Morono ﻿Progress in Earth and Planetary Science(2023) 10:18
https://doi.org/10.1186/s40645-023-00551-5

Progress in Earth andPlanetary Science

Open Access

REVIEW

Accessing the energy‑limited and sparsely
populated deep biosphere: achievements
and ongoing challenges of available
technologies
Yuki Morono1*   

Abstract
Microbes in marine sediments detected and counted by direct observation of membrane-filtered sediment samples
stained with acridine orange. This technique can still be applied to high-biomass (> ­105 cells/cm3) sedimentary habitats, such as organic-rich sediments collected in shallow areas near the seafloor. However, to further explore the nutrients and energy turnover under extremely low energy flux conditions, or in habitats that are close to the lower limitof the biosphere, technological breakthroughs have been required to increase the detection sensitivity for microbial
life at densities of a few cells/cm3 of sediment. These technological developments contributed to increasing fundamental information on microbial life at the fringes of the subseafloor biosphere and led to the discovery of revivablemicrobes in sediments aged up to 101.5 million years old. More recently, chemical detection methods have revealed
the existence of spores in the deep biosphere that are impermeable to conventional DNA stains. Previous applicationsof molecular biology-based approaches have been limited to relatively higher biomass samples, potentially because
the cells surviving in these very low energy flux environments have less integrated genomes. Here, I review the contribution and importance of the technological developments that have been made in the study of microbes from thesubseafloor biosphere, recent developments of alternative methods to microscopically detect microbial spores andtheir application to deep subseafloor sediments, and the challenges associated with applying molecular biologicalapproaches to study low-biomass samples.
Keywords Subseafloor biosphere, Low energy habitat, Technical developments, Limits of life
1 Introduction
Our understanding of the distribution of microbial life on
Earth has expanded continuously as research activities that
focused on the discovery of life in new environments haveprogressed. The subseafloor environments are among the*Correspondence:
Yuki Morono
morono@jamstec.go.jp
1
Geomicrobiology Group, Kochi Institute for Core Sample Research,
Extra‑cutting‑edge Science and Technology Avant‑garde Research(X‑star), Japan Agency for Marine-Earth Science and Technology(JAMSTEC), Monobe B200, Nankoku, Kochi 783‑8502, Japan

most explored natural habitats to date by numerous studies that have been conducted in recent decades. In 1955,
Morita and ZoBell (1955) conducted a pioneering study ofthe subseafloor biosphere, in which they used cultivationbased techniques to suggest that the biosphere extends
only several meters below the seafloor (mbsf). Several decades later, the Ocean Drilling Program (ODP: 1983–2003)
significantly increased our understanding of the deep
subseafloor biosphere (water depth from 151 to 3773 m,
maximum sediment depth from 80.2 to 748.49 mbsf
[meters below seafloor], age to 10 million years, and variety of physical/chemical environments) (Parkes et al. 1994,
2000b). The findings of the ODP accelerated attempts to© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

Morono P﻿ rogress in Earth and Planetary Science(2023) 10:18

explore the subseafloor biosphere and were followed by the
Integrated Ocean Drilling Program (2003–2013) and theInternational Ocean Discovery Program (2013–2023) (e.g.,
Ciobanu et al. 2014; Inagaki et al. 2015; Roussel et al. 2008),
which showed that the biosphere extended to more than
2.4 km below the seafloor. The accumulation of knowledge
about subseafloor microbial abundance facilitated estimates of the global microbial biomass in the subseafloor
biosphere, which is considered to account for 12–45% of
the total microbial biomass on Earth, or 0.6–1.8% of thetotal living biomass on Earth (Bar-On et al. 2018; Kallmeyer
et al. 2012; Magnabosco et al. 2018; Whitman et al. 1998).
Even though the entire population of subseafloor
microbes is massive, volumetric estimates indicated thatthe microbial cells constitute only a minor component(< 0.01% by volume) of the sediments, even at the seafloor
where the highest microbial densities are typically found(Morono and Kallmeyer 2014). Consequently, the general distribution of microbial cells in the sediment matrix
is considered to be relatively sparse. However, their realin situ distribution has not yet been directly observed. The
majority (> 90%) of marine sediments are composed offine particles measuring less than 1 μm to several tens of
micrometers (Diesing 2020; Dutkiewicz et al. 2015). This
fine nature of subseafloor sediments restricts both active
and passive transport of even small microbial cells andresults in the formation of depth-stratified microbial communities (Inagaki et al. 2006). In addition, the fine nature ofsubseafloor sediments can complicate the analysis of subseafloor communities, such as identifying and enumerating
microbial cells and performing DNA/RNA-based molecular analyses.
The accumulation of knowledge about the subseafloor
biosphere has been achieved through extensive development of subseafloor-targeted technical and analytical
approaches; using preexisting processes can produce variable (non-reproducible) and misleading results. The subseafloor environment, in which microbial communities
constitute a very small and volumetrically minor component, contains numerous substances that can interfere withanalysis and sampling, both of which are challenging using
existing technologies. Consequently, extensive optimization of, or improvements in, analytical techniques has beennecessary. This review focuses on technological aspects ofsubseafloor biosphere exploration, in particular, on effective solutions to previous challenges and areas that require
further technological development.

