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Geochemical Journal, Vol. 43, pp. 331 to 341, 2009
Thermal stability of amino acids in biogenic sediments and aqueous solutions
at seafloor hydrothermal temperatures
M IHO I TO,1* KYOKO YAMAOKA ,2 HARUE MASUDA ,1 HODAKA KAWAHATA1 and L ALLAN P. GUPTA 3
1
Department of Geosciences, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
2
Graduate School of Frontier Sciences and Ocean Research Institute, University of Tokyo,
1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan
3
Kochi Institute for Core Sample Research (JAMSTEC), B200 Monobe, Nankoku, Kochi 783-8502, Japan
(Received December 3, 2008; Accepted March 16, 2009)
Siliceous ooze was reacted with NaCl solution at temperatures of 100–250°C to evaluate the effect of the mineralogical and chemical properties of host sediments on the thermal stability of amino acids (AAs). Results were compared
with those previously reported from calcareous ooze. The siliceous ooze preserved more AAs than did the calcareous
ooze, and the solution with the siliceous ooze preserved the AAs for a longer time than did the solution with the calcareous
ooze. When siliceous ooze was reacted under hydrothermal conditions, the AAs were released easily and were more stable
in alkaline solution than in the NaCl solution. Solubility of silica was greater in alkaline solution than in NaCl solution,
and an excess of hydroxyl ion and/or carbonate species in the alkaline solution underwent exchange more frequently with
AAs in the siliceous ooze than in the NaCl solution. The thermal stability of neutral AAs was enhanced significantly in
alkaline solution at temperatures higher than 200°C. When montmorillonite and saponite were heated in NaCl solution
with a known amount of AAs at 250°C, some AA concentrations increased, probably due to negatively charged AAs
binding to cations in the clay minerals. The results suggest that the AAs are dissolved into the solution from the sediments
primary via ion exchange, and that polymerization of silica that included AAs in its framework stabilized AAs in siliceous
ooze.
Keywords: hydrothermal system, siliceous ooze, NaCl solution, clay mineral, seafloor
Previous hydrothermal experiments examining the stability and synthesis of pure AAs have yielded contradictory results about the decomposition and synthesis of AAs
at temperatures greater than 100°C (reviewed by Holm
and Andersson, 2005). According to Yanagawa and
Kobayashi (1992), AAs could be synthesized after 1.5
hours in hydrothermal solution that contained
Fe(NH4)2(SO4)2, MnCl2, ZnCl2, CuCl2, CaCl2, BaCl2, and
NH4C at 325°C and 200 atm under methane and nitrogen
gas. Oligomers of glycine were formed when glycine solution was heated at 200–350°C for 2 minutes in a
supercritical water flow reactor (Islam et al., 2003). According to Cox and Seward (2007a, b), aspartic acid,
alanine and glycine in aqueous solutions exhibited highly
complex reactions, such as decarboxylation, polymerization and cyclization, when the solutions were heated at
120–170°C in a custom-built spectrophotometric reaction
cell. In contrast, aqueous AAs were decomposed at temperatures greater than 240°C (Bada et al., 1994), and AAs
were unstable under seafloor hydrothermal conditions
based on hydrothermal experiments at high temperature
and pressure (250°C and 265 bar; Bernhardt et al., 1984;
Miller and Bada, 1988). Terashima (1991) demonstrated
that decomposition of acidic AAs (aspartic and glutamic
INTRODUCTION
The seafloor hydrothermal system is a region where
life may have originated and evolved (Holm and
Andersson, 1995). Collecting samples without contamination from seafloor hydrothermal venting fields located
several thousands meters below sea level is difficult,
therefore laboratory-based hydrothermal experiments are
performed to simulate ideal hydrothermal conditions without concern about contamination. However, since amino
acids (AAs) are fundamental organic compounds necessary for life, controversy has surrounded laboratory-based
hydrothermal experiments simulating biological activity
under hydrothermal conditions (Bernhardt et al., 1984;
White, 1984; Miller and Bada, 1988; Qian et al., 1993;
Marshall, 1994; Bada et al., 1994; Andersson and Holm,
2000).
*Corresponding author (e-mail: [email protected])
*Present address: Osaka Training Center, Advantec Co., Ltd., 303 Saito
Bio Hills Center, 7-7-18 Saito Asagi, Ibaraki, Osaka 567-0085, Japan.
Copyright © 2009 by The Geochemical Society of Japan.
331
acids) in carbonates and muddy sediments obeyed firstorder kinetics at 180 and 220°C, and that the rate at 220°C
was three-fold greater than that at 180°C. Qian et al.
