Isaji et al. Progress in Earth and Planetary Science
https://doi.org/10.1186/s40645-019-0306-x(2019) 6:60

Progress in Earth andPlanetary Science

RESEARCH ARTICLE

Open Access

Biomarker records and mineralcompositions of the Messinian halite and
K–Mg salts from Sicily
Yuta Isaji1* , Toshihiro Yoshimura1, Junichiro Kuroda2, Yusuke Tamenori3, Francisco J. Jiménez-Espejo1,4,
Stefano Lugli5, Vinicio Manzi6, Marco Roveri6, Hodaka Kawahata2 and Naohiko Ohkouchi1

Abstract
The evaporites of the Realmonte salt mine (Sicily, Italy) are important archives recording the most extreme conditions of
the Messinian Salinity Crisis (MSC). However, geochemical approach on these evaporitic sequences is scarce and little is
known on the response of the biological community to drastically elevating salinity. In the present work, we investigatedthe depositional environments and the biological community of the shale–anhydrite–halite triplets and the K–Mg salt
layer deposited during the peak of the MSC. Both hopanes and steranes are detected in the shale–anhydrite–halite
triplets, suggesting the presence of eukaryotes and bacteria throughout their deposition. The K–Mg salt layer iscomposed of primary halites, diagenetic leonite, and primary and/or secondary kainite, which are interpreted to haveprecipitated from density-stratified water column with the halite-precipitating brine at the surface and the brineprecipitating K–Mg salts at the bottom. The presence of hopanes and a trace amount of steranes implicates thateukaryotes and bacteria were able to survive in the surface halite-precipitating brine even during the most extreme
condition of the MSC.
Keywords: Messinian Salinity Crisis, Evaporites, Kainite, μ-XRF, Biomarker

Introduction
The Messinian Salinity Crisis (MSC) is one of the most
massive evaporation events in Earth’s history (Hsü et al.
1973; Rouchy and Caruso 2006; Ryan 2009; Roveri et al.
2014; and references therein). More than 1 million km3
of salts precipitated over the Mediterranean basin between 5.97 and 5.33 Ma (Krijgsman et al. 1999), primarily driven by tectonically induced restriction of theMediterranean–Atlantic gateway and gradual isolation of
the Mediterranean Sea (Duggen et al. 2003). The MSC is
divided into three stages, which correspond to the
gypsum (CaSO4·2H2O)–halite (NaCl)–gypsum trilogy
observed mainly in the Eastern Mediterranean basin(Fig. 1a; Roveri et al. 2014), among which the halite
phase accompanying occasional intercalation of highly
soluble K–Mg salt layers represents the most extreme* Correspondence: isajiy@jamstec.go.jp
1
Biogeochemistry Program, Japan Agency for Marine-Earth Science andTechnology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan
Full list of author information is available at the end of the article

hypersaline condition between 5.60 and 5.55 Ma (Manziet al. 2009).
Previous studies have explored the relationships
among the climate, the hydrological conditions, and theprecipitating evaporites during the MSC peak, based on
the distributions of mineral phases and elements in theevaporites (Garcia-Veigas et al. 1995; Lugli et al. 1999;
Yoshimura et al. 2016), rhythmical alternation of theevaporitic sequences (Manzi et al. 2012), chemistry of
the fluid inclusions in halite crystals (Rigaudier et al.
2011), and Os isotopic records (Kuroda et al. 2016). By
contrast, much less is known on the responses of thebiological communities, partly because evaporites generally do not contain microfossils commonly used in biogeochemical studies (Bertini et al. 1998). Based on thenitrogen isotopic compositions of geoporphyrins, which
are derivatives of chloropigments, it has been suggested
that the nitrogen cycle oscillated dynamically during the
MSC peak, between a phase of active ammonium uptake
by phototrophs and that dominated by evaporative degassing of ammonium under suppressed biological© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.

Isaji et al. Progress in Earth and Planetary Science(2019) 6:60

aPage 2 of 15bFig. 1 a Map of the Mediterranean Sea indicating the distribution of the Messinian evaporites (after Ryan 2009; Manzi et al. 2012). The location ofRealmonte salt mine investigated in this study is also indicated. b Stratigraphic column of the Realmonte salt deposits (modified after Lugli etal. 1999)

activity (Isaji et al. 2019a). Despite such a clear indication of substantial fluctuations in the biological activity,
insights into the compositions of microbial communities
are lacking. Such investigation is necessary in constraining the habitable limit of life during the massive evaporation events. Moreover, the transitions in the state andcompositions of the microbial communities may in turnstrongly affect the chemical characteristic of the evaporating brine. For example, biological activity strongly influences the CO2 exchange between the brine and theatmosphere (Lazar et al. 1992; Isaji et al. 2017), and organic matter concentrated in the brine reduces the evaporation rate and the degree of evaporation that the brine
can reach (Shalev et al. 2018).
While the massive halites deposited during the MSC
peak are widely distributed in the deep Mediterraneanbasins (Fig. 1a; Lofi et al. 2011), the accessible archive islimited, one of which is the on-shore salt deposits of theRealmonte salt mine in the Caltanissetta Basin (Sicily,
Italy). Here, we present and discuss the results of microscale elemental mapping and biomarker (i.e., source-specific organic molecules) distributions of the K–Mg salt
layers and the shale–anhydrite–halite cyclical deposits.
The K–Mg salt layers are among the most soluble evaporite found in the Realmonte salt mine, recording one of
the most extreme evaporitic conditions during the MSC
peak. The shale–anhydrite–halite triplets are the mostrepresentative cyclical deposits of the Realmonte saltmine (Decima and Wezel 1973; Lugli et al. 1999). Because the mineralogy of the evaporites is dependent on
the chemical composition of the brine, the distribution
patterns of the evaporite minerals provide fundamental
information on the hypersaline environments. Amongthe biomarkers preserved in the evaporites, we specifically focused on lipid biomarkers, including long-chain(C25–C36) n-alkanes produced mainly by terrestrial