2 Review
2.1 Fluorescence staining: Are those tiny blobs reallythe ones you are looking for?

Detection and enumeration of microbial life are themost fundamental steps for exploring the biosphere.

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Conventionally, these steps were conducted by cultivation techniques, such as by direct plating andcounting colonies. A pioneering study on counting
microbes from the subseafloor biosphere by Morita
and ZoBell (1955) described the microbial life foundin deep marine pelagic sediments of the Pacific Ocean.
They conducted direct plating of diluted subseafloor
sediments and succeeded in culturing colonies fromseveral meters below the seafloor (mbsf ). However,they did not detect any countable colonies from samples collected at 3.86–7.47 mbsf. Although they noted
the potential for underestimation of microbial abundance due to the limitations of the culture media, thelimit of the marine biosphere at that time was considered to be 7.47 mbsf. Beginning in the late 1980s, John
Parkes’ group began quantifying microbial biomass indeeper subseafloor sediments using fluorescence staining of intracellular DNA. They used the acridine orange
direct count (AODC) method for their counts and biomass estimation (see review in Parkes et al. (2000a)).
Acridine orange is a widely used DNA intercalator that
efficiently stains the genomic DNA in microbial cells.
Initially, all the cell counts of ODP core samples were
performed by Parkes’ research group, mainly by Barry
Cragg (Cragg 1994; Cragg et al. 1992, 1997, 1998, 1999,
1990, 1995a, 1995b, 2000, 2002; Cragg and Kemp 1995;
Cragg and Parkes 1993, 1994; Wellsbury et al. 2000).
The fact that all of the measurements were performed
by a single research group (and primarily by the same
person) was of primary importance in creating a dataset of almost unrivaled consistency.
Although DNA-specific staining and fluorescencemicroscopy methods have been successfully applied toaquatic habitats (the most common examples include
4’,6-diamidino-2-phenylindole (DAPI; Porter and Feig
(1980)), acridine orange, (AO; Daley and Hobbie (1975)),
and SYBR Green I (SYBR-I; Noble and Fuhrman (1998)),
the application of these methods to marine subseafloor
sediment analysis was not straightforward. These difficulties were primarily related to the presence of fine particles
that constitute the majority (> 90%) of marine sediments.
For example, the native fluorescence of mineral grains,
especially under UV excitation (Robbins 1994), caused
problems with microbial cell detection using DAPI, oneof the most widely used cell stains. As a result, DAPI
is not commonly used for marine sediment analysis.
Another problem is the non-specific adsorption of dyesto the fine particles that are present in marine sediments.
Figure 1A shows an example of AO staining of marine
sediments in which the fluorescence from many noncellular particles was comparable to that of the microbial
cells. The non-specific binding of the fluorescent dye tosediment particles made microbial cell detection difficult,Morono P﻿ rogress in Earth and Planetary Science(2023) 10:18

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Fig. 1 Examples of fluorescence microscopy images. Sediment samples obtained by International Ocean Discovery Program (IODP) Expedition 370(T-limit) at 207.5 mbsf (3F-3) was stained with AO (A) and SYBR-I (B, C). B and C show the same position of the prepared membrane observed with
optical emission filter that passes only wavelength around 525 nm (B) and without emission filter (C). It is noted that only single cell (pointed bywhite arrows) is present in the area of each image. Bars represent 5 µm

and investigators had to be trained intensively to produce
reliable and consistent data.
The problem was more significant when SYBR-I
was used to stain microbes in sediments. The fluorescence intensity of SYBR-I has been reported to increaseby ~ 1000-fold upon binding to DNA (Dragan et al. 2012).
Since free SYBR-I dye has extremely faint fluorescence,
it is often regarded as a DNA-specific stain that fluoresces brighter than other DNA stains. While this may
be true for most samples, SYBR-I is preferentially boundby marine sediment particles (Fig. 1B), which meansthat the take-up of SYBR-I by microbes only occurs
once sediment particle adsorption has reached saturation levels (Mori et al. 2021; Morono and Inagaki 2010;
Morono et al. 2008). Even though the fluorescence intensity is enhanced after binding to DNA, the fluorescenceintensity of the SYBR-I dye that accumulated on thesediments was similar to that taken up by the microbes;
consequently, the problems associated with discriminating between microbes and sediments using SYBR-I weresimilar to those encountered with AO. Microscopes forfluorescence observations are equipped with optical filters, which allow only light at the excitation or emission