(1993) showed that the decomposition rates of glycine,
alanine, and glutamic acid obeyed first-order kinetics in
aqueous solutions at 100–220°C, and that those increased
exponentially at increasing temperatures.
The main sources of AAs in present seafloor sediments
are thought to be calcareous and siliceous biogenic debris, from which AAs decompose during early diagenesis
(Henrichs and Farrington, 1987; Kawahata and Ishizuka,
1993). For example, biogenic opal and calcium carbonates occupied 85% of the mass of sinking materials collected in sediment traps at 1000 m in the southern part of
the Western Pacific ocean along 170°W (Honjo et al.,
2000). The AAs found in seafloor hydrothermal solution
must have a biological origin based on the D/L ratio
(Horiuchi et al., 2004). Although the major portion of AAs
in natural oceanic sediments are considered to be of biogenic origin, migration paths and thermal stability of those
AAs during later diagenesis and hydrothermal alteration
are not well understood.
In a previous experiment (Ito et al., 2006), the thermal stability of AAs was examined by reacting calcareous ooze, a common organic matter-rich sediment in the
ocean, with artificial seawater at temperatures between
100 and 300°C. The results indicated that the AAs could
not survive nor be synthesized in solution at temperatures
greater than 250°C; a temperature of about 150°C was
optimal for long-term stability of AAs. We also reported
that a small amount of AAs remained in the reacted ooze
after heating at temperatures greater than 250°C for 240
hr, suggesting that minor clay minerals could protect AAs
from decomposition by adsorption. Naidja and Huang
(1994) observed that aspartic acid was adsorbed on natural Ca-montmorillonite surfaces within 2 hours at 25°C
and that the d001 spacing increased due to the formation
of aspartate-montmorillonite complexes. These complexes
were maintained after heating at 170°C for 2 hours. Such
studies indicate the importance of clay or other silicate
minerals to preserve AAs in hydrothermal systems.
In the present study, the effect of host sediment on the
stability of AAs with a biological origin was investigated
under hydrothermal conditions. First, siliceous ooze was
reacted with NaCl solution under the same experimental
conditions as reported by Ito et al. (2006). The experimental result examining the effect of solution pH reported
by Yamaoka et al. (2007) was combined with the results
of this study. Second, montmorillonite and saponite were
reacted with NaCl solution containing AAs at 250°C under similar conditions. Montmorillonite and saponite belong to the smectite group, which is one of clay mineral
groups having large cation exchange capacity (CEC).
Results are discussed in terms of the effect of mineral
332 M. Ito et al.
assemblages and solution chemistry on the thermal stability of AAs in seafloor hydrothermal systems.
MATERIALS AND METHODS
Materials
The starting material was siliceous ooze sampled from
the deepest part (43.5–44.7 mbsf) of a sediment core
(MD01-2409-30) collected offshore Shimokita (41°N,
141°E) during the IMAGES (International Marine Global Change Study) cruise in 2001. The ooze was freezedried and manually ground to fine powder using an agate
pestle and mortar. X-ray diffractometry (Rigaku
Geigerflex RAD-IA) revealed that the most abundant
mineral involved silica minerals (mostly quartz and minor biogenic opallin silica), with moderate amounts of
calcite and minor amounts of smectite and illite.
Montmorillonite (JCSS-3102, Mikawa, Japan) and
synthesized saponite (JCSS-3501, Kunimine Industries
Co., Ltd.), distributed by the Clay Science Society of Japan, were used for the other experiments. The
montmorillonite was ground and sieved with a no. 300
mesh (45-µm sieve pore size) and pre-heated in a muffle
oven at 400°C to remove organic matter while maintaining mineral structure since this mineral structure changed
at 600°C. X-ray diffractometry confirmed that the mineral structure was unchanged during the heating, however, the AAs could not be completely decomposed (Fig.
8). Saponite, synthesized by Kunimine Industries Co., Ltd.
and containing a small amount of AAs (Fig. 8), was used
without pretreatment.
Hydrothermal experiments
The siliceous ooze was reacted with 3.5% NaCl solution, which contained no detectable amount of AAs, under two different experimental conditions as previously
described by Ito et al. (2006).
In one series of experiments, 5 g of powdered sample
was mixed with 150 ml NaCl solution in a titanium vessel (160 cm3). The vessel was closed tightly after flushing with argon gas, and heated in a mantle heater at 100–
200°C for 240 hr. While maintaining constant temperature, aliquots of the solution were sampled at predetermined time intervals. The samples were filtered through
a polytetrafluoroethylene (PTFE) membrane filter, and
stored in a glass ampoule at –20°C until analysis. After
completing the experiment, residual sediment in the vessel was freeze-dried and ground to fine powder.
