Concentrations and distributions of amino acids in black and white ...

Organic Geochemistry 66 (2014) 98–106

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Concentrations and distributions of amino acids in black and white smoker fluids at temperatures over 200 °C Shigeshi Fuchida a,⇑, Yuki Mizuno a, Harue Masuda a, Tomohiro Toki b, Hiroko Makita c a

Department of Geoscience, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan c Subsurface Geobiology and Advanced Research Project, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan b

a r t i c l e

i n f o

Article history: Received 27 August 2013 Received in revised form 8 November 2013 Accepted 9 November 2013 Available online 16 November 2013

a b s t r a c t The distribution of amino acids in seafloor hydrothermal systems was investigated through the determination of the concentrations of total hydrolysable amino acids (THAAs), dissolved free amino acids (DFAAs) and cell density in > 200 °C black and white smoker fluids sampled from the Mariana Trough. THAA concentrations of > 10 lM were detected in the black and white smoker fluids, which are higher than those of low temperature (< 53 °C) fluids and ambient seawater (< 1 lM). 1.4 104–8.6 105 cell/ml of microbe was detected from low temperature hydrothermal fluids (< 100 °C) and ambient seawater. The concentration of THAAs increased with increasing temperature, although the cell density decreased in high temperature fluid (> 150 °C). The bioactivity would be restricted under the high temperature condition. Levels of DFAAs (< 0.7 lM) were very low, suggesting that the amino acids existed mainly as polymers in these hydrothermal fluids. The amino acid polymers plausibly derive from biological protein and dissolve during the reaction of hydrothermal fluids along flow paths around the hydrothermal vents. Amino acids are considered to be unstable under hydrothermal condition (> 200 °C). However, labile amino acids (e.g., Asp and Ser) were abundant in high temperature fluids. These amino acids would be protected by reaction with inorganic compounds. The behavior of the amino acids derived from organisms around hydrothermal vents would be constrained more by abiotic physico-chemical reactions than biological activities in hydrothermal systems. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Many hydrothermal systems have been discovered in the world’s oceans since the first discovery at the Galapagos rift in 1977 (Weiss et al., 1977; Corliss et al., 1979). Seafloor hydrothermal fluids are physico-chemically distinct from seawater by having high temperatures, strongly reducing conditions and being enriched in heavy metal ions (Von Damm et al., 1985). Highly diverse ecosystems develop around hydrothermal vents, commonly in temperatures > 300 °C. The hydrothermal environment is postulated to have been the cradle of life on the primitive Earth (e.g., Miller and Bada, 1988; Holm, 1992). Previous studies revealed that the amino acids necessary to form life can be synthesized in laboratory-replicated hydrothermal conditions: large amounts of glycine, alanine and serine were produced when a solution containing aldehyde and ⇑ Corresponding author. Tel.: +81 6 6605 2591; fax: +81 6 6 6605 2522. E-mail address: [email protected] (S. Fuchida). 0146-6380/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.orggeochem.2013.11.008

ammonia was heated to 100–325 °C (Kamaluddin et al., 1979; Marshall, 1994; Aubrey et al., 2009). Protein formation processes have also been investigated based on the behavior of amino acids under hydrothermal conditions. Polymer balls of amino acids (0.3–2.5 lm) called marigranules were observed when amino acids were heated to 105 °C in an artificial hydrothermal solution that contained dissolved Mg, Ca, Mo, Zn, Fe, Cu, Mn and Co (Yanagawa and Egami, 1978). Short chained peptides were synthesized when a solution containing highly concentrated amino acids was heated in a flow reactor that simulated rapid heating and cooling conditions similar to those of hydrothermal vents (Imai et al., 1999; Cleaves et al., 2009). Although amino acid synthesis and oligomerization are promoted under hydrothermal conditions, the formation rates are very small and the concentrations of amino acids and peptides decrease because of rapid hydrolysis (Qian et al., 1993; Bada et al., 1995). Thermodynamic theories suggest that hydrothermal conditions are unfavorable for the growth of life (Miller and Bada, 1988). In contrast to experimental studies, research on amino acids recovered from natural hydrothermal systems including fluids,