wavelengths of the target fluorophore to pass through,and dichroic mirrors, which reflect the excitation wavelength and allow light at the emission wavelength to passthrough. With the proper filter set, target fluorescencecan be precisely observed; however, some specialized filter sets can hide important information. Conventionally,
the green-fluorescent dye-stained (i.e., AO- or SYBRstained) microbes are observed with an emission filter
that only allows the green color of the fluorescence (generally around 525 nm) to pass through. However, problems can arise when identifying the signals of microbial
cells from SYBR-I dye in situations where there is an
overaccumulation of non-cellular particles (Fig. 1B).
Morono et al. (2008) reported that the fluorescence frommicrobial cells and non-cellular particles can be distinguished by the fluorescence color when observations
are performed without an emission filter (Fig. 1C). They
proposed that even though high levels of SYBR-I adsorption to sediment particles results in the fluorescenceintensity being comparable to that of the SYBR-I-DNA
complex, the overaccumulation of SYBR-I may haveinduced a shift in the color of the fluorescence throughintermolecular energy transfer. Based on their findings, aMorono P﻿ rogress in Earth and Planetary Science(2023) 10:18

computer-based recognition scheme was established toautomatically detect “green” microbial cells by calculating the fluorescence of two images taken using emission
filters through which only green (525 nm) and orange(605 nm) light could pass (SYBR-DiCE method; Morono
and Inagaki 2010; Morono et al. 2009). According totheir data, the “false-positive” counts from non-cellularparticles were almost constant with subseafloor depth.
However, the “actual” cell abundance decreased withan increase in depth; as a result, the scatter of two plots
increased with sediment depth (Fig. 2). These findings
provided a way to accurately distinguish between microbial cells and non-cellular particles in the sediment without intensive training of the investigator. Unfortunately,a similar shift in fluorescence color due to overaccumulation was also observed with several other green fluorescent dyes, but no shift due to overaccumulation wasobserved when using dyes of other colors.
There are currently more than several commerciallyavailable cell-permeable DNA stains on the market and
SYBR-I is only one of them. However, there are certain
types of microbial cells that do not take up many ofFig. 2 Cell abundance plot for the samples obtained from offshore
Shimokita Peninsula. Increased deviation of observed cell abundance
was found between counts conducted without (gray) and with(red) SYBR-DiCE method along the depth of subseafloor sediments.
Modified from Morono et al. (2009)

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these stains; spores, which are a type of microbial cells
having almost impermeable cell wall structure. Microbial endospores, especially the spores of thermophilic
microbes, or thermospores, are considered to be widelydispersed in the marine environment, and thus, in thesubseafloor biosphere as well (Hubert et al. 2009). The
abundance of spores in subseafloor sediments is thought
to be enormous, and unlike their vegetative counterparts, the relative abundance of endospores is reported
to increase with depth below the seafloor (Fichtel et al.
2007; Lomstein et al. 2012; Wörmer et al. 2019). Indeed,a spore-dominated horizon was discovered in the subseafloor sediments of the Nankai Trough (Heuer et al.
2020). Quantification of these microbial spores has
been typically performed by chemical detection of thespore-specific compound, dipicolinic acid (DPA) (Fichtel
et al. 2007; Lomstein et al. 2012) as well as estimates of
microbial metabolic activity after germination (Hubert
et al. 2009). However, to the author’s knowledge, direct
observations of microbial spores from marine sediments
have not been reported to date. The reason for the lack ofsuch observations is likely attributable to the impermeable nature of the spore coat. Heuer et al. (2020) reported
that the SYBR-I stain was ineffective for staining cultured Bacillus spores. After SYBR-I staining, the Bacillus spores showed yellow fluorescence (Fig. 3), indicatingthat SYBR-I was adsorbed by the spore surface and unable to penetrate the outer surface of the spores. Although
the abundance of spores is expected to be similar to theirvegetative counterparts (Wörmer et al. 2019), they cannot be visualized using current technologies.
There is a large variety of fluorescence staining techniques, some of which use fairly complicated reactions
and/or steps to detect microbial cells. Fluorescencein situ Hybridization (FISH) (Amann et al. 1990) is used
to stain 16S rRNA molecules using fluorescent DNA
probes that have been specifically designed to bind totaxon-specific sequences. There are also a variety ofderivative techniques with enhanced detection sensitivity, such as horseradish peroxidase-catalyzed depositionof fluorescent molecules (catalyzed reporter deposition,
CARD) (Pernthaler et al. 2002a) and cascade hybridization of fluorochrome-labeled DNA (hybridization chain
reaction, HCR) (Yamaguchi et al. 2015). Schippers et al.
(2005) successfully detected bacterial cells in subseafloor sediment samples using CARD-FISH. Their findings showed the existence of 16SrRNA molecules, which
are easily degraded in dead cells, and indirectly showedthat the microbes in subseafloor sediments were living
cells and not remnants of dead cells. FISH-based microbial cell detection methods have several advantages over
simple DNA stains; for example, they can be used to infer
the phylogeny and physiology of microbes based on theMorono P﻿ rogress in Earth and Planetary Science(2023) 10:18

