita National Park, on the southwestern Atlantic coast of Costa Rica. The well is now a stagnant pool, with the new species being taken from an anaerobic layer 30 cm beneath the surface. 

Ancient oil well in the Cahuita National Park, Costa Rica, original sampling site of strain CaP3V-MF-L2AT. Dengler et al. (2022).

The new species, initially identified as strain CaP3V-MF-L2AT, was found to be able to metabolise propanol with acetate producing methane. It was unable to metabolise acetate on its own, nor acetate with methanol, ethanol, butanol, or cyclopentanol. It was found by genetic analysis to be a new species within the genus Methanofollis, and given the specific name propanolicus, in reference to its obligate metabolising of propanol.

(a) Transmission electron micrograph of freeze-etched cells of strain CaP3V-MF-L2AT indicating (b) proteinaceous S-Layer (SL) and an archaellum (AR). Scale bars are 200 nm. Dengler et al. (2022).

Cells of Methanofollis propanolicus are irregularly coccoid (roughly spherical),  0.8–1.8 μm in diameter, and found singularly or in pairs. They are have two types of appendages; archaella (a filament attached to the cell membrane by a molecular motor, used to propel some forms of Archaea in a swimming-like motion) with a diameter of about 12 nm, and pili (hair-like structures found on both Bacteria and Archaea, which can have a variety of functions), with diameters of about 8 nm.

Transmission electron micrograph of Pt/C shadowed cells of strain CaP3V-MF-L2AT show the great variability in cell-shape as well as archaella. Scale bar is 400 nm. Dengler et al. (2022).

Although only propanol and acetate were necessary for the survival of Methanofollis propanolicus, the species did better in the presence of yeast-extract. It was able to grow at temperatures of 25-40°C, with optimum growth at 37°C. The species was able to tolerate salinities in the range 0-2.5% weight/volume sodium chloride, with optimum growth at 0.2-1.5%. Methanofollis propanolicus was able to grow at pH values in the range 6.0-7.5, with optimum growth in the range 6.5-7.0.

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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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