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sediments and rocks has been limited. Haberstroh and Karl (1989) determined the concentration of dissolved free amino acids (DFAAs) in hydrothermal fluids and pore waters of sediments collected from the Guaymas Basin in the East Pacific Rise, where beneath 500 m of sediment, hydrothermal systems actively discharge high temperature fluids > 300 °C. Small amount of DFAAs were detected (< 1 nM) in the hydrothermal fluids. The sediment pore water contained 5–445 lM DFAAs, with glutamic acid, glycine, serine and alanine being the most abundant amino acids. Andersson et al. (2000) measured the concentrations of amino acids in the hydrothermally altered sediments cored from the Juan de Fuca Ridge in the northeastern Pacific Ocean; the total hydrolysable amino acids (THAAs) in their samples was 100–2000 nmol/g, with glycine, alanine, serine and histidine as the major components. Takano et al. (2005a) studied amino acids in solid samples taken from the seafloor around a hydrothermal vent at Suiyo Seamount in the western Pacific Ocean; the concentrations of THAAs in the cored rocks and the chimneys were 26–107 nmol/g and 11–64 nmol/g, respectively. Hydrothermal fluids > 300 °C collected from the Suiyo Seamount hydrothermal vents contained 246– 1163 nM THAAs, with abundant glycine and serine (Horiuchi et al., 2004). Highly concentrated amino acids were detected from low temperature hydrothermal fluids (42–89 °C) collected at Vulcano Island in Italy, and the highest amino acid concentrations were found at sites with relatively high temperature (82 °C), acidic (pH 2.0) and reducing conditions (Svensson et al., 2004). Understanding the behavior of amino acids in natural hydrothermal systems is important to evaluate the thermal response of biomolecules and bioactivity in nature. In this study, samples from black and white smokers with temperatures > 200 °C were collected from active hydrothermal systems in the southern Mariana Trough. We determined the composition of THAAs and DFAAs, and cell density in these fluids and discuss the origins of amino acids.

of subduction of the Pacific plate beneath the Philippine Sea plate, which also led to formation of chains of volcanic islands and submarine volcanoes. The Mariana Trough is a back-arc basin that developed behind the Mariana volcanic island chain. Several hydrothermal vents associated with back-arc spreading were found in this area (Tsunogai et al., 1994; Kato et al., 2010). In this study, hydrothermal fluids were collected from the southern Mariana Trough at 12°550 N, 143°390 E (Urashima site), located on an off-axis seamount during the NT12-24 cruise (14–22 September 2012, R/V Natsushima). This site is in the area where the sampling area is shown in Fig. 1(a). Black smoker fluids > 280 °C were discovered during the 2010 cruise (Nakamura et al., 2013). Hydrothermal fluid samples were collected from five different active vents (Fig. 1b). Sampling of the smoker fluids and in situ measurements of temperature were performed by the remotely operated vessel, HYPER-DOLPHIN 3K, of JAMSTEC (Fig. 2). The pH of the fluids was measured on the tender ship soon after sample recovery. The sampling points, temperature, pH and purity of hydrothermal fluids analyzed in this study are summarized in Table 1. The purity of the fluids was calculated from Mg2+ concentrations of hydrothermal fluids relative to that of ambient seawater, as Mg2+ is not found in high temperature hydrothermal fluids (Von Damm et al., 1985). The given temperature of the sample fluids is the maximum value recorded during sampling. Hydrothermal fluid samples for cell count measurement are summarized in Table 2. Samples were preserved shortly after sampling at 80 °C in glass vials that had been soaked in HNO3 overnight, washed with ultrapure water, and annealed at 500 °C for 2 h.

3. Methods 3.1. Extraction of amino acids from liquid samples

2. Geological setting and sampling All the glass equipment used for the amino acid extraction was soaked in alkaline reagent overnight, then overnight in 6 N HNO3, washed with ultrapure water, and annealed at 500 °C for 2 h to completely remove organic matter. The samples were hydrolyzed

The sampling site is located in the Izu-Bonin-Mariana (IBM) arc trench system, which is an outstanding example of a plate tectonics convergent boundary. IBM has been formed as a result

(a)

(b)

Japan

Pacific Plate Izu-Bonin Arc

1440 1441 1435, 1436

1438

Mariana Arc

Philippine Plate

Off-axis seamounts

Mariana Trough

Studied area Fig. 1. (a) Sampling area of black and white smokers in the Mariana Trough at 12°550 N, 143°390 E. (b) Topographic map around Urashima site and locations of the hydrothermal vents.