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Fig. 3 SYBR Green I staining of vegetative cells (a) and endospores (b) of Bacillus subtilis NBRC13719. This figure illustrates the difficulty in
identifying spores even of pure cultures of organisms that are mixed into sterilized sediments. In (c), endospores were mixed in sterilized sedimentprior to staining by SYBR Green I. Several representative yellowish endospores, which are difficult to distinguish from sediment particles, areindicated by white arrows as examples. Bars represent 10 μm. Adopted from Heuer et al. (2020)

16S rRNA expression. However, because of difficulties
in delivering probes that are larger than DNA dyes, thedetection efficiency varies depending on the methods
used to permeabilize the cells (Lloyd et al. 2013). In addition, the probes are also highly susceptible to non-specificadsorption to sediment particles, and this effect is exacerbated with CARD or HCR signal amplification. If it is
the simple adsorption of DNA probes onto sediment particles, then green fluorochromes, such as FITC-labeledprobes, can be discriminated by the fluorescence color
shift induced by overaccumulation like SYBR-I (data notshown). However, in the case of signal amplification, thecolor shift does not occur, probably because the deposited tyramides or amplification probes dilute the local
concentration of fluorochromes and thereby, prevent
energy transfer. Therefore, preventing the adsorption ofprobes to sediment particles is extremely important for
ensuring the efficacy of the FISH-based approach. The
use of hybridization buffers containing a high concentration of EDTA has been found to be effective for reducing adsorption onto non-cellular particles (Morono et al.
2020). Jia et al. (2021) reported that the EDTA buffer was
effective in reducing non-specific signals when using the
HCR-FISH protocol.

As shown in the above examples, even the moststraightforward DNA staining methods have pitfalls
when they are applied to the analysis of marine sediments. It is clear that simply applying an existing technique can cause serious problems and lead to misleading
results. There are many fluorescence staining techniques,
each of which varies in its complexity and what it detects.
In all of the techniques, obtaining either false positives orfalse negatives is possible, and a careful understanding of
the detection principles and methodologies is required tocorrectly interpret the obtained results. Therefore, continuously asking whether the fluorescent blob that you
are looking at really is the target or something different is
one of the most basic strategies for avoiding mistakes andmisleading results.
2.2 
Requirements/prerequisites for cell detection
and analysis: separation of microbial cells
from the surrounding matrix and subsequent analysis

Given that microbial cells constitute only a minor fraction of marine subseafloor sediments (< 0.01% in volume), the ability to detect microbial cells is largely
restricted to be insensitive. For fluorescence microscopic observations, subseafloor cells need to be trappedMorono P﻿ rogress in Earth and Planetary Science(2023) 10:18

onto a membrane; however, these membranes can easily become clogged, which restricts the volume of sediment samples that can be filtered/analyzed. Generally,
the lower detection limit of subseafloor microbial cells is­104–105 cells per c­ m3. However, the separation of microbial cells from other sedimentary particles is the simplest
and most effective way to improve microbial cell detection. Kallmeyer et al. (2008) first reported a method forphysically separating microbes and sedimentary particles in deep biosphere samples using differences in density. They first carefully tested a variety of approaches for
detaching microbial cells from sedimentary particles andfound that the combination of chemical and mechanical treatments yielded high and reproducible recovery of
microbial cells with minimal lysis. They started with Percoll, a colloidal silica-based gradient agent that is widelyused in medical and biochemical fields (Pertoft et al.
1978), to separate microbial cells from sediment samples.
Even though it was effective for separating microbes fromshallow or coastal sediments and anaerobic methaneoxidizing aggregates (Orphan et al. 2009), cell recovery
after separation with Percoll was insufficient for deep
subseafloor samples. Kallmeyer et al. subsequently used
Nycodenz, a non-ionic density gradient agent (Rickwood
et al. 1982), which increased the recovery of microbial
cells from subseafloor sediments. At that time, separation was performed by layering the sediment suspension
onto a cushion of 50% (w/v) Nycodenz. Later, Morono
et al. (2013) modified this separation technique froma bilayer method to a multilayered method using different density agents and achieved higher recovery ofPage 6 of 13