Two experiments at 150°C (150°C-1 and 150°C-2)
were conducted to estimate the reproducibility of AA concentrations, since this temperature was critical for the
thermal stability of amino acids in solution (Ito et al.,
2006). Figure 1 shows the amount of dissolved total
hydrolyzable amino acids (THAA; nmol) after comple-
Soluble silica concentration
The amount of soluble silica after heating at 150°C
was measured by molybdenum yellow absorbance spectrophotometry. Approximately 3–5 ml solution was dried
in a platinum crucible on an electric hot plate (ca. 70°C).
Dried sample was mixed with 0.2 g anhydrous Na2CO3.
The mixture was heated until a clear melt formed (about
900°C), then the fused residue was dissolved with 50 ml
of 0.1N HCl.
Four ml of sample solution was mixed with 0.2 ml of
HCl (1:3) and 10% ammonium molybdate solution in a
cuvette. After 15 min, soluble silica was measured using
spectrophotometry at 410 nm. Standard solutions containing 0, 10, 20, and 25 mg/l of soluble silica also were
measured for calibration.
Fig. 1. The amount of total hydrolyzable amino acids (nmol)
in solution heated at 150 °C to evaluate the reproducibility of
amino acid concentration.
tion of the experiment. The amount of dissolved THAA
is calculated using Eq. (1) (see Section “Results”). Reproducibility of THAA after heating for 72 and 240 hr
was ±13% and ±5%, respectively, in Fig. 1. Since the sample sediment and solution are not stirred during heating,
the sediment was not enough reacted in 72 hrs. However,
the reaction between sediment and solution reaches equilibrium after 240 hrs.
In another series of experiments at 250°C, 0.5 g of
powdered sediment was reacted with 15 ml NaCl solution in a tightly closed Teflon jar (25 cm3) contained in a
stainless steel vessel. The vessel was heated in a muffle
oven at 250°C for 240 hr. After cooling, the solution and
sediment were processed in a manner similar to that described above.
To examine the ability of clay minerals to protect AAs
from thermal decomposition, a known amount of standard AAs (aspartic acid, ASP; glutamic acid, GLU; lysine,
LYS; serine, SER; glycine, GLY; alanine, ALA; leucine,
LEU; methionine, MET and γ-aminobutyric acid, GABA;
Fig. 9) were dissolved in 15 ml of aqueous NaCl. The
AAs used in this experiment were selected on the basis
of thermal stability (Ito et al., 2006). The solution containing the AAs was heated with 0.5 g montmorillonite
or saponite in a Teflon jar at 250°C for 240 hr.
Before and after the hydrothermal experiments, solution pH was measured with a pH meter using a glass electrode (Horiba D-55). Approximate total pressure in the
vessels was equal to hydrostatic pressure at each temperature: 0.5, 1.5, and 3.9 MPa at 150, 200, and 250°C,
respectively (Haas, 1971).
Amino acid analysis
The procedure used for analysis of AAs has been described in detail by Gupta et al. (2006). For AA analysis,
1 ml of solution was treated with 1 ml concentrated HCl
to hydrolyze the AAs, while approximately 30–40 mg of
sediment was reacted with 3 ml of 6 N HCl. The HCltreated solution and sediment were placed together in a
glass ampoule with 50 µl internal standard (L-norleucine)
and sealed under an argon atmosphere, followed by heating for 22 hr at 110°C. After cooling to room temperature, the hydrolyzate of the sediment was filtered through
a PTFE membrane filter (0.45 µ m pore size) using a disposable syringe. The ampoule was rinsed twice with AAfree Milli-Q water, and the rinse filtered and added to the
sample solution. The solution was transferred into a pearshaped flask and unreacted HCl was removed by evaporation under vacuum at 40°C. The dried residue was dissolved in 1 ml Milli-Q water.
After completing the hydrolysis of initial siliceous
ooze, the residue was rinsed with Milli-Q water three
times. Then, concentrated hydrofluoric acid (HF) was
added to the sample to release silicate-bound amino acids (King, 1974), and was slowly evaporated on a hot plate
at a temperature below the boiling point of HF (108°C;
Ingalls et al., 2003). Once the HF completely evaporated,
the residue was hydrolyzed with 6 N HCl, and the resulting solution analyzed to quantify the silicate-bound AAs
by HPLC.