higher plants, insects, and fungi (Oro et al. 1966; Eglinton and Hamilton 1967), steroids constituting a major
component in the cell membrane of eukaryotes (Volkman 2005; Summons et al. 2006), and hopanoids abundantly produced by bacteria as a cell membranecomponent (Rohmer et al. 1984; Ourisson and Albrecht
1992). This study will better constrain the biological andhydrological conditions during the MCS peak in the Caltanissetta Basin, which will be an important comparative
data for understanding the environmental conditions in
the entire Mediterranean basin during the MSC.

Geological setting
The Realmonte salt mine cuts through evaporites of theCaltanissetta Basin located in the southwest of Sicily(Fig. 1a), deposited during the peak of the MSC between
5.60 and 5.55 Ma (Manzi et al. 2009; Roveri et al. 2014).
It belongs to the Gessoso-Solfifera Formation and lies
above a gypsiferous/anhydritic unit considered to have deposited during stage 1 of the MSC (5.97–5.60 Ma) and isoverlain by a marly succession that contains up to six gypsum beds (the Pasquasia Gypsum Member) (Decima andWezel 1973; Garcia-Veigas et al. 1995; Lugli et al. 1999).
The salts in the Realmonte salt mine are divided into
four main lithological units (A–D) from the base to the
top (Fig. 1b; Decima and Wezel 1973; Lugli et al. 1999).
Unit A is composed of plate cumulates of halite precipitated in a relatively deep-water setting. The overlying
Unit B is composed of plate cumulates of halite in ashallowing upward sequence containing as many as 12
layers of K–Mg salts near the top of the unit. Theselayers are mainly composed of fine- to coarse-grained
kainite (MgSO4·KCl·3H2O) that are cumulitic in origin(Garcia-Veigas et al. 1995). They have undergone deformation by tectonics with slump-like folding during or
after the deposition, which acted differently on the saltsIsaji et al. Progress in Earth and Planetary Science(2019) 6:60

having specific rheological behavior compared to thesurrounding rocks (Lugli et al. 1999). The halite layers inthis unit show no evidence of bottom overgrowth,current structure, or dissolution and/or truncation surfaces, indicating that the halite precipitation occurred ina relatively deep-water setting below the wave base
(Lugli et al. 1999). Upward shallowing of the basin occurred only in the uppermost halite layers of unit B,
which is evidenced by large halite rafts together withlocalized dissolution pits filled by mud. The contacts
between Unit B and C are characterized by the localized dissolution pits filled with mud, features that suggest desiccation of the basin and the formation of large
expansion–contraction salt polygons (Lugli et al. 1999).
Unit C, deposited after the basin had undergone desiccation, is composed of more than 120 lithological cycles, each one represented by the superposition of threemain facies: (1) a millimeter-thick shale layer, (2) a millimeter–centimeter-thick anhydrite (CaSO4) or polyhalite(K2MgCa2(SO4)4·2H2O) layer, and (3) a decimeter-thickhalite layer with white patchy polyhalite grains (Lugli et al.
1999; Yoshimura et al. 2016). The highly pure halite cumulates of skeletal hoppers with chevron overgrowths indicate that the precipitation occurred in a relatively
shallow, non-stratified water body during the dry seasonof the year, whereas the shale layers are considered to havedeposited under a stratified water body during the wet
season (Lugli et al. 1999; Manzi et al. 2012). Unit D is discontinuous and comprises halite and minor anhydrite, but
it is not crossed by the mine and could not be describedin detail.

Materials and methods
Sample collection

Rock samples of the Unit C shale–anhydrite–halite triplets
were collected with a chisel and hammer from the 37th to
38th cycles above the erosional surface between Unit B and
Unit C at “Church section” of the Realmonte salt mine
(Manzi et al. 2012; Fig. 2h, i), which is located below 100 mfrom the ground level and over 1 km in horizontal distancefrom the exit of the mine. We also collected two fallen rocksamples of K–Mg salts near the “Church section” where
the outcrop of K–Mg salt layer in the upper part of Unit B
is exposed (Fallen rock A and B; Table 1). The yellowish
mineral surrounding the fallen rocks were similar in appearance to the K–Mg salt layer in the outcrop, indicatingthat these rocks originated from this layer. This layer was
highly deformed, and the sedimentological features werelost (Fig. 2a–d).