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D1441 CW-2

D1438 CW-1

D1441 CW-3

Fig. 2. Conditions during sampling of black (D1438 CW-1, D1441 CW-2) and white (D1441 CW-3) smokers.

Table 1 Sampling location and characteristic of hydrothermal fluids and seawater.

a

Sample name

Latitude

Longitude

Fluid type

Depth (m)

Temperature (°C)

pH

Purity (%)

D1435 CW-1 D1435 CW-2 D1436 CW-1 D1438 CW-1 D1440 N-2 D1440 CW-2 D1441 CW-2 D1441 W-3

12°55.2600 N 12°55.2640 N 12°55.2680 N 12°55.2410 N 12°55.3190 N 12°55.3190 N 12°55.2800 N 12°55.2560 N

143°38.8820 E 143°38.8960 E 143°38.8720 E 143°38.9220 E 143°38.9260 E 143°38.9260 E 143°38.8530 E 143°38.8640 E

Black smoker Clear smoker Black smoker Black smoker Seawater Clear smoker Black smoker White smoker

2908 2912 2895 2870 2940 2947 2935 2898

208 39 160 271 n.d.a 53 270 243

3.4 5.9 3.3 2.8 7.7 5.5 3.1 3.3

49 2 74 87 n.d. 21 60 73

n.d. = no data.

Table 2 Sampling location and characteristic of hydrothermal fluids and seawater for cell density analysis. Sample name

Latitude

Longitude

Depth (m)

Temperature (°C)

D1435 Bag-l D1435 Bag-2 D1436 Bag-l D1438 Bag-l D1438 CW-2 D1440 Bag-1 D1440 CW-1 D1441 CW-1 D1441 W-l

12°55.2600 N 12°55.2640 N 12°55.2680 N 12°55.2640 N 12°55.2640 N 12°55.3190 N 12°55.3190 N 12°55.2900 N 12°55.2700 N

143°38.8820 E 143°38.8960 E 143°38.8720 E 143°38.8640 E 143°38.8640 E 143°38.9260 E 143°38.9260 E 143°38.8540 E 143°38.8930 E

2908 2912 2895 2899 2899 2947 2947 2939 2908

110 16 39 6 8 45 35 70 178

Same location as D1435 CW-1 Same location as D1435 CW-2 Same location as D1436CW-1

0

10

20

30

40

His

NH3

Val

Gly Ala

Glu

Asp Thr

Ser

Fluorescence (mV)

D1435 CW-1

50

60

Retention time (min) Fig. 3. Chromatograms of hydrolyzed sample (D1435 CW-1). The concentration of each amino acid in the standard solution is 10 lM.

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Table 3 Concentrations of THAAs (a) and DFAA (b) in the hydrothermal fluids at Urashima site in southern Mariana Trough. Met, Tyr, Leu, Ile and Phe were not detected in any of the THAA samples. Sample

a

Amino acids (uM) Asp

Thr

Ser

Glu

Gly

Ala

Val

Phe

Lys

His

Arg

Total

(a) THAAs D1435 CW-1 D1435 CW-2 D1436 CW-1 D1438 CW-1 D1440 N-2 D1440CW-2 D1441 CW-2 D1441 W-3

1.46 0.07 0.51 0.43 0.10 0.14 1.10 1.85

0.89 BDa 0.31 0.29 BD BD 0.70 0.84

4.31 BD 1.27 1.03 0.02 0.18 3.73 4.18

0.51 0.16 0.64 0.31 BD BD 0.39 0.42

2.99 0.29 1.62 1.27 0.10 0.39 3.04 3.43

1.74 BD 0.48 0.39 BD BD 1.83 2.67

0.61 BD 0.29 BD BD BD BD 0.85

BD BD BD BD BD BD BD BD

BD 0.11 0.15 BD 0.06 BD 0.27 BD

0.45 0.30 0.27 0.10 BD BD 0.21 BD

BD BD BD BD BD BD BD 1.00

12.96 0.94 5.55 3.82 0.28 0.72 11.26 15.24

(b) DFAAs D1435 CW-1 D1435 CW-2 D1436CW-1 D1438 CW-1 D1440 N-2 D1440CW-2 D1441 CW-2

0.11 BD BD BD BD BD BD

BD BD BD BD BD BD BD

BD 0.06 0.11 BD BD BD 0.07






0.14 BD 0.11 BD 0.07 BD BD



0.69 0.06 0.22 BD 0.07 BD 0.07

BD = below detection limit (10 nM).