The AAs were quantitatively analyzed according to
the Waters AccQ-Tag method (Cohen and Michaud, 1993),
which has been described in detail by Gupta et al. (2006).
Twenty µl of the sample solution mixed with 60 µl borate buffer solution was derivatized with 20 µl Waters
AccQ-Fluor Reagent at 55°C for 10 min. Five µl of the
derivatized solution were injected into a Waters 2695
Separations Module with an ion-exchange column (AccQTag amino acid analysis column, Waters). The AA derivatives were detected using fluorescence detection at
Effect of sediment composition on stability of amino acids 333
Fig. 2. Mol% of amino acids in the initial siliceous ooze and calcareous ooze. Data on calcareous ooze obtained from Ito et al.
(2006).
395 nm (Waters 2475 multi λ fluorescence detector).
Chromatograms were processed using Empower software
(Waters). Concentrations of individual AAs were calculated from peak area using calibration curves of each AA
made with a standard solution (0.1–10.0 pmol/µ l). Recovery of the internal standard (L-norleucine) was >90%.
The detection limit was 0.1 nmol/ml, with a standard deviation of ±10% for absolute concentration (nmol/ml or
nmol/mg) and ±5% for relative mole concentration
(mol%), which was calculated by dividing absolute concentration with total hydrolyzable amino acids (THAA).
RESULTS
Changes in concentrations of amino acids in sediment
Total concentration of AAs adsorbed onto the surface
of siliceous ooze was 14.9 nmol/mg, and that of silicatebound AAs was 2.2 nmol/mg. Among the silicate-bound
AAs, LYS was the most abundant (20.9 mol%), followed
by SER (19.1 mol%) and GLY (16.9 mol%). Relative mole
concentrations (mol%) of AAs in the initial siliceous ooze
334 M. Ito et al.
Fig. 3. Changes in the proportion of total hydrolyzable amino
acid concentration remaining in siliceous ooze and calcareous
ooze after heating for 240 hr at 100–250°C when the percentage of THAA in the initial oozes was 100%. Data on calcareous ooze obtained from Ito et al. (2006).
Fig. 4. Concentration (nmol/mg) of individual amino acids in siliceous ooze and calcareous ooze after heating for 240 hr at 100–
250 °C. Data on calcareous ooze obtained from Ito et al. (2006).
is shown in Fig. 2 along with those in the initial calcareous ooze. Among the AAs, GLY was the most abundant
(21.4 mol%), followed by ALA (9.1 mol%) and ASP (7.8
mol%). The neutral AAs were the most abundant (65.3
mol%), followed by acidic (14.1 mol%), basic (10.7
mol%), aromatic (4.8 mol%), non-protein AAs (2.9
mol%), and sulfur-containing AA (2.2 mol%). Mol% concentrations of acidic AAs (ASP and GLU), non-protein
AAs [β-alanine (BALA) and GABA], histidine (HIS), and
ALA were lower, while mol% of neutral AAs [threonine
(THR), SER, GLY, isoleucine (ILE), LEU, and proline
(PRO)] except ALA and valine (VAL), aromatic AAs [tyrosine (TYR) and phenylalanine (PHE)] and sulfurcontaining AAs (MET) were higher in the siliceous ooze
than in the calcareous ooze.
Total hydrolyzable AA (THAA; total sum of AAs
adsorbed onto surface of siliceous ooze and silicate-bound
AAs) concentration in siliceous ooze decreased after reaction for 240 hr to 10.0, 8.3, and 5.0 nmol/mg at 100,
120, and 150°C, respectively, and reached a constant value
of 2.8 nmol/mg at temperatures greater than 200°C. Figure 3 shows changes in the proportion of THAA concentration in the two oozes after heating for 240 hr at 100–
250°C relative to THAA in the initial oozes (CT/C0 × 100;
C0 and CT are concentrations (nmol/mg) before and after
heating at T (°C), respectively). The proportion of THAA
in siliceous ooze decreased more slowly than that in the
calcareous ooze.
Figure 4 shows changes in the concentration (nmol/
mg) of individual AAs in siliceous ooze and calcareous
ooze reacted at 100–250°C. GLY was the most thermally
tolerant in the siliceous ooze. GLU, SER, PRO, and
GABA remained in siliceous ooze after reaction at 200°C,
while ASP, HIS, LYS, arginine (ARG), and THR did not
remain after reaction at 150°C. GLY in the calcareous ooze
was not thermally tolerant compared to that in siliceous
ooze, and only GLU, GLY, and GABA remained in the
calcareous ooze after reaction at 200°C.
