Hydrogen isotope fractionation in lipid biosynthesis by the piezophilic bacterium Moritella japonica DSK1

Chemical Geology 367 (2014) 34–38

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Hydrogen isotope fractionation in lipid biosynthesis by the piezophilic bacterium Moritella japonica DSK1 Jiasong Fang a,b,⁎, Chao Li c, Li Zhang d, Tara Davis a, Chiaki Kato e, Douglas H. Bartlett f a

State Key Laboratory of Marine Geology, School of Ocean and Earth Sciences, Tongji University, 1239 Siping Road, Shanghai 200092, China Department of Natural Sciences, Hawaii Pacific University, 45-045 Kamehameha Hwy., Kaneohe, HI 96744, USA State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074, China d State Key Laboratory of Geological Processes and Mineral Resources, Faculty of Earth Sciences, China University of Geosciences, Wuhan 430074, China e Research Program for Marine Biology and Ecology, Extremobiosphere Research Center, JAMSTEC, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan f Scripps Institution of Oceanography, University of California, San Diego, Mail Code: 0202, 9500 Gilman Drive, La Jolla, CA 92093, USA b c

a r t i c l e

i n f o

Article history: Received 23 October 2013 Received in revised form 27 December 2013 Accepted 27 December 2013 Available online 7 January 2014 Editor: Michael E. Böttcher Keywords: Fatty acids Hydrogen isotopes Piezophilic bacteria Biosynthesis Moritella japonica DSK1 Hydrostatic pressure

a b s t r a c t The δD of fatty acids is emerging as an important marine biogeochemical proxy, but the microbiological and environmental factors controlling the variations of δD of the lipids are not fully constrained. We report here the first measurement of D/H ratios of fatty acids in a piezophilic bacterium and show that hydrostatic pressure and the lipid biosynthetic pathway probably exerts dominant control over the δD of fatty acids. Piezophilic bacterium Moritella japonica DSK1 was grown at a pressure of 30 MPa with glucose as substrate. Fatty acids in DSK1 showed vastly varied δD, ranging from +44.4 to −171‰. Short-chain fatty acids (SCFA), which are synthesized by the fatty acid synthase (FAS) pathway, had positive δD (average +3‰), whereas long-chain polyunsaturated fatty acid (LC-PUFA) synthesized via the polyketide pathway exhibited much depleted δD (−171‰). Our results suggest that the lipid biosynthetic pathways can exert first-order control on the hydrogen isotope signature of bacterial membrane lipids under elevated pressure. Our findings have important implications in marine biogeochemistry. D-depleted fatty acids in marine sediments and in the water column may be derived from piezophilic bacterial reworking and resynthesis of organic matter at high pressure condition. Thus, caution must be exercised in the interpretation of hydrogen isotope signatures of lipids in, e.g., deducing sources of organic matter and tracing microbial biogeochemical processes in the deep ocean and the deep biosphere. © 2014 Elsevier B.V. All rights reserved.

1. Introduction There has been a proliferation of the use of hydrogen isotope ratios (δD) of lipid biomarkers as proxies for studying modern biogeochemical cycles (Jones et al., 2008; Li et al., 2009), paleohydrological processes (Pagani et al., 2006; Sachse et al., 2009), and paleoenvironmental and paleoecosystem changes (Xie et al., 2000; Sauer et al., 2001; Huang et al., 2002). Early studies focus mostly on δD of lipids of terrestrial plants preserved in peat bogs and lake sediments (Chikaraishi et al., 2005; Smith and Freeman, 2006). The rationale is that the δD of plant lipids primarily reflect the δD of environmental water, which ultimately provides hydrogen atoms in the biosynthesis of plant lipids and exerts dominant control over the δD of the lipids (Zhang and Sachs, 2007; Sachse et al., 2012). In addition, the δD signature of lipids can be preserved reasonably well over geologic time scales (Sessions et al., 2004). As a result, the δD of lipids can also be useful tracers for deciphering paleo-biogeochemical processes (Chikaraishi and Naraoka, 2006). ⁎ Corresponding author at: Department of Natural Sciences, Hawaii Pacific University, 45-045 Kamehameha Hwy., Kaneohe, HI 96744, USA. Tel.: + 1 808 236 3555; fax: +1 808 236 5880. E-mail address: [email protected] (J. Fang). 0009-2541/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemgeo.2013.12.018