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cycle 37th and 38th halite layers (~ 70 g each), andshale–anhydrite layers in between in Unit C (~ 10 g)
were analyzed for hydrocarbon biomarkers. The outerparts of the rock samples were carefully eliminated witha chisel and hammer. The remaining inner parts were
washed in Milli-Q water, methanol, and dichloromethane for 1 min each with sonication to eliminate any possible contaminants on the surface of the rock.
Each pulverized sample was extracted three times bysonication in dichloromethane:methanol (7:3, v/v) for
15 min. The total lipid extracts were saponified with 0.5
M KOH in methanol:H2O (95:5, v/v) at 80 °C for 2 h.
This was extracted then with n-hexane three times toobtain neutral lipids. Free hydrocarbon fraction (i.e., organic compounds not bound to macromolecules) was
obtained by silica gel column chromatography using nhexane:dichloromethane (95:5, v/v) as the solvent. The
remaining saponified fraction was reacted with nickel
boride to release S-bound compounds following themethodology of Schouten et al. (1993). First, the dried
fraction was eluted in 4 mL methanol:tetrahydrofuran (1:1, v/v). A total of 200 mg NiCl2 were added and stirredwith a magnetic stirrer. Then, 100 mg NaBH4 was added
and reacted for 1 h at room temperature. This was extracted in n-hexane:NaCl-saturated-H2O (1:1, v/v) todissolve the released organic compounds in the n-hexane fraction. Sulfur-bound hydrocarbon fraction (desulfurized N-1) was obtained by silica gel column
chromatography using n-hexane:dichloromethane (95:5,v/v) as the solvent. Both free and desulfurized N-1 fractions were eluted in n-hexane prior to the injection togas chromatography/mass spectroscopy (GC/MS).
The GC/MS analysis was carried out using an Agilent
Technologies (Santa Clara, CA, USA) model 7890A GC
connected to a 5975C mass–selective detector. We useda VF-5ms fused silica capillary column (30 m × 0.25 mm
i.d.; film thickness 0.1 μm) and helium as the carrier gas.
The column oven temperature was programmed as follows: hold at 40 °C for 2 min, raise to 120 °C at 30 °C
min−1, then to 320 °C at 6 °C min−1, and hold at 320 °C
for 20 min. The PTV injector temperature was programmed as follows: hold at 50 °C for 0.3 min, raise to350 °C at 600 °C min−1, and hold at 350 °C for 10 min.
Also, selected ion monitoring (SIM) of m/z = 217 (steranes) was performed for free and desulfurized N-1 fractions of the Fallen rock B sample. Identification of eachcompound was based on its retention time and mass
spectrum. Blank experiments performed concurrently
with the sample analysis showed no contamination thatwould affect the results.

Analytical procedures
Hydrocarbon biomarker analysis

μ-XRF, XANES, and XRD analyses

Two fallen rock samples from K–Mg salt layer in Unit B(~100 g of Fallen rock A and ~ 70 g of Fallen rock B),

The microscale elemental mapping of geological material
is a commonly employed approach for identifying theIsaji et al. Progress in Earth and Planetary Science(2019) 6:60

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Fig. 2 a The outcrop of the Unit B salt deposits containing the yellowish K–Mg salt layer above the halite–mud laminae near “Church section”(Manzi et al. 2012). b A close-up image of the highly deformed structure found in the K–Mg salt layer. c, d The fallen rock samples A and B
analyzed in this study originated from the yellowish K–Mg salt layer. e–g The polished slabs of the collected samples (K-1 to -3). K-1 and -2 wereprepared from Fallen rock A and K-3 from Fallen rock B. The μ-XRF measurements were performed at the areas indicated in red rectangles. The
blue rectangle represents the area where thin section picture is shown in Additional file 1: Figure S4. Arrows indicate the orientation of the slabs.
h The outcrop of Unit B and Unit C in “Church section”. Unit C consists of shale–anhydrite–halite cycles deposited as a result of the dry–wet
alternation in the climate. i A sample taken from cycles 37th to 38th for the biomarker analysis

depositional environments (e.g., Yoshimura et al. 2016).
The high-sensitivity scanning X-ray fluorescence (XRF)
that utilizes synchrotron radiation sources with X-ray focusing optics (i.e., μ-XRF) has the advantage of generating trace element distributions with a micrometer-scalespatial resolution (e.g., Sutton et al. 2002; Tamenori etal. 2014). In addition, the use of energy-tunable synchrotron radiation allows chemical speciation by X-ray absorption near edge structure (XANES) (e.g., Spiro et al.
1984; Yoshimura et al. 2015), which can be used to constrain the phases involved in compositionally complex
materials like geologic materials.
Three 5 mm-thick slabs of the K–Mg salts were prepared from two fallen rock samples with a saw for μXRF measurement (herein referred to as K-1, K-2, andK-3; K-1 and K-2 prepared from Fallen rock A and K-3
from Fallen rock B; Fig. 2c–g). The cut surfaces were
polished and then cleaned in an ultrasonic bath withhexane. After the X-ray analysis, thin sections of K-2
were prepared to describe the sedimentary structures.
The sample was mounted on a glass slide and groundsmooth.
All X-ray analysis was conducted at the b-branch of thesoft X-ray photochemistry beamline (BL27SU) at theSPring-8 synchrotron radiation facility (Hyogo, Japan),using monochromatized soft X-ray beam with a silicon
drift detector (SDD) (Tamenori et al. 2011; Tamenori etal. 2014). The radiation from the undulator was monochromatized by using a double crystal Si(111) monochromator ensuring an energy resolution of 350 meV.
Photon flux on the sample was 1 × 1011 Ph s−1 at sulfur
K-edge. The Kirkpatrick–Baez mirror focused the photon
beam on the sample, and the horizontal and vertical beam
size at focus point were 16.3 and 13.7 μm, respectively.
For μ-XRF measurements, the polished slab was fixedon an aluminum sample holder that was then installed
in a vacuum chamber and fixed on a motorized XYZstage. The μ-XRF measurements were taken at a photon
energy of 3000.0 eV with an acquisition time of 1 s at
100 μm intervals for both the horizontal and verticalaxes. All XRF data were obtained at each mapping point,
and the elemental imaging data were extracted from the
XRF dataset. The reproducibility for signal intensities
based on repeated analysis on the standard material was
typically less than 5% (± 2σ) over ten replicates.
Sulfur K-edge μ-XANES measurements were performed using a two-dimensional approach for the partial
fluorescence yield (PFY) measurement with an SDD in

Table 1 Samples from Realmonte salt mine analyzed in this study
Location

Deposits

Sample position

Subsamples

AnalysesUnit B near “Church section”