microbial cells regardless of the sediment type (Fig. 4).
They improved two points in bilayer separation: (i) coprecipitation of microbial cells with sediment particles,and (ii) precipitation of microbial cells with a higher density. The first point was considered to occur due to theturbulent flow caused by the precipitating particles as
they moved through the density boundary and captured
and co-precipitated with microbial cells floating on thedensity boundary. The second point was attributed tothe decreased cell recovery observed in cell samples that
had been fixed in formalin and stored for several months.
After considering that the high viscosity of Nycodenz
can cause turbulent flow, they used multiple layers of
the Nycodenz density agent in combination with the lessviscous density agent, sodium polytungstate. The combination of viscous Nycodenz and the less viscous sodium
polytungstate resulted in recovery rates that were 8–46
times higher than the recovery rates obtained using thebilayer protocol. To further improve recovery, linear gradient density agents have been developed and applied tocell separation, which has facilitated the ultrasensitive
detection and enumeration of microbial cells from a variety of subseafloor environments, including those near thefringe of the subseafloor biosphere (D’Hondt et al. 2015;
Früh-Green et al. 2018; Heuer et al. 2020; Inagaki et al.
2015; Templeton et al. 2021).
2.3 Necessity of separation of microbial cells for obtaining
reliable data on the single‑cell‑level analyses

Once the cell detection was optimized, further exploration of the subseafloor biosphere was possible. Indeed,

Fig. 4 Scheme of cell separation by multiple density layers. By using multiple density layers, better recovery of microbes was achieved than bilayer
protocolMorono P﻿ rogress in Earth and Planetary Science(2023) 10:18

not only was ultrasensitive detection or enumeration
possible, but the combination of fluorescence staining
and cell separation was also effective for analyzing thecommunities found in subseafloor sediments. Determination of the living status of microbes from subseafloor
environments by Schippers et al. (2005) further stimulated interest in the physiology of microbes in deeply
buried, energy-starved sediments (comprehensive
reviews can be found in Hoehler and Jørgensen (2013),
Orcutt et al. (2011), and Orsi (2018)). Based on the availability of energy sources (organic matter), microbes were
speculated to exist under extremely hostile and nutrientdeficient conditions, even though cell division undersuch conditions may only occur once over thousands ofyears after entrapment in the subseafloor environments(Jørgensen and Boetius 2007). Further modeling analyses suggested that the availability of energy sources forthe microbial cells in subseafloor sediments was less thanthat was previously thought, considerably lower than theminimum requirements of any microbes that had been
cultured to date (Bradley et al. 2020; LaRowe and Amend
2015). However, the detection of microbial life and geochemical evidence of very low, but detectable, respiration
activity demonstrated that the subseafloor biosphere wasindeed capable of sustaining biological life, even though
the physiological and metabolic mechanisms employedby life under such extreme conditions were not yet fully
understood (LaRowe and Amend 2015). In addition,
many aspects related to the ecological physiology of
microbes, such as what proportions of these microbes
were alive, actively growing, just surviving, dormant, ordead fossils, were unknown. To address this fundamental question regarding the physiological status of subseafloor microbial life, single-cell-level probing of microbial
metabolic activity by nanoscale secondary ion mass spectrometry (NanoSIMS) was used to clarify whether carbon and nitrogen substrates were being assimilated by
microbes. The basic strategy for this approach is that the
microbes in the subseafloor biosphere samples are supplied with substrates in which the carbon and/or nitrogen atoms have been replaced with stable isotopes (e.g.,2
H, 13C, and 15N). If the modified substrates are metabolized and assimilated into the microbial cells, then therelative abundance of the stable isotope(s) increases andbecomes detectable by NanoSIMS analysis. Upon NanoSIMS analysis, a ­Cs+ primary ion beam is directed at the
samples, producing secondary ions that are separated bymass spectroscopy, detected by an electron multiplier (inthe case of NanoSIMS 50L, parallel acquisition of up toseven masses is possible), and then recorded as images ofthe selected ions. To detect stable isotope-labeled microbial cells, 12C+, 13C+, 12C14N+, and 12C15N+ ions are typically used (Morono et al. 2020, 2011), but 1H+ and 2H+