to extract the amino acids from the hydrolysable peptides and proteins as follows. Approximately 3 ml of liquid sample were put into a glass ampoule (10 ml) with 3 ml of concentrated 12 N HCl. Air was flushed out with argon and the ampoule was sealed and heated in a drying oven at 110 °C for 22 h. After cooling to room temperature, the solution was filtered through a PTFE membrane filter (0.2 lm) using a disposable syringe and transferred into a pear shaped flask. The unreacted HCl in the solution was removed by evaporation under vacuum at 40 °C, and the residue was dissolved in 0.1 N HCl. The concentration of the amino acids was quantified using high performance liquid chromatograph (HPLC) as described below. 3.2. Speciation and quantification of amino acids by HPLC The amounts of THAAs in the treated sample solutions and DFAAs in the untreated samples were measured by HPLC using a postcolumn ortho-phthalaldehyde (OPA method, e.g., Benson and Hare, 1975). Fifteen types of a amino acids[aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), glycine (Gly), alanine (Ala), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys), histidine (His) and arginine (Arg)] were separated through a cation exchange resin (Hitachi, #2619PH, 4.0 150 mm i.d.), derivatized with ortho-phthalaldehyde, and then each amino acid derivative was detected using a GL-7453 fluorometric detector (excitation wavelength: 360 nm; emission wavelength: 440 nm). The eluent was a citrate buffer solution (3-sodium citrate, citric acid, sodium chloride and ethanol), and the flow rate was 0.4 ml/min at 60 °C. Fig. 3 shows examples of the chromatograms of hydrolyzed sample solutions (D1435 CW-1). Detection limits of each amino acid were < 10 nM in this analysis. All chemicals used were analytical grade and obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 3.3. Microscopic observation Water samples were fixed with 3% formaldehyde. Prokaryotic cells were stained with 40 ,60 -diamidino-2-phenylindole dihydrochloride (DAPI) (Porter and Feig, 1980), and were filtered through a 0.2 lm Isopore membrane filter. Then, the cells on the filters were counted under fluorescence using an Olympus BX51 microscope. At least 50 microscopic fields for each sample were examined to determine the microbial community density.

4. Results 4.1. Chemical characteristics of hydrothermal fluids In this study, samples from black and white smokers with temperatures > 200 °C were collected at D1438 CW-1, D1441 CW-2, D1435 CW-1 and D1441 W-3. The black smoker fluids > 160 °C were acidic, with pH between 2.8 and 3.4, and contained a large amount of silica (> 10 mM). The purity of the high temperature fluids was 6–87%. Hydrothermal fluids < 52 °C (clear smoker fluids) were collected at D1435 CW-2 and D1440 CW-2. The pH values of these fluids were weakly acidic and those of ambient seawater obtained near the seafloor at D1440 N-2 for reference was neutral. The details of these characteristics will be reported elsewhere. 4.2. THAAs in the hydrothermal fluids Table 3(a) shows the concentration of each hydrolysable amino acid in the sample fluids and Fig. 4 shows the distributions of each amino acid constituting the THAAs. The concentrations of THAAs were between 0.28 and 0.94 lM in the clear smoker fluids and ambient sea water. Gly was the most abundant amino acid and accounted for > 30% of the THAAs, followed by Ser, Asp and Lys. The white and black smoker fluid THAA concentrations were between 3.82 and 15.24 lM, with abundant Ser and Gly. Unlike the clear smoker fluids and seawater, only small amounts of Thr, Glu and Val were detected. Met, Tyr, Leu, Ile and Phe were not detected in any of the samples. 4.3. DFAAs in the hydrothermal fluids Table 3(b) shows the concentration of each DFAA in the sample fluids. Very small amounts of DFAAs were detected in the samples. Ambient sea water contained only a small amount of Lys and the black smoker fluids contained some Ser. The white smoker fluid contained the highest total DFAAs, with Asp, Gly, Lys and His detected in these fluids. No DFAAs were detected in the clear smoker fluids of samples D1440 W-2 and D1438 CW-1. 4.4. Cell density of hydrothermal fluids Cell densities of hydrothermal fluids and ambient seawater are shown in Table 4. The cell densities were between 1.4 104 and