K–Mg salts

Fallen rock A

K-1, K2

XAFS, XRD, biomarker, thin section

Fallen rock B

K-3

Unit C “Church section”

Halite-anhydrite-mud

Cycle 37th–38th

37th halite
37/38th shale-anhydrite
38th halite

Biomarker

Isaji et al. Progress in Earth and Planetary Science(2019) 6:60

the soft X-ray region (Tamenori et al. 2011). The sample
and detector layout for S-XANES was the same as that
employed for the μ-XRF measurements. The selectedenergy range for S-XANES measurements was 2460–
2510 eV with an energy step of 0.2 eV, and an acquisition time of 1 s step−1. Five spots were analyzed for K-1(spots 1-1–1-5) and four spots each for K-2 and -3(spots 2-1–2-4 and 3-1–3-4).
The evaporite minerals composing the samples were further confirmed by X-ray diffraction (XRD) analysis using
PANanalytical X’Pert Pro X-ray diffractometer (PANalytical
B.V., Almelo, the Netherlands). Single crystals of each evaporite mineral, which were determined by μ-XRF and
XANES, were collected from K-2 with a dental drill, andpowdered using agate mortar prior to the analysis.

Results and discussion
Sedimentary facies and spatial distribution of elements ofUnit B K–Mg salt layer

While the outer parts of the fallen rock samples and thesurface of the outcrop of the K–Mg salt layers in Unit B
were both covered by yellowish mineral ~ 1 cm-thick, thecut surface of the K–Mg salt slabs showed transparent
and whitish crystals in the inner part (Fig. 2c–g). Elemental mapping by μ-XRF revealed a similar distribution pattern of elements for all slabs (Fig. 3, Additional file 1:
Figures S1 and S2). Compared to the main peaks of thesulfur K-edge XANES spectra of the reference compounds
shifting toward higher energies with increasing valent state(Fig. 4), all spectra of the K–Mg salts exhibited a pronounced peak at around 2482 eV, indicating that sulfur is
predominantly in a fully oxidized state (S6+). The evaporite
minerals comprising the slabs are identified as follows:
1. The major elements composing the most abundant
mineral of the inner part of the slabs were Mg, O,
S, and Cl (Fig. 3, Additional file 1: Figures S1 andS2). The S μ-XANES spectra obtained from themeasurement points 1-1, 1-2, 2-2, and 3-4 exhibiteda pronounced peak appearing at 2482 eV and someresonances at around 2486.5, 2491, 2494.5, and2499 eV in the post-edge regions (Fig. 4), which is
identical with the spectrum of kainite reported
previously (Yoshimura et al. 2016). The XRD
analysis also confirms this mineral as kainite(Additional file 1: Fig. S3).
2. Minerals composed of Na and Cl were also
distributed in the inner part of the K–Mg salt rocks(Fig. 3, Additional file 1: Figure S1 and S2), which
was identified as halite (Additional file 1: Figure S3).
Although any primary sedimentary structures withdetrital textures and fabrics were not recognized in
the thin section of K-2, the halite crystals contained
fluid-inclusion-rich bands alternating with fluid-

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inclusion-poor bands (Additional file 1: Figure S4),
a primary subaqueous texture indicating that they
are of primary origin precipitated at the brine
surface as the halite rafts (Lowenstein and Hardie
1985; Garcia-Veigas et al. 1995; Lugli et al. 1999).
The cross plots of Al, Si, and P indicate that thesignal intensities of these elements are higher andvaries within a narrow range in the halite phasecompared to other mineral phases (Fig. 5). The
high correlations between these elements imply thatthese signals derive from aluminosilicate clay
minerals, with P present as adsorbed inorganic andorganic P on the surface of the clay minerals and/or
as aluminum oxides (e.g., Shen et al. 2011). The S
μ-XANES spectra obtained at the measurement
spots 2-1 and 3-1 unambiguously indicate the coexistence of sulfate compounds in the halite crystals(Fig. 4). However, a low signal-to-noise ratio inthe μ-XANES analyses did not permit us to findconclusive evidence concerning the average local
structural and compositional environment of
sulfate in halite.
3. The minerals surrounding the outer part of the slab
which are found as vertically oriented needle
crystals mostly having euhedral to subhedral shapes(Additional file 1: Figure S4) contained O, S, andMg (Fig. 3, Additional file 1: Figures S1 and S2).
The XRD analysis identified this mineral as leonite(K2Mg(SO4)2·4H2O) (Additional file 1: Figure S3).
The S-XANES spectra of leonite at themeasurement spots 1-4, 1-5, 2-3, 2-4, 3-2, and 3-3
exhibited a pronounced peak in the post-edge
region appearing at 2494 and 2499 eV, andresonance on the high-energy side of highest peaks
appeared at 2485.5 and 2488.5 eV (Fig. 4).
Thus, kainite, halite, and leonite are the main minerals
composing the K–Mg salt slabs, among which halite isof primary origin. Both kainite and leonite can be precipitated from the seawater as primary mineral orformed as secondary mineral by dissolution–reprecipitation of the bittern salts (Stewart 1963; Bąbel and Schreiber 2014). The distribution of leonite surrounding theouter rims of our rock samples suggests them as the secondary diagenetic mineral. On the other hand, although
the kainite grains of the Unit B K–Mg salt layers showpressure–dissolution feature (Garcia-Veigas et al. 1995;
Lugli et al. 1999) indicating that at least the rims of thekainite crystals have undergone post-burial alteration,
the observations in this study could not provide conclusive evidence on their origin. Thus, kainite may be ofprimary origin precipitated from the brine, or they may
have been recrystallized from other K–Mg salt mineralssuch as sylvite (KCl), carnallite (KCl·MgCl2·6H2O), orIsaji et al. Progress in Earth and Planetary Science(2019) 6:60