Changes in amount and composition of amino acids in
solution
Figure 5 shows the amount of dissolved THAA (nmol)
versus reaction time. The amount of dissolved THAA
(M(t); nmol) is calculated by:
M(t) = C(t){150 – 10 × (n – 1)}
(1)
where C(t) is concentration (nmol/ml) of THAA in 10 ml
Effect of sediment composition on stability of amino acids 335
Fig. 5. The amount of total hydrolyzable amino acids (nmol)
in solution heated at 100–200 °C. Data on calcareous ooze obtained from Ito et al. (2006).
solution including the dead volume sampled after reaction time t (hr). Total amounts of AAs in the initial siliceous and calcareous oozes were 86 × 103 nmol and 16 ×
10 3 nmol, respectively.
The amount of dissolved THAA removed from the siliceous ooze gradually increased with time up to 100 hr
during heating at 100 and 120°C. When heated at 150°C,
the fraction quickly increased to 24 × 103 nmol after 5 hr,
and did not significantly change with additional time.
When heated at 200°C, the fraction quickly reached 35 ×
103 nmol after 3 hr, then decreased with time. The amount
of dissolved THAA released from the calcareous ooze
gradually increased with time until 100–150 hr at 100 and
120°C, and then decreased slightly with time. The dissolved fraction quickly reached 5 × 103 nmol after 6 hr
when heated at 150°C and 6 × 10 3 nmol after 3 hr at
200°C, then decreased with time. The rate of decrease in
THAA was greater at 200°C than at 150°C.
Based on the thermal response of each AA dissolved
in siliceous ooze, three groups of AAs, represented by
336 M. Ito et al.
Fig. 6. The amounts of ASP, GLY, and GLU (nmol) in solution
upon heating at 150 and 200°C.
ASP, GLY, and GLU, could be identified. Figure 6 shows
the amounts of dissolved ASP, GLY, and GLU (nmol) after heating at 150 and 200°C (calculated using Eq. (1)):
(1) ASP type AAs, which were increasingly soluble up to
120°C, decreased with time at 150°C and above (ASP,
LYS, ARG, THR, and SER); (2) Soluble GLY type AAs,
which increased with temperature up to 150°C, dramatically decreased with time at 200°C after reaching maximum concentration in the first 3 hr (HIS, GLY, VAL, ILE,
LEU, PRO, TYR, PHE, MET, and BALA); and (3) Soluble GLU type AAs increased in concentration at temperatures up to 150°C. The amounts of these AAs decreased
slightly at 200°C (GLU, ALA, and GABA). The thermal
stability of AAs in solution increased from (1) to (3). A
previous study (Ito et al., 2006) showed that the concentration of all AAs dissolved in the calcareous ooze gradually increased with time up to 130 hr at 100 and 120°C,
and then decreased slightly. They also showed that the
AA concentrations increased rapidly with time until about
48 hr at 150 and 200°C, and then decreased. Thus, the
AAs remain in solution with siliceous ooze longer than
with calcareous ooze.
Fig. 7. Concentration of soluble silica (mg/l) after heating at
150 °C.
pH and soluble silica concentration in reaction solutions
The pH decreased from an initial value of 8.0~8.3 to
6.4~6.6 after reaction with siliceous ooze. The final pH
of the solutions reacted with siliceous ooze was lower
than those reacted with calcareous ooze (8.0~9.5). The
concentration of soluble silica (mg/l) was monitored during heating of siliceous ooze in artificial seawater at
150°C. The experiment was conducted two times under
the same conditions; results are shown in Fig. 7. The concentration of soluble silica increased from 0 to 72 hr (96
to 611 mg/l in 150°C-1 and 335 to 423 mg/l in 150°C-2),
followed by a decrease to 385 and 376 mg/l, respectively.
Soluble silica concentrations after reaction for 72 hr are
within the analytical error, and appear to be nearly at
equilibrium controlled by α -cristobalite (400 mg/l at
150°C; Walther and Helgeson, 1977).
Adsorption effect by montmorillonite and saponite
Figure 8 shows the concentration (nmol/mg) of AAs
in montmorillonite and saponite before and after reaction at 250°C for 240 hr. Initially, montmorillonite contained a small amount of GLY (0.04 nmol/mg), and
saponite initially contained small amounts of GLU (0.008
nmol/mg), GLY (0.02 nmol/mg), and PRO (0.04 nmol/
mg). The concentration of GLY increased, and GLU, LEU,
and GABA were detected from the montmorillonite after
the reaction. In saponite, ALA, LEU, and GABA were
detected in addition to GLU and GLY, whose concentrations increased during the reaction. PRO was not detected
after the reaction. All of the AAs that increased in concentration in montmorillonite and saponite were added
to the solution when the reaction started (Fig. 9). However, ASP, LYS, SER, and MET, which also were added
to the solution, were not detected in either clay mineral
Fig. 8. Initial concentrations (nmol/mg) of amino acids in
montmorillonite and saponite and after heating at 250°C for
240 hr.