There have also been attempts in utilizing δD of lipids in marine chemistry and biogeochemistry. Chikaraishi et al. (2005) measured δD of sterols in marine sediments to distinguish marine and terrestrial organic matter. Englebrecht and Sachs (2005) used δD of alkenones to track their sources and the water masses in which they were produced. In other studies, Schouten et al. (2006) and van der Meer et al. (2008) employed δD values of alkenones to delineate paleo-salinity and temperatures. More recently, Jones et al. (2008) and Li et al. (2009, 2011) measured δD of fatty acids and other lipids in particulate organic matter and sediments in the California Borderland Basins to infer the source of sedimentary organic matter and microbial biogeochemical activities. However, the major limitation of using δD as a proxy in defining the sources of sedimentary organic matter and trace biogeochemical processes is our limited knowledge about the variations of lipid δD of source organisms in the marine environment. Laboratory studies have been mainly focused on the variability of δD of lipids in marine autotrophs. For instance, Sessions et al. (1999) investigated the D/H ratios of lipids of marine phytoplankton and algae. The authors observed that there were little isotopic variations within a lipid class (b50‰) of the plankton. Hydrogen isotope fractionation in heterotrophic bacteria is inherently more complex because of multiple sources of hydrogen and their transfer during lipid biosynthesis. There are only a few reported studies

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investigating hydrogen isotope fractionation in lipid biosynthesis in heterotrophs: the aerobic methanotroph Methylococcus capsulatus (Sessions et al., 2002), the acetogen Sporomusa sp. DSM 58 (Valentine et al., 2004), and the sulfate-reducing bacterium Desulfobacterium autotrophicum (Campbell et al., 2009). Zhang et al. (2009) investigated hydrogen isotope fractionation in lipid biosynthesis in bacteria with various modes of metabolism (photoautotrophic, chemoautotrophic, and heterotrophic growth). A similar study on hydrogen isotope fractionation and lipid biosynthesis of a halophilic archaea, Haloarcula marismortui, was reported recently by Dirghangi and Pagani (2013). The authors conclude that the D/H ratios of lipids are controlled primarily by microbial metabolic pathway rather than by the biosynthetic pathway of lipids. Many questions remain to be addressed before lipid δD can be used as a marine biogeochemical proxy. An important first-order question is: Does lipid biosynthetic pathway influences D/H ratios of lipids? In this study, we cultured a piezophilic bacterium, Moritella japonica strain DSK1, at hydrostatic pressure of 30 megapascal (MPa) to examine the impact of lipid biosynthesis on D/H ratios of fatty acids and the implications for marine biogeochemistry. M. japonica DSK1, like other piezophilic bacteria, possesses dual biosynthetic pathways of fatty acids (the FAs and the PKS) and is, therefore, an ideal microorganism for exploring the control of lipid biosynthesis on D/H ratios of lipids under environmentally relevant conditions. 2. Materials and methods 2.1. Growth of M. japonica strain DSK1 M. japonica strain DSK1 is a piezophilic bacterium isolated from the Japan Trench sediment taken at a depth of 6356 m (40′06.8″N, 144′ 11.0″E) (Kato et al., 1995). Strain DSK1 was grown in a custom-built high-pressure bioreactor (Fang et al., 2006). Growth media was prepared using sterile-filtered natural seawater (δD = 6.2‰; Sigma Chem. Co.) with glucose as the source of carbon (concentration = 64 mM), supplemented with yeast extract (0.08%) (Fang et al., 2006). Enriched glucose solution was prepared by dissolving normal glucose (δD = 18.3‰) and minute amount of 6,6-d2-glucose (98% atom D, Aldrich) in sterilized seawater, and the δD of the final glucose solution was 34.7‰. The media was distributed into airtight pouches. To each pouch 15 mL of fluorinert was added to provide oxygen for the cultures. Fluorinert was saturated with oxygen (25% of the total volume) by bubbling high-purity oxygen for 2 h at 4 °C and filtered prior to usage. The media was inoculated with M. japonica strain DSK1 initially grown on agar plates (marine agar 2216, Difco, Detroit, MI) at 7 °C and atmospheric pressure. Strain DSK1 was cultured at 7 °C and 30 MPa in the bioreactor. Cultures were removed from the bioreactor at stationary phase (based on optical density measurements at 600 nm). Cell pellets were collected by centrifugation at 10,000 g for 20 min for fatty acid and isotope analyses. 2.2. Fatty acid and hydrogen isotope analyses Total bacterial lipid was obtained by extracting bacterial cells using a one-phase solvent system containing phosphate buffer (50 mM, pH = 7.4), dichloromethane, and methanol (Fang and Findlay, 1996). The total lipids were separated into neutral lipids, glycolipids, and phospholipids using miniature champagne columns (Supelco Inc., Bellefonte, PA) eluted with 5 mL of chloroform, acetone, and methanol, respectively. Fatty acid methyl esters (FAMEs) were formed by methanolic HCl hydrolysis (6.0 M HCl:methanol, 1:0.8, v/v; 80 °C, 15 min) of phospholipids. FAMEs were analyzed on an Agilent 6890 GC interfaced with an Agilent 5973N Mass Selective Detector using a 30 m × 0.25 mm i.d. DB-5 MS fused-silica capillary column (J&W Scientific, Folsom, CA). The oven temperature was programmed from 50 °C to 120 °C at 10 °C/min, then to 310 °C at 5 °C/min, and then held for 20 min. Individual fatty acids were identified from their mass spectra.