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Fig. 3 The μ-XRF intensity maps of O, S, Na, Cl, Mg, Al, P, and Si, together with the picture of the measured area in K-2. Color scales indicate
element concentrations in signal counts per second. Black crosses in the μ-XRF intensity map of S denote μ-XANES measurement points

kieserite (MgSO4·H2O) (Stewart 1963). Either primary or
secondary in origin, their presence at least indicates thatthe salinity of the brine in the Caltanissetta basin
exceeded the saturation point of the K–Mg salts during
the MSC peak, which is over 70 times concentration of
the seawater (Warren 2016; Shalev et al. 2018).
The biomarker assemblages and their origins
Unit C shale–anhydrite–halite triplets

The distribution of organic compounds in the halite
phases of Unit C cycles 37th and 38th were similar, thus

only the chromatograms of the cycle 37th halite layer
are indicated (Fig. 6 and Table 2). All samples analyzed
contained abundant S-bound organic compounds, suggesting the presence of reduced sulfur species (e.g., sulfide, elemental sulfur, polysulfides) in the water column
and/or porewater generally supplied by sulfate reduction
driven under anoxic condition (Werne et al. 2004).
Gammacerane, derived from tetrahymanol produced bybacterivorous anaerobic ciliates (Sinninghe Damsté et al.
1995a; Takishita et al. 2012), was indeed detected fromthe shale–anhydrite layer of the Unit C (Fig. 6c; peakIsaji et al. Progress in Earth and Planetary Science(2019) 6:60

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Fig. 4 a–c Sulfur K-edge XANES spectra of the K–Mg salt slabs. Micro-XANES measurements were performed at the focused mode at eachmeasurement point. Black crosses in the μ-XRF intensity map of S denote μ-XANES measurement points. The spectra for reference materials (bluelines) are from Yoshimura et al. (2013)12), which suggests the formation of bottom water anoxia as a result of density stratification between brines
with different salinity (Manzi et al. 2012). The lack of
gammacerane in the Unit C halite layers and the Unit B
K–Mg salt layer do not necessarily indicate the absenceof anoxic bottom water, because the salinity may havebeen too high for bacterivorous anaerobic ciliates to inhabit. Considering the upper salinity limit of the sulfatereducing bacteria in culture (240 g L−1: Oren 2002), thereduced sulfur species used to form S-bound organic
compounds in the halite and K–Mg salts may have beenoriginally produced when freshwater–brine stratification
was formed.
While n-alkanes were not detected in free N-1 fractionof the Unit C halite layer, the desulfurized N-1 fraction
contained C18–C29 n-alkanes characterized by a strong
even carbon-number predominance for C18–C22 (Fig. 6a,b). Because a functional group or unsaturated sites arenecessary for an organic compound to become S-bound,these C18–C22 n-alkanes are derived probably from alkyl
lipids such as fatty alcohols and fatty acids of marine organisms (Naraoka and Ishiwatari 2000; Schouten et al.

Isaji et al. Progress in Earth and Planetary Science(2019) 6:60

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Fig. 5 The cross plots of the fluorescence signals of Al, Si, and P (in counts per second) of K-1, K-2, and K-3. Halite (red circles), kainite (graycircles), and leonite (yellow circles) phases were separated based on the signal intensity of Cl

2001; Bergé and Barnathan 2005). By contrast, a strong
odd carbon-number predominated long-chain (i.e., C25–
C36) n-alkanes in the free N-1 fraction of the shale–anhydrite layer of the Unit C (Fig. 6a) are derived from terrestrial organisms such as higher plants, insects, andfungi (Oro et al. 1966; Eglinton and Hamilton 1967),
which are likely to have been transported together withthe continental water inflow supplied during the deposition of shale layers (Manzi et al. 2012).
Hopanes and steranes preserved in the deposits are potentially informative biomarkers for paleoenvironmental
studies. Hopanes are degradation products of hopanoids,
which are pentacyclic triterpenes abundantly produced bybacteria as a cell membrane component (Rohmer et al.
1984; Ourisson and Albrecht 1992). Steranes, on the other
hand, are molecular indicators of eukaryotes, as their biosynthetic process requiring molecular oxygen is found almost exclusively in eukaryotes (Volkman 2005; Summons
et al. 2006). Because of the ubiquity of eukaryotes and bacteria in normal marine environments, hopanes and steranes are preserved in almost any geological materials.
However, their presence or absence under extreme environments could provide valuable insights, although it

should be kept in mind that such discussions are based on
the assumptions that the degradation rate and sulfur-binding efficiency of the steranes and hopanes are comparable.
The chromatograms of the fragment ion m/z = 191
showed that both free and desulfurized N-1 fractions of
the Unit C halite and shale–anhydrite layers contained17α(H)- and 18α(H)-isomers of trisnorhopanes, 17α(H),21β(H) isomers of norhopane and hopane, C31 to C33
17α(H),21β(H)-homohopanes with both 22S and 22R
configurations (22S:22R = 3:2), and 17β(H),21β(H) isomers of C34 and C35 homohopanes (Fig. 6c). The chromatograms of the fragment ion m/z = 217 indicated thepresence of C27–C29 sterane homologs with both 5α(H)and 5β(H) isomers in the desulfurized N-1 fractions of
the halite and shale–anhydrite layers of Unit C (Fig. 6d).
Hopanes and steranes have similarly been reported fromother MSC deposits, such as carbonate deposits and organic-rich marls (e.g., Sinninghe Damsté et al. 1995b;
Guido et al. 2007; Birgel et al. 2014; Natalicchio et al.
2017), and gypsums deposited during stage 1 of the
MSC (ten Haven et al. 1985; Schouten et al. 2001; Isaji2018). Our study reports here for the first time the presence of steranes in evaporites precipitated above theIsaji et al. Progress in Earth and Planetary Science(2019) 6:60