Fig. 9. Initial concentration (nmol/ml) of amino acids in solution and after heating with montmorillonite and saponite at
250°C for 240 hr.
after reaction.
The concentration (nmol/ml) of AAs in solution before and after heating with montmorillonite and saponite
are given in Fig. 9. The solution pH changed from an initial value of 4.6 to 5.3 after reaction with montmorillonite,
Effect of sediment composition on stability of amino acids 337
and from 4.6 to 7.8 after reaction with saponite. The
THAA concentration decreased from 1698 to 122 nmol/
ml (7% of the initial THAA concentration) when heated
with montmorillonite. GLU (50% of initial concentration),
GLY (0.2%), ALA (0.2%), LEU (0.6%), GABA (24%),
and a small amount of BALA, which was not added to
the initial solution, were detected in the reaction solution. THAA concentration decreased to 198 nmol/ml (12%
of initial concentration) when heated with saponite. GLU
(84% of the initial concentration), LYS (0.1%), SER
(0.2%), GLY (10%), ALA (4%), LEU (2%), and GABA
(16%) were present in solution after the reaction.
DISCUSSION
Effect of sediment composition on amino acids
The amount of THAA was greater in calcareous ooze
than in the siliceous ooze at 100°C, however, it decreased
dramatically with increasing temperature, and only small
amounts could be detected at temperatures greater than
200°C (Fig. 3). By contrast, 17% of the THAA were preserved in siliceous ooze at 250°C. As shown in Fig. 5, the
amount of THAA in solution with siliceous ooze at 150°C
was nearly constant for 72 hr. However, the amount of
THAA with calcareous ooze under the same conditions
decreased with time. Solutions with the same chemical
composition were used for both experiments, indicating
that the sediment composition affected the stability of AAs
in both sediment and solution.
The amounts of HIS, ARG, THR, and SER in calcareous ooze decreased dramatically with increasing temperatures from 100 to 120°C. The AAs were readily oxidized
and decomposed via deamination in neutral or alkaline
solution to release ammonia (Fearon and Montgomery,
1924). Thus, basic AAs, such as HIS and ARG, must decompose more easily in solution with calcareous ooze (pH
8.0~9.5) than in solution with siliceous ooze (pH 6.4~6.6).
Andersson and Holm (2000) heated ASP, SER, LEU, and
ALA in KCl solution (pH 6.0) with and without controlling redox conditions at 200°C and 50 atm in Tefloncoated autoclaves. Pyrite-pyrrhotite-magnetite (PPM) and
K-feldspar-muscovite-quartz (KMQ) buffer reagents were
added to control oxygen fugacity and hydrogen ion activity, respectively. They observed that the decomposition rates of ASP, LEU, and ALA were lower in the solution containing redox buffer than that without the buffer.
Only SER decomposed faster in the buffered solution,
which promoted dehydration through the higher hydrogen fugacity. Although THR behavior cannot be evaluated based on their experiments due to lack of examination of this AA, in the present study SER and THR decomposed more readily when reacted with calcareous ooze
than with siliceous ooze, due to control of oxygen fugacity by carbonate minerals (Stagno and Frost, 2008), which
338 M. Ito et al.
reduces oxygen fugacity by carbonate dissolution rather
than by silicate dissolution.
In siliceous ooze reacted at 250°C, 17% of the THAA
was preserved (Fig. 3), although GLU, SER, GLY, PRO,
and GABA were the only AAs remaining after the reaction (Fig. 4). Previous studies showed that SER decomposed more rapidly in high-temperature solutions (Miller
and Bada, 1988; Andersson and Holm, 2000), and this
study confirmed these results (i.e., SER increased in solution in 5 hr at 150°C and 3 hr at 200°C, then decreased
with time). However, SER remained in siliceous ooze
reacted at temperatures greater than 150°C (Fig. 4). GLY
did not decrease at elevated temperatures in siliceous
ooze, a result that differed from that in calcareous ooze
(Fig. 4). Hecky et al. (1973) showed that SER, THR, and
GLY were abundant in the diatom cell wall due to
stabilization within the silica framework (i.e., SER and
THR contain hydroxyl groups, which bind with silicic
acid, and GLY is the simplest amino acid, which can act
as a spacer among protein molecules). Andersson et al.