102


60

60

Mol%

40

50

Black smoker 208°C

40

30

Mol%

50

D1435 CW-1

20

10

10 0

Asp Thr Ser Glu Gly Ala Val Phe Lys His Arg

D1435 CW-2 Clear smoker 39°C

Mol%

40 30

10

10 0



60

60

D1436 CW-1 Black smoker 160°C

40 30

D1441 CW-2

50

Black smoker 270°C

40

Mol%

50

Mol%

30 20

30

20

20

10

10 0


60


60

D1438 CW-1 Black smoker 271°C

40 30

White smoker 243°C

40 30

20

20

10

10 Asp Thr Ser Glu Gly Ala Val Phe Lys His Arg

D1441 W-3

50

Mol%

50

0

Clear smoker 53°C

40

20

0

D1440 CW-2

50

Mol%

50

0


60

60

Mol%

30

20

0

D1440 N -2 Ambient seawater

0


Fig. 4. Concentrations of THAAs (Mol%) in the hydrothermal fluid samples.

8.6 105 cell/ml in the ambient seawaters and low temperature hydrothermal fluids (< 100 °C). On the other hand, the cell densities decreased to < 104 cell/ml in high temperature fluids (> 150 °C). 5. Discussion 5.1. Origins of amino acids in hydrothermal fluids Amino acids and short chain peptides can be abiotically synthesized under hydrothermal conditions. When a solution containing 0.1 M ammonium hydrogen carbonate (NH4HCO3) was heated under the simulated hydrothermal conditions of 50–292 °C and 150 atm for 20 min through a flow reactor, Gly was the dominant amino acid formed (Aubrey et al., 2009). Other experiments synthesizing abiotic amino acids also confirmed the high concentration of Gly formation (Kamaluddin et al., 1979; Marshall, 1994; Islam et al., 2001). In Red Sea brine samples, Ingmanson and

Dowler (1980) detected high concentrations of Gly, which was formed by the Strecker reaction between aldehydes and cyanide. Gly was the most abundant amino acid in both the black and white Table 4 Cell density in hydrothermal fluids and ambient seawater. Sample

Cell density (cell/ml)

D1435Bag-l D1435 Bag-2 D1436Bag-l D1438Bag-l D1438 CW-2 D1440 Bag-1 D1440CW-1 D1440 CW-2 D1441 CW-1 D1441 CW-2 D1441 W-l D1441 W-3

2.9 104 8.6 105 1.4 104 2.6 104 2.4 104 2.3 104 2.3 104 7.3 104 4.4 104 5.6 103 2.4 103 2.4 103

103

smoker fluids of this study. The high concentration of Gly would suggest that amino acids are created abiotically in those hydrothermal systems. However, Horiuchi et al. (2004) concluded that most of the amino acids in hydrothermal fluids collected from the Suiyo Seamount were formed biologically because the D/L ratios of Ala, Glu and Asp were very low, whereas those of abiotically formed amino acids is close to 1. In addition, the concentration of DFAAs was low in the all samples, indicating that most of the amino acids existed in polymer forms in the studied hydrothermal fluids. It is usually presumed that amino acid polymers are derived from organisms and bio-debris (Cowie and Hedges, 1992; Kawahata and Ishizuka, 1993; Sigleo and Shultz, 1993). Thus, most of the amino acids would be biologically derived in natural hydrothermal environments. As shown in Fig. 5, the concentration of THAAs increased with increasing temperature until 200 °C. It is known that the concentration of THAAs is correlated with the bioactivity (Takano et al., 2005a, 2005b). However, we observed that the cell density in the fluids decreased with increasing temperature (Fig. 6). Bioactivity would likely be restricted under high temperature conditions, because the survival temperature of life was estimated to be limited to 122 °C (Takai et al., 2008a). Although a few microbial cells were detected in > 200 °C hydrothermal fluids, they could be contamination from ambient seawater. The range of cell density in low temperature hydrothermal fluids (< 100 °C) and ambient seawater (1.4 104–8.6 105 cell/ml) of this study is consistent with the results in previous studies: 1.2 104 cell/ml in borehole fluid (65 °C) located on Juan de Fuca Ridge (Jingbluth et al., 2013); 8 103–9 104 cell/ml in deep seawater (Karner et al., 2001; Santelli et al., 2008). Even though biological activity is constrained under high temperature conditions, hydrothermal vent fields on or near midocean ridges are thought to be hot spots of biology in the Earth’s crust (Amend and Teske, 2005). Kato et al. (2009) detected abundant and active Bacteria and Archaea in hydrothermal fluids ( plume (0.6 lM) > seawater (0.3 lM) (Lang et al., 2013). This inference suggests that high temperature hydrothermal fluids are important as a supply source of amino acids. The concentrations of amino acids in the clear smokers (D1435 CW-2 and D1440 CW-2) were lower than in the high temperature fluids (Fig. 5). Purities of these fluids were very low (< 22%), suggesting that these fluids were diluted with seawater, which contained a very small amount of amino acids. Amino acids are important as common sources of carbon and nitrogen for microorganisms (Fuhrman and Ferguson, 1986). Marine bacteria use amino acid monomers and polymers in seawater; bacteria (Vibrio sp. and Pseudomonas) obtained from Otsuchi (Iwate, Japan) efficiently used Asp, Thr, Ser, Gly, Ala and Arg (Amano et al., 1982). A high concentration of microorganisms in hydrothermal plumes has been observed (e.g., Winn et al., 1986). The hydrothermal plumes can be nutrient source of chemolithoautotrophs because the mixing of reduced hydrothermal chemical products with oxic seawater provides abundant thermodynamic energy (McCollom, 2000; Lang et al., 2013). Thus, the distribution