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can also be included (Trembath-Reichert et al. 2017).
The ability to trace stable isotope incorporation using
NanoSIMS has opened up a vast new field of researchin single-cell environmental microbiology (Kuypers andJøgensen 2007; Morono et al. 2014; Musat et al. 2012).
The application of NanoSIMS to the analysis of microbial ecophysiology was pioneered by McMahon et al.
(2006). Although they were not the first to demonstrate
analysis of microbes using a SIMS-based approach, theintroduction of NanoSIMS was a technical breakthroughin terms of the ability to observe stable isotope incorporation at a high spatial resolution (50 nm), a level ofresolution that is theoretically not possible using microscopic techniques. To date, more than 90 reports on theuse of NanoSIMS in biology and microbiology have been
published in high-profile journals, indicating that the
introduction of NanoSIMS has been a groundbreaking
development for bio-analysis and has led to numerous
novel findings in previously unexplored natural systems.
Despite the extensive application of NanoSIMS to theanalysis of metabolic activity in microbes, its application to microbes from the subseafloor biosphere has
been limited, primarily because of the analytical problems associated with the sediment matrix. During
NanoSIMS analysis, secondary ions generated by thebombardment of the primary ion are detected. By scanning with the primary ion, the produced secondary ions
are used to generate an image of the scanned target
area showing element distributions. The informationthat is obtained is the two-dimensional distribution ofeach element. Analyzing microbes in aquatic habitats
is relatively straightforward, as microbial cells constitute the major source of solids with elemental compositions that can be distinguished with ease. However,subseafloor sediments contain large quantities of noncellular particles, including particulate organic matter(POM), which contains carbon and sometimes nitrogen. In such cases, distinguishing between the organic
matter in microbial cells and POM in images can bedifficult using NanoSIMS. The sample area in NanoSIMS isotope imaging analysis typically encompasses
an area of 30 µm × 30 µm. If samples are prepared byconventional filtration (i.e., suspending the sample inbuffer and passing it through a membrane filter), thenumber of single cells per analysis area will be < 1 cell
if the cell abundance in the sediment is < ­107 cells per­cm3. Considering that the CCD view of microbial cells
in the NanoSIMS system is obscured by the presence oflarge quantities of sediment grains, the analytical efficiency goes low since targeting microbial cells rarely
found in low-abundance samples to capture in the fieldof analysis is almost impossible. To overcome these difficulties, identifying microbial cells after density cell

Morono P﻿ rogress in Earth and Planetary Science(2023) 10:18

separation became more widely used. As described in
the previous section, microbial cells and sediment particles can be recognized based on differences in theirfluorescence characteristics after staining with SYBR-I.
Further, a combination of density-based cell separationand selective sorting using the fluorescence-activatedcell sorting (FACS) approach enabled the purificationof microbial cells from subseafloor samples and concentrating them to a spot of ~ 200 µm on the membrane(Morono et al. 2013) (Fig. 5). Through these technicaldevelopments, substrate incorporation by subseafloor
microbes, regardless of their abundance in the sediments, could be analyzed by NanoSIMS. In NanoSIMS
analyses shown in Morono et al. (2011), in which no cell
separation was conducted on the sediment incubation
slurry of ~ ­107 cells/mL, there were just a few cells per
field of analysis. However, in another study, sorting andmicrobial substrate incorporation analysis successfullyincreased the number of microbes per field of analysis
even using a sample containing 4 × ­102 cells/gram ofsedimentary rock collected from subseafloor shale andcoal deposits sampled from as deep as 2 km below theseafloor (Trembath-Reichert et al. 2017). In the case ofultraoligotrophic subseafloor sediments, syringe-subsampled sediment mini-cores were incubated without
preparing a slurry and drops of concentrated substratesolution were placed onto the mini-cores for incubation. Although the cell concentration varied ­(102–106
cells/cm3 sediment), separation, purification, and concentration of cells were sufficient for NanoSIMS analysis (tens of cells per NanoSIMS analysis) (Morono
et al. 2020). The findings of that study revealed thePage 8 of 13

persistence of revivable aerobic microbial communities
in sediments as old as 101.5 million years (Fig. 6).
The use of the cell sorting approach is not only for
microbial cell separation from sediment particles, but
the wider application is possible. Cell sorter utilizes
laser irradiation, with a wavelength of typically 488 nm,
but a number of lasers with other wavelengths are available to obtain optical information of laser-scattering andfluorescence. Approaches that are used to tag microbial
cells, such as FISH and its derivatives (e.g., BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT)(Hatzenpichler et al. 2014) and BrdU (Steward and Azam
1999)(Pernthaler et al. 2002b)/EdU (Yamakoshi et al.
2011)), can be incorporated into sample preparation for
NanoSIMS analyses. However, it should be noted that themore processing the microbial cells have for tagging, themore exogenous carbon (and nitrogen and hydrogen) areintroduced into the microbes, which dilutes the naturally
abundant isotopes and alters the ratios of isotopes in the
microbes (Meyer et al. 2021). Although the isotope incorporation itself can be detected, as the increase in the ratioof 13C or 15N following stable isotope incubation, quantitative analyses of isotope incorporation and rate analysis
are compromised because of the uncertainty introducedby sample processing. Thus, minimizing the number ofprocessing steps is highly recommended.
2.4 Remaining challenges for deciphering microbial
dynamics in the subseafloor biosphere