(2000) observed that the relative abundance of SER increased with depth in a hydrothermally altered sediment
core. They explained that SER condensated in kerogenlike polymers, which was an important process for stabilizing SER under hydrothermal conditions. Thus, in addition to incorporation of AAs into the silica framework,
maturation of organic matter such as kerogen formation
also promotes stabilization of SER and GLY in siliceous
ooze.
Effect of solution pH on amino acids
To evaluate the effect of pH on thermal stability of
AAs under hydrothermal conditions, siliceous ooze was
reacted in alkaline solution (0.55 M NaCl and 0.2 M
Na2CO3) under the same PT conditions (Yamaoka et al.,
2007). The pH of the solution decreased from an initial
value of 11.4~11.5 to 9.6~10.3 after reaction.
In Fig. 10, changes in the amount of THAA are compared in solutions reacted for 240 hr and at various temperatures. The proportion of THAA in solution was calculated by MT/M 0 × 100 (where M0 and MT are THAA
amounts before and after heating for 240 hr at T, respectively).
The proportion of THAA reached a maximum at 150°C
in NaCl solution and at 100°C in alkaline solution. During reactions with siliceous ooze, alkaline solutions dissolved more THAA than did the NaCl solution over the
entire range of temperatures examined. Thus, AAs are
more easily released into, and preserved more readily in,
alkaline solution than in NaCl solution. The concentrations of all AAs, except ARG, were greater in alkaline
solution at 100°C. Under alkaline conditions (pH
9.6~10.3), all AAs except ARG are negatively charged
and exist as anions rather than as cations or zwitterions.
Fig. 10. Proportion of total hydrolyzable amino acids in NaCl
and alkaline solutions after reaction for 240 hr. Data under
alkaline conditions obtained from Yamaoka et al. (2007).
Therefore, excess hydroxyl ions and/or carbonate species
in an alkaline solution are exchanged more frequently with
AAs in sediment at temperatures between 100 and 120°C
than in NaCl solution. The concentrations of neutral AAs,
except SER, were higher in alkaline solution at temperatures greater than 200°C (Yamaoka et al., 2007), and the
anionic and cationic forms of neutral AAs were more stable than zwitterions (Snider and Wolfenden, 2000; Li et
al., 2002). Thus, the thermal stability of neutral AAs is
significantly enhanced in alkaline solution at temperatures greater than 200°C.
Figure 11 shows the change in the proportion of THAA
remaining in the sediments studied after 240 hr reaction.
The proportion of THAA decreased with increasing temperature regardless of the compositional differences in
solution and sediment. The THAA in siliceous ooze was
more readily released into alkaline solution than in NaCl
solution even at temperatures less than 150°C. About 17%
and 6% of THAA remained in siliceous ooze after reaction with the NaCl and alkaline solutions, respectively,
at temperatures between 200 and 250°C. These observations suggest that the AAs are more easily released into
alkaline solution from the sediments, and that a certain
silica phase plays a role in protecting AAs from dissolution and decomposition even at temperatures between 200
and 250°C. The THAA that remained in the siliceous ooze
reacted more readily with the NaCl solution than did the
silicate-bound AAs (about 13% of the THAA). The concentration of GLY (Fig. 4) was higher than that of
silicate-bound GLY (0.62 nmol/mg). Silica is present in
aqueous solution primarily as a monomer species, and
aqueous silica becomes polymerized to polymeric silica
(Walther and Helgeson, 1977). The GLY may be incor-
Fig. 11. Changes in AA amounts in sediments after heating for
240 hr at different temperatures (THAA in the starting sediment is 100%). No data is reported from experiments using NaCl
solution after heating at 230 and 280°C. Data under alkaline
conditions obtained from Yamaoka et al. (2007).
porated into the polymeric silica when polymerization
occurs. Consequently, polymeric silica could protect GLY
against dissolution and decomposition.