104


of amino acids could be important for the circulation of organic carbon in the ocean and the growth and maintenance of marine ecosystems. 5.2. Behavior of amino acids under hydrothermal conditions Amino acids were isolated from both black and white smoker fluids with temperatures > 200 °C in this study. It is notable that the THAAs are more abundant in the high temperature fluids than in the low temperature fluids. It is commonly recognized that organic compounds, including amino acids, become labile with increasing temperature under hydrothermal conditions and that they do not exist in significant concentrations at temperatures > 200 °C (Bernhardt et al., 1984; White, 1984; Miller and Bada, 1988). In this study, however, the maximum THAA concentration was found in the 243 °C white smoker fluids. The THAA concentrations decrease in black smoker fluids > 270 °C, suggesting that the stability of amino acids decreases rapidly above 250 °C. Kinetic calculations indicate that amino acids rarely survive under hydrothermal conditions > 200 °C for long periods of time because they rapidly decomposed according to first order kinetics (Qian et al., 1993; Cox and Seward, 2007). However, Shock (1990) noted that amino acids could exist under hydrothermal conditions for some time because they attain a state of metastable equilibrium and he suggested that in natural hydrothermal systems metastable thermodynamic equilibrium was constrained by redox conditions rather than by kinetic reaction rates. Andersson and Holm (2000) heated amino acids with and without pyrite-pyrrhotite-magnetite (PPM) and K-feldspar-muscovite-quartz (KMQ) to buffer hydrogen fugacity at 200 °C, and they observed that amino acid decomposition was related in the redox buffered systems rather than in the non-redox buffered systems. High temperature seafloor hydrothermal systems evidently must maintain reducing conditions to preserve the high concentrations of amino acids in a metastable equilibrium state. In contrast to most of the hydrothermal laboratory experiments, which are performed in closed system (batch type) reactors, natural hydrothermal systems are open and occasionally at a non-equilibrium state (Imai et al., 1999). Flow type reactors can produce rapid heating–cooling and supercritical conditions (e.g., Islam et al., 2003). The flow type experiments can simulate natural conditions more closely than can batch type experiments. When Gly solution was heated in a flow reactor for 10 s, much of the Gly remained even when the solution reached > 350 °C and 40 MPa (Alargov et al., 2002). This finding indicates that amino acids can survive in an instantaneous subcritical condition. Stability of amino acids is also controlled by their ionic form, which is dependent on pH. In general, the cationic and anionic forms of amino acids are more stable than the zwitterionic form (Snider and Wolfenden, 2000), which means that an acidic hydrothermal environment is more favorable than a neutral environment for the survival of high concentrations of amino acids. The pH of the high temperature hydrothermal fluids of this study was 2.8–3.3. Although the pH of natural hydrothermal fluids ranges from acidic to alkaline, acidic conditions are more common for black smoker fluids (e.g., Shibuya et al., 2010). The stability of amino acids also depends on their functional group. Among acidic amino acids, Glu is stable under hydrothermal conditions as it adopts a stable cyclic conformation (Kawamura and Shimahashi, 2008), while Asp is unstable and rapidly decomposes under similar conditions (Andersson et al., 2000). In the hydrothermal fluids of this study, the concentration of Asp was higher than that of Glu. Higher concentrations of Asp than Glu in high temperature fluids were observed in a previous study (Horiuchi et al., 2004). Although the Glu monomer is stabilized via the cyclic conformation in hydrothermal fluids (Kawamura and Shimahashi,