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a

c

b

d

Fig. 6 a m/z = 99 mass chromatograms of the free N-1 fraction of the cycle 37 halite phase and the shale layer between cycle 37th and 38th. Nalkane peaks are labeled with black circles. b m/z = 99 mass chromatograms of the desulfurized N-1. Peaks identified are labeled with numbers
and listed in Table 2. c m/z = 191 mass chromatograms of the desulfurized N-1 fraction. d m/z = 217 mass chromatograms of the desulfurized
N-1 fraction

saturation point of halite (concentrated over 10 times ofthe seawater).
Several criteria must be met before hopanes and steranes can be used as biomarkers of organisms inhabiting
the water column. Due to their wide occurrence in thecontemporary natural environment, hopanes andsteranes could be “contaminants” added after the deposition of evaporites. Thus, the criteria for their use as thebiomarkers of eukaryotes and bacteria is whether theseorganic compounds are syngenetic with the associateddeposits (Hayes et al. 1983). In the face of such a problem, the stereochemistry of hopanes can be used toTable 2 Hydrocarbon compounds identified in this study
Peak

CompoundPeak

Compound118α(H)-trisnorhopane*

11

Phytane217α(H)-trisnorneohopane*

12

Gammacerane317α(H), 21β(H)-norhopane

13

C34-17β(H), 21β(H)-homohopane

417α(H), 21β(H)-hopane

14

C35-17β(H), 21β(H)-homohopane

5

C31-17α(H), 21β(H)-homohopane (22S)15

C27-5β(H)-sterane6

C31-17α(H), 21β(H)-homohopane (22R)

16

C27-5α(H)-sterane7

C32-17α(H), 21β(H)-homohopane (22S)

17

C28-5β(H)-sterane8

C32-17α(H), 21β(H)-homohopane (22R)

18

C28-5α(H)-sterane9

C33-17α(H), 21β(H)-homohopane (22S)

19

C29-5β(H)-sterane10

C33-17α(H), 21β(H)-homohopane (22R)20

C29-5α(H)-sterane*Tentative assignmentsIsaji et al. Progress in Earth and Planetary Science(2019) 6:60

distinguish the hopanoids of living organisms and theirfossil counterparts. The biological stereochemistry of
hopanoids (22R-17β(H),21β(H) isomer) are progressively
degraded and stereochemically altered upon burial, leading to the dominance of 17α(H),21β(H) configuration
with 22S/22R ratio ~ 3:2 at advanced levels of maturity(Ourisson and Albrecht 1992). Thus, the stereochemicalpatterns of C29 to C33 hopanes in all of our samples
clearly eliminate their origin from the recent bacterial
source. The presence of biological isomers of hopanes(C34 and C35 17β(H),21β(H)-homohopanes) in the halite
layers of Unit C is enigmatic. Although speculative, they
may originate from bacteria trapped in the fluid inclusions of the halite crystals, which are reported to survive
on a geological timescale (McGenity et al. 2000; Lowenstein et al. 2011).
Another point that should be evaluated is whether
these biomarkers are of terrestrial or aquatic origin. The
contribution of terrestrially-derived steranes can be distinguished by the relative abundance of C29 steranes to
C27 counterparts, because β-sitosterol, which degrades
to C29 sterane, is abundantly produced by various terrestrial higher plants (Huang and Meinschein 1979). Thus,
the higher abundance of C27 steranes relative to C29
counterparts in the Unit C shale–anhydrite layer (C27/
C29 ratio ~ 1.1) implies aquatic eukaryotes as their main
biological source (Fig. 6d), which is consistent with thecontinental water inflow during its deposition (Manzi etal. 2012) because the lowering of salinity is likely to haverelieved the salinity stress of eukaryotes which are lesstolerable to a hypersaline condition compared to bacteria and archaea. The C27/C29 sterane ratio was lower,~ 0.5, for the Unit C halite layers. However, the abundance of terrestrial long-chain n-alkanes in these layers
were below detection limit, which argues against terrestrial higher plants as the major origin of steranes. A possible source of steranes in the halite layers is the green
alga Dunaliella salina, which is the only eukaryotic algaein the modern solar saltern ponds tolerable to salinity
above the saturation point of NaCl (Oren 2002). Interestingly, D. salina grown at 0.6, 1.4, and 2.1 M predominantly produce C29 sterols (C27/C29 ratio ~ 0.01;
Francavilla et al. 2010), while C27 sterols are mainly synthesized by that grown at ~ 4 M NaCl (Kelly 2009). Although speculative, the relatively low C27/C29 steraneratio may reflect the predominance of D. salina among
eukaryotic microorganisms during the halite deposition.
The relative peak intensities of hopanes and steranes
were comparable for the shale–anhydrite layer (Fig. 6c,d). Inhabitance of eukaryotes during its deposition isreasonable considering the presence of less-saline layer
in the surface of the water column. Clear peaks ofhopanes and steranes are similarly detected from MSC
gypsums (ten Haven et al. 1985; Schouten et al. 2001;

Page 10 of 15

Isaji 2018). By contrast, the relative peak intensities ofsteranes with respect to hopanes were substantially reduced in the Unit C halite layers. This implies that the
inhabitance of eukaryotes was gradually suppressed as
the salinity increased from the saturation point of gypsum to that of the halite.
Unit B K–Mg salt layer