In NaCl solutions, dissolved THAA became a peak
maximum at 150°C. The THAA concentrations at different pH values with siliceous ooze coincidentally are similar at this temperature (Fig. 10). THAA concentration
decreased with temperatures above 150°C, and the decrease was much greater in the NaCl solution than in the
alkaline solution. Thus, AAs are more stable in alkaline
solution when reacted with siliceous ooze than in NaCl
solution. The solubility of amorphous silica increased
steeply at pH > 8 (e.g., Faure, 1991). Thus, silica would
be significantly dissolved in alkaline solution, which also
results in a greater concentration of AAs in alkaline solution than in NaCl solution. In alkaline condition, siloxane
bonds in clay minerals also would break more easily than
in NaCl solution. AAs would bind to the siloxane in clay
minerals like hydrocarbon molecules (Sposito et al.,
1999). If so, they would be released from clay minerals
more easily in alkaline solution than in NaCl solution. In
contrast, a greater concentration of AAs was found in siliceous ooze in NaCl solutions than in alkaline solutions.
Role of clay minerals in protecting AAs
The concentration of dissolved AAs was only 7 and
12% of the concentration of initial solutions after heating with the montmorillonite and saponite, respectively,
at 250°C for 240 hr (Fig. 9). However, THAA concentration of the montmorillonite did not change after reaction,
while that of the saponite increased 17.5-fold relative to
that of the initial saponite.
Concentrations of GLU, GLY, ALA, LEU, and GABA
Effect of sediment composition on stability of amino acids 339
increased in montmorillonite and saponite after reaction
(Fig. 8). Adsorption of AAs on clay minerals is due to
ionic attraction and chemical reaction (Wang and Lee,
1993; Naidja and Huang, 1994). If ionic attraction was
the dominant process for adsorption of AAs, the adsorption of positively charged AAs onto negatively charged
clay minerals is expected. Wang and Lee (1993) demonstrated that the adsorption of positively charged AAs onto
montmorillonite is essentially a cation exchange process.
They showed that LYS, a positively charged basic AA at
neutral pH, was adsorbed to a greater extent than acidic
GLU and neutral ALA on montmorillonite at pH 6.3~7.2.
In this study, the pH of solution changed from an initial
value of 4.6 to 5.3 after reaction with montmorillonite,
and to 7.8 after reaction with saponite, a pH range at which
all AAs were negatively charged. Therefore, the adsorption of the AAs on the clay minerals cannot be explained
simply by ionic attraction. Henrichs and Sugai (1993)
suggested that the negatively charged carboxyl group
would bind with sedimentary organic matter via a bridging cation. They found calcium ions tightly bound to the
AAs. The saponite examined in the study described here
contained high amounts of magnesium as interlayer cations. Although no study has reported magnesium as a
bridging cation for AAs to silicate minerals, they could
function as binding cations to stabilize AAs in saponite.
ASP, LYS, SER, and MET, which were added to the
solution, were not detected in either clay minerals after
reaction (Figs. 8 and 9). These AAs were unstable at the
high temperatures to which the solutions were exposed
(Andersson and Holm, 2000; Sato et al., 2004; Ito et al.,
2006). Thus, these AAs must decompose immediately in
solution before they bind to the clay minerals. In conclusion, negatively charged AAs are protected from decomposition under seafloor hydrothermal conditions by clay
minerals.
CONCLUSIONS
Negatively charged amino acids (AAs) were released
into solution more easily under alkaline conditions than
in aqueous NaCl, implying that ion exchange with excess hydroxyl and/or carbonate ions is a primary mechanism for the release of AAs from sediments. The AAs
remain stable longer in alkaline solution reacted with siliceous ooze than in NaCl solution reacted with siliceous
ooze or in NaCl solution reacted with calcareous ooze.
The thermal stability of neutral AAs is enhanced significantly in alkaline solution at temperatures greater than
200°C. This stability indicates that polymerized SiO2 protects AAs from thermal decomposition. The AAs tend to
remain stable longer in siliceous ooze than in
carbonaceous ooze, indicating that clay minerals have a
high capacity to stabilize AAs, especially negatively
340 M. Ito et al.
charged ones, via binding to interlayer cations. Thus, AAs
are protected from thermal decomposition by amorphous
silica and silicate minerals via adsorption and/or binding
in both solution and sediment under hydrothermal conditions. Research results suggest that the optimal temperature of hydrothermal solutions for AAs was below 150°C
(Ito et al., 2006). However, AAs remain stable longer at
higher temperatures when associated with silicate, such
as hydrothermal sediments or surfaces of rock fissures in
hydrothermal systems.
Acknowledgments—This study was financially supported by
the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan; through a special coordination fund
(Archaean Park Project: International Research Project on Interaction between Sub-Vent Biosphere and Geo-Environments),
and Grant-in-Aid 17253006 and 16340161 for scientific research funded by the Japan Society for the Promotion of Science (JSPS). We thank to two anonymous reviewers, who gave
many suggestions to improve the discussion.
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