2008), Glu in amino acid polymers and proteins might not be stabilized due to the lack of a cyclic conformation. Asp could be stabilized via combination with calcium ion which was abundant in hydrothermal fluids (Ito, 2008). Such a complexation of Asp may enhance the stability in the studied hydrothermal fluids. Gly and Ser were abundant in the black and white smoker fluids. These amino acids are also found at high concentrations in other submarine hydrothermal fluids (Guaymas Basin, Haberstroh and Karl, 1989; Suiyo Seamount, Horiuchi et al., 2004; Logatchev field, Klevenz et al., 2010). Gly, the simplest achiral amino acid, is stable under hydrothermal conditions and is readily formed via decomposition of other labile amino acids (Andersson et al., 2000). Thus, high concentrations of Gly could easily occur under hydrothermal conditions. In contrast, Ser is unstable and rapidly decomposes under these conditions (Sato et al., 2004). Amorphous silica is also found in high concentrations in high temperature fluids (Von Damm et al., 1985) and may protect labile amino acids from thermal decomposition. Ito et al. (2009) observed that the stability of Ser increased in hydrothermal fluids when heated with siliceous ooze. This behavior was most likely because bonding between the hydroxyl group of Ser in proteins and silica increased the stability of Ser (Hecky et al., 1973). Metal ions can also stabilize labile amino acids via complexation. When six different amino acids (Asp, Ser, Pro, Gly, Ala and Val) were heated with metal ions (Cu2+, Fe2+ and Mn2+) in a hydrothermal flow reactor at 200–250 °C for 120 min, stabilities of these amino acids increased compared to the same reaction carried out without the metal ions (Chandru et al., 2013). In fact, > 90% of organic molecules form complex components with metals (Sander et al., 2007; Klevenz et al., 2010). The preference of amino acids to form a metal complex or to be adsorbed onto mineral particles may also affect the concentration of amino acids in hydrothermal fluids. Coexisting chemical components and the chemical properties of individual amino acids are important factors controlling their distribution in hydrothermal fluids. In particular, labile amino acids are protected by reaction with inorganic compounds. The behavior of the amino acids derived from organisms and bio-debris around a hydrothermal vent would be constrained more by abiotic physicochemical reactions than biological activities in hydrothermal systems. 6. Conclusions Concentrations and distributions of amino acids in fluids > 250 °C from black and white smokers were measured. THAAs were more abundant in the high temperature fluids than in the low temperature fluids and ambient seawater, with high concentrations of Gly and Ser in particular. Very low levels of DFAAs were detected and amino acids were found in polymer forms in the hydrothermal fluids. Our findings suggest that the amino acid polymers in hydrothermal fluids were derived from organisms and bio-debris around hydrothermal vents via dissolution along the circulation paths of the hydrothermal water. The release of organic matter, including amino acids, during hydrothermal reactions would be effective for the development of bacteria in hydrothermal systems, including hydrothermal plumes, in which high densities of microbial cells were estimated (Winn et al., 1986). The dissolution process of polymer amino acids along the hydrothermal systems and the factors that influence the stability of amino acids potentially play important roles in the oceanic organic carbon cycle the growth of marine ecosystems. Acknowledgements We thank Nakamura K. (chief scientist) and the other members of the NT12-24 cruise. We would also like to thank Takahashi, Y.


and Kikuchi, S. for providing the hydrothermal fluid chemistry data. This study was financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan through a special coordination fund (Project TAIGA: Trans-crustal Advection and in situ reaction of Global subseafloor Aquifer). We thank Philip A. Meyers, University of Michigan and the reviewers for their thoughtful comments that helped us to improve this contribution.

Associate Editor—Phil Meyers

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