Among the biomarkers detected from the K–Mg salts,
n-alkanes showed clearly different distribution patterns
between the two fallen rock samples (Figs. 7, 8 andTable 2). While only C18–C27 n-alkanes were detectedfrom the Fallen rock B, an odd carbon-number predominated long-chain n-alkanes were present in the free N-1
fraction of the Fallen rock A, implying terrigenous organic matter input. On the other hand, the distributionsof hopanes in free and desulfurized fractions of the tworock samples were similar to the Unit C samples, except
for the absence of C34 and C35 17(H)β,21β(H)-homohopanes. Both fallen rocks also contained trace amount of
C27–C29 steranes, although those in Fallen rock B were
only detectable with the more-sensitive SIM analysis
mode (Fig. 8). The homologous distributions of steranes
in these K–Mg salts were similar to that of the Unit C
halite layers.
Therefore, while the distribution of some classes ofbiomarkers (e.g., n-alkanes) are inhomogeneous, we consider that the clearly detectable amount of hopanes anda contrastingly lower amount of steranes are the characteristics of the K–Mg salt layers near the “Church section” in the Realmonte salt mine. The steranes detectedin the Fallen rock A sample could potentially derivefrom terrestrial eukaryotic organisms such as plants, because long-chain n-alkanes indicative of terrigenous organic matter were also contained in this sample. On theother hand, steranes present in trace amount in Fallenrock B could have derived from aquatic eukaryotes, inparticular, D. salina as similarly discussed in the previous section for the Unit C halite layers. Thus, our results
imply that not only bacteria but also eukaryotes were
present at least temporarily in the brine during the deposition of the K–Mg salt layer.
Depositional environment of Unit B K–Mg salt layer

Theoretically, the halites and K–Mg salts composing theUnit B K–Mg salt layer could have co-precipitated froma homogenous brine, because both halite and K–Mg
salts can precipitate from brine concentrated over 70
times upon evaporation from the normal seawater (Warren 2016; Shalev et al. 2018). However, this is unlikely
considering the presence of hopanes and steranes (Figs.
7 and 8), because the MgCl2 concentration of the brine
will be well above the habitable limit (~ 2.3 M: Hallsworth et al. 2007). Thus, the possible mechanisms

Isaji et al. Progress in Earth and Planetary Science(2019) 6:60

Page 11 of 15abFig. 7 Total ion chromatogram, m/z = 99, m/z = 191, and m/z = 217 mass chromatograms of a the free N-1 and b desulfurized N-1 fraction of
the K–Mg salt Fallen rock A. N-alkane peaks are labeled with black circles and peaks identified are labeled with numbers and listed in Table 2.

explaining the mineral composition of the K–Mg salt
layer include (1) allochthonous origin of either halite or
K–Mg salts, (2) progressive evaporation of the brinefrom the saturation point of halite to that of K–Mg salts,and (3) the formation of temporal or persistent density
stratification between halite-precipitating brine at thesurface and brine-precipitating K–Mg salts at the bottom. Allochthonous transport of the K–Mg salts fromthe shallow margins to the halite-precipitating innerbasin has indeed been proposed for the Na–Mg sulfate
layer in Unit B of the Realmonte salt mine (Yoshimura
et al. 2016). Nevertheless, this mechanism involves detrital materials filling the intergranular matrix (Yoshimura et al. 2016), which is not observed in our samples(Fig. 3, Additional file 1: Figsure S1, S2, and S4). Regarding possibility (2), it is highly improbable that a complete
mineralization sequence from halite to K–Mg salt

saturation would be recorded in the analyzed area (< 1cm, Fig. 2) considering the abundance of halite that precipitates during the course of seawater evaporation(Friedman and Sanders 1978).
Therefore, the most likely explanation is possibility (3),with eukaryotes and bacteria inhabiting in the halite-precipitating surface brine formed under temporal or persistent density stratification. An extremely low
concentration of steranes implies that the salinity of thisbrine was much higher than the saturation point of halite, substantially reducing the population of eukaryotes.
In such an extreme salinity, the evaporation rate and theamount of halite precipitated from the brine are low(Friedman and Sanders 1978). The resulting low precipitation rate of the halite crystals in the surface brine may
explain the elevated concentration of Al, Si, and P derived from aluminosilicate clay minerals in the haliteIsaji et al. Progress in Earth and Planetary Science(2019) 6:60

Page 12 of 15abFig. 8 Total ion chromatogram, m/z = 99, m/z = 191, and m/z = 217 mass chromatograms of (a) the free N-1 and (b) desulfurized N-1 fraction of
the K–Mg salt Fallen rock B. Mass chromatograms of m/z = 217 were obtained by selected ion monitoring (SIM) mode. N-alkane peaks arelabeled with black circles and peaks identified are labeled with numbers and listed in Table 2.

phase (Figs. 3 and 5, Additional file 1: Figures S1 andS2). In addition, this halite-precipitating surface brine
may have been further diluted by episodic freshwatersupply such as storms, which must have temporarily
relived organisms from salinity stress. Such an occasional event may have produced inhomogeneous distribution of some biomarkers among different K–Mg
salt rock samples, including terrestrial long-chain nalkanes (Figs. 7 and 8). Indeed, blooming ofeukaryotic phototroph D. salina have been reported
in the upper layers of the Dead Sea after the storm
events (Oren 2014).
The obtained data suggest for the first time the presence of eukaryotes and bacteria during the deposition of
the K–Mg salt layer. They were able to survive even themost extreme condition of the MSC in the halite-

precipitating brine at the surface formed as a result ofdensity stratification and/or possibly an episodic dilutionof surface brine by freshwater. As the stratigraphic correlation between the marginal and deep Mediterraneanbasins is still inconclusive, it is difficult, at present, to extrapolate our conclusions to the entire Mediterraneanbasin. Nevertheless, our results will be an importantcomparison for the future drilling of cores from deep
Mediterranean basin (e.g., MEDSALT initiative, https://
medsalt.eu). Similar investigations for the cores fromdeep Mediterranean basin would reveal a lateral transition in key biogeochemical parameters such as salinity,
microbial communities, and water column structurefrom the on-shore to off-shore basins. Such geochemical
constraints on the water column structures are essential
in understanding the hydrology and the spatial