The aforementioned technical developments have facilitated the exploration of areas close to the fringe of the
deep subseafloor biosphere (D’Hondt et al. 2015; FrühGreen et al. 2018; Heuer et al. 2019; Inagaki et al. 2015;

Fig. 5 Scheme of microbial cells purification for NanoSIMS analyses. Density-separated samples were applied to cell sorter after staining with SYBR-I(green). Then, the microbial cells (green) were specifically sorted onto polycarbonate membrane. The speed of sorting ranges few to hundreds ofcells per second depending on the cell abundance in the original samples. Modified from Morono et al. (2014)

Morono P﻿ rogress in Earth and Planetary Science(2023) 10:18

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Fig. 6 Detection of substrate incorporation into microbial cells revived in 101.5 Ma sediment. Cells from incubations with 13C-bicarbonate and15
N ammonium (a, b, c, d) and 13C,15N-Amino acid mix (e, f, g, h) are shown. a, e SYBR Green I-stained cells under fluorescence microscopy. b, c,
f, g Ratio images of 13C/12C (b, f) and 12C15N/12C14N (c, g) ratios of the same regions imaged in a and e, demonstrating locations of 13C and 15N
incorporation. Color scale range of the ratios are shown as number appearing at top and bottom of the color bar. The background membraneregion, which is identified by fluorescence images, is excluded from the ratio calculation and shown as black background. d, h Secondary electron(NanoSIMS) images of the same region in a and e. Bars represent 5 µm. Adopted from Morono et al. (2020)

Li et al. 2020). These areas are characterized by very low
cell abundance and a partially undetectable region has
been reported (Heuer et al. 2020). Studies have unveiled
the extreme resilience and survivability of microbes
in extreme environments, which were previously considered to be too hostile for life (e.g., areas with hightemperatures and/or low energy fluxes). Nonetheless,environments, where life cannot survive, are yet to bediscovered. “Defining the rules of microbial life” and“Exploring the limits of life” are among the most important future scientific goals of the global research community involved in scientific ocean drilling (Koppers and
Coggon 2020). However, how these goals can be achieved
is one of the remaining technical challenges in subseafloor research.
It has been said that the absence of evidence is not evidence of absence (Altman and Bland 1995; Sagan 1997).
In the case of deep biosphere research, the existence of
microbial life in subseafloor environments directly gives
direct evidence of the presence of life, but in cases wheresuch evidence is absent, researchers need to consider the
possibility that the results of absence are from methodological limitation being below detection limits. The sameis true for measurements of catabolic activity using radioisotopes as tracers; all carefully designed experiments
have controls, which are used to provide statistical evidence that observations are beyond noise. Life is affectedby a variety of environmental factors, including temperature, bioavailable energy flux, pressure, dissolved metal

concentrations, water availability, and pH. Although
identifying the limits of life cannot be ascertained withcertainty when examining a limited number of samples,a series of samples can help to identify the trend of environmental factors and biological signatures and provide
practical evidence of the limits of the biosphere. Given
that subseafloor environments are saturated with water,
they usually exhibit gradients of physical and chemical
conditions; for example, temperature gradually increases
with depth, and the compacted nature of subseafloor sediments restricts conductive flow and diffusion forms gradients of chemical species. The sampling of subseafloor
substrates along these gradients should make it possible
to identify the practical limits of the biosphere, althoughsome inhabitable “spots” may exist close to the interfacial region. At around the depths where life appears to
be absent, abiotic chemical reactions continue to takeplace. If the location of the drilling zone is appropriately
selected, then the critical zone, i.e., the transition fromhabitable to inhabitable conditions, could be identified.
In this regard, factors such as continuous sedimentation and plate movements which gradually change thephysical environment and make it less habitable overgeological time also need to be considered. The potential for chemical reactions involving leftover biochemicals occurring at different rates has been suggested (Orsiet al. 2020). For example, peptide hydrolysis, nucleic acidhydrolysis (RNA should decay faster than DNA), aminoacid racemization, sugar decay, and lipid hydrolysis,