Isaji et al. Progress in Earth and Planetary Science(2019) 6:60

distribution of evaporites in the Mediterranean basin(e.g., Simon and Meijer 2017). Moreover, while the impact of MSC on global climate has been discussed focusing on its influence on the ocean circulation (Ivanovic etal. 2014; Capella et al. 2019), changes in the microbialcommunities and water column structures within the
Mediterranean basin may also have global impact
through their influences on the biogeochemical cycle.
For example, efficient nutrient recycling and density
stratification during the MSC is suggested to have resulted in efficient carbon burial (Isaji et al. 2019a,b),
which have the potential to influence the global carbon
cycle. Such investigations for both on-shore and offshore deposits will lead to comprehensive understandingof the global impact of MSC.
Conclusions The μ-XRF elemental mapping and mineral identification by S K-edge XANES and XRD, as wellas thin section observation revealed that the K–Mg salt
layer in Unit B of the Realmonte salt mine is composedof primary halite, diagenetically-formed leonite, and primary and/or secondary kainite. These minerals are interpreted to have precipitated from a density-stratified
water column, with halite-precipitating brine at the surface and brine-precipitating K–Mg salts at the bottom.
Eukaryotes and bacteria, the source of steranes andhopanes detected from the K–Mg salt layer, should havebeen inhabiting the halite-precipitating brine in the surface, because the MgCl2 concentration in the bottom
brine-precipitating K–Mg salts is well above their habitable limits. Temporal density stratification between
brines with different salinity may have formed a temporal refuge for the microbial communities during themost extreme conditions of the MSC. Both steranes andhopanes are present in the Unit C shale–anhydrite–halite triplets, suggesting that both eukaryotes and bacteria
persisted throughout this unit. Future deep-sea drillingof MSC evaporites would resolve whether the obtained
conclusions are representative of the entire Mediterranean or just applicable to shallow restricted basins.

Additional files
Additional file 1: Figure S1. The μ-XRF intensity maps of O, S, Na, Cl,
Mg, Al, P, and Si, together with the picture of the measured area in K-1.
Color scales indicate element concentrations in signal counts per second.
Black crosses in the μ-XRF intensity map of S denote μ-XANES
measurement points. Figure S2. The μ-XRF intensity maps of O, S, Na, Cl,
Mg, Al, P, and Si, together with the picture of the measured area in K-3.
Color scales indicate element concentrations in signal counts per second.
Black crosses in the μ-XRF intensity map of S denote μ-XANES
measurement points. Figure S3. The XRD spectra of spot 2-1, 2-2, and 23, together with the reference spectra of halite, kainite, and leonite. The
presence of kainite peaks in spot 2-1 is probably due to contamination of
kainite during the sampling by dental drill, because halites only occur as
small crystals, making the sampling procedure difficult. Figure S4. (a)Page 13 of 15

Thin section of K-2 under transmitted light. Area filled with resin isindicated as R. (b) Close-up of the red rectangle showing fluid inclusion
bands in a halite crystal. (DOCX 4305 kb)
Abbreviations
GC/MS: Gas chromatography/mass spectroscopy; MSC: Messinian Salinity
Crisis; XANES: X-ray absorption near edge structure; XRD: X-ray diffraction;
XRF: X-ray fluorescenceAcknowledgements
We appreciate Italkali S.P.A. for the permission to sample the Realmontemine. We gratefully acknowledge Dr. K. Nagashima of JAMSTEC for XRD
analysis. We thank the handling editor and two anonymous reviewers forconstructive comments. This study was conducted with the approval of theSPring-8 Proposal Review Committee.
Authors’ contributions
YI, HK, JK, TY, and NO designed the study. YI, HK, JK, TY, FJJE, SL, VM, and MR
collected the samples. YI, TY, JK, and YT conducted the experimental workand data analysis. YI wrote the paper with contributions from TY, YT, andNO, and all authors edited and approved the final manuscript.
Funding
This work was performed with the support of Japan Society for the
Promotion of Science (JSPS) Research Fellowship (16 J07844) to YI and
JAMSTEC President Fund to NO.
Availability of data and materials
The datasets supporting the conclusions of this article are included within thearticle and its additional files.
Competing interests
The authors declare that they have no competing interest.
Author details
1
Biogeochemistry Program, Japan Agency for Marine-Earth Science andTechnology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan.
2
Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5
Kashiwanoha, Kashiwa, Chiba 277-8564, Japan. 3Japan Synchrotron Radiation
Research Institute/SPring-8, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan. 4The
Andalusian Earth Sciences Institute, The Spanish Research Council and the
University of Granada, Avda de las Palmeras 4, 18100 Armilla, Spain.
5
Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi diModena e Reggio Emilia, Via Campi 103, 41125 Modena, Italy. 6Physics andEarth Science Department, University of Parma, Parco Area delle Scienze 157/
A, 43124 Parma, Italy.
Received: 2 March 2019 Accepted: 12 August 2019

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