Morono P﻿ rogress in Earth and Planetary Science(2023) 10:18

could potentially occur at different rates. Consequently,
analyses that combine the detection of cells and biomolecules would provide a finer and clearer indication of thelimits of the biosphere.
Despite the high sensitivity of microscopic methods for
detecting microbial cells—down to several cells per cubic
centimeter of sediment (Heuer et al. 2020)—the ability
to accurately detect biomolecules such as DNA and/or
RNA is partially left behind. Although there have been
a number of reports describing subseafloor microbialcommunity structure (e.g., Hoshino et al. 2020; Inagaki
et al. 2006; Orsi 2018), only two studies have successfully described the composition of very deep (> 1000
mbsf ) subseafloor microbial communities (Ciobanu et al.
2014; Inagaki et al. 2015). The reason for these difficulties
often involves DNA quantity and quality. In general, theapproach for investigating microbial community structure employs 16S rRNA gene sequences amplified fromextracted DNA pool by PCR amplification. There are
numerous steps where contamination can be introduced
from exogenous sources, such as during sample retrieval(shipboard sampling), subsampling at onshore laboratories, DNA extraction (from working environments andthe kits used), and PCR amplification (from the environment and previously used PCR enzymes). Even though
the working environments can be DNA-clean (Morono
et al. 2018), the extreme sensitivity of PCR amplification means that even the slightest contamination canadversely affect results. The problem is exacerbated whenusing samples containing small amounts of DNA, such as
the samples from deeper areas of the subseafloor environment where cell abundance is low. In order to resolve
the problem and obtain molecular signals from indigenous microbial communities, intensive sequencing ofvarious controls and probabilistic approaches are implemented to identify contaminating sequences (Hoshino
et al. 2020; Inagaki et al. 2015; Morono et al. 2020).
However, for omics approaches, such as metagenomics
or metatranscriptomics, the low recovery of DNA/RNA
is more problematic. Although there have been morethan several attempts to conduct metagenomics studies
using subseafloor samples, the samples are usually fromshallower areas of the subseafloor biosphere. Nonetheless, there is a need for further technical developmentsthat will facilitate investigations in deeper subseafloor
regions. Particularly, the quality of the biomolecules in
the retrieved samples should be a concern. Although
not reported in the literature, there is a general feeling among some microbiologists that DNA amplification is more challenging for subseafloor microbes than
it is for microbes in other habitats. Humic substances,which are abundant in subseafloor sediments, are generally considered to be the main cause of this problem, asPage 10 of 13

they interfere with, or inhibit, enzymatic DNA amplification. However, even after density separation or selective sorting of microbial cells, when there are few humic
substances, DNA amplification is often difficult. While
this has not yet been empirically demonstrated, thisbelief seems to be shared among researchers in the field.
Given the energy-limited conditions in subseafloor environments, microbes might not be able to meet the energetic requirements that are necessary to keep their DNA
intact, causing problems associated with damaged DNA
upon amplification.

3 Conclusions
Technological developments have driven and accelerated
the biological exploration of the subseafloor biosphere.
To date, a variety of subseafloor environments, such as
ultradeep (~ 2 km below the seafloor (Trembath-Reichert
et al. 2017)), extremely oligotrophic and old (~ 1 million
years (Morono et al. 2020)), and hot (~ 120 °C (Heueret al. 2020)) regions, have been investigated for microbial
survival and living status. The findings of these studies
have revealed that heterotrophic metabolism is dominant, regardless of the degree of availability of organic
compounds in the surrounding environment. In addition
to the biosphere regions mentioned above, there is also
the rock biosphere. Despite the fact that relatively few
studies have been conducted on the microbial abundanceof this part of the biosphere (Früh-Green et al. 2018; Liet al. 2020; Suzuki et al. 2020; Wee et al. 2021) and on
microbial activity in crustal fluids (Trembath-Reichert
et al. 2021), further exploration will likely present investigators with yet additional challenges in future.
The unique environmental setting of the subseafloor
biosphere provides critical information on the factorsthat constrain the extent of biosphere, the conditions
that limit survival, and the evolution and adaptability oflife. However, the limited amounts of biomass in samples
necessitate the ability to sensitively detect biosignatureswith appropriate techniques while minimizing contamination. In order to develop optimal techniques, it is ofutmost importance to know the limitations and characteristics of the techniques employed. Based on an indepth knowledge of the underlying principles, limiting
factor(s), and sources of noise and interference, investigators can apply the most appropriate techniques to theanalysis of particular samples and obtain reliable data asa result.
The accumulation of knowledge and development of
optimal procedures to detect, characterize, and understand life below the seafloor will directly benefit the
search for extraterrestrial life, where similar barriers
to discovery exist. To overcome these challenges, theexchange of information, especially on “failures” andMorono P﻿ rogress in Earth and Planetary Science(2023) 10:18

negative results, is essential as failure often contains anabundance of important information.
Abbreviations
ODP	Ocean Drilling Program
AODC	Acridine orange direct counting
AO	Acridine orange
POM	Particulate organic matter
FISH	Fluorescence in situ hybridization
Acknowledgements
The author thanks Dr. Tatsuhiko Hoshino for sharing his experience of extracting DNA from deep biosphere samples.
Author contributions
The author read and approved the final manuscript.
Funding
This work was supported by Grants-in-Aid from JSPS KAKENHI (Grant Numbers
19H00730, 20K20952, and 22K18426).
Availability of data and materials
Not applicable.

Declarations
Competing interests
The author has no competing interests to declare.
Received: 19 December 2022 Accepted: 22 March 2023

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