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Hydrogen isotope analysis was done at the Division of Geological and Planetary Sciences at Caltech. The δD of FAMEs was determined on a ThermoFinnigan Trace GC coupled to a Delta + XP mass spectrometer via a ThermoFinnigan GC/TC pyrolysis interface operated at 1440 °C. Two n-alkanes (C22 and C23) with known hydrogen isotopic composition were co-injected with each sample; one was used as reference peaks to calibrate isotopic analyses, the other as an unknown sample to determine analytical accuracy. Instrument system error was corrected for by using a standard mixture of C16–30 even carbon numbered n-alkanes inserted every four analyses in the sequence. The δD values of FAMEs were corrected for the added methyl hydrogen by isotopic mass balance, with the δD value of methyl hydrogen derived from analysis of phthalic acid methyl ester of known isotopic composition (Sessions, 2006). Phthalic methyl ester was prepared in the same way as fatty acid methyl ester. Each sample was measured three times, and the standard deviation ranged from 0.34 to 1.9‰ for C16–18 fatty acids, and 2.8–4.7‰ for C14–15 fatty acids. The reported δD values are the average of replicate measurements. For C18:0 and C22:6 fatty acids, the sample was concentrated for δD measurements. All isotopic ratios are reported in the δD notation, in per mil relative to the VSMOW standard. Hydrogen isotope analysis of glucose and water was done at Isotech Laboratories, Inc., Champaign, Illinois. Glucose was analyzed by using a Finnigan Thermal Conversion/-Elemental Analyzer (TCEA) interfaced to Finnigan Delta Plus XL isotope-ratio mass spectrometer. Water was analyzed by CRDS (cavity ringdown spectrometer) model L1102-i fitted with a Leap autosampler. Two reference water samples were used to verify accuracy and reproducibility. These reference waters were analyzed approximately every tenth analysis. The system was calibrated by analysis of primary reference standards (SMOW and SLAP) obtained from IAEA or NIST. At a minimum, every tenth sample analysis is a replicate. Precisions for δD measurements are ±2‰.

3. Results M. japonica strain DSK1 contains short-chain fatty acids (SCFA) typically found in surface bacteria (C14–19), which include saturated, monounsaturated, and cyclopropane fatty acids (Table 1; Fig. 1). Like other piezophilic bacteria, DSK1 synthesized long-chain polyunsaturated fatty acids (LC-PUFA), mainly DHA (all cis-4,7,10,13,16,19docosahexaenoic acid) with trace amount of EPA (eicosapentaenoic acid, 20:5ω3) (Fig. 1). This is consistent with the distribution patterns Table 1 δD values of fatty acids (FAs) isolated from piezophilic bacterium Moritella japonica DSK1. Compoundsa

δD (Stdevb)

αFA/wc

εFA/wd

αFA/se

εFA/sf

14:0 a15:0 15:1 16:1 16:0 cy17:0 18:1 18:1 18:0 22:6 Water Glucoseh

4.7 (4.7) 44.3 (2.8) −44.0 (3.3) 2.4 (1.9) 23.2 (1.4) −10.2 (1.9) −12.2 (–g) −5.8 (0.3) 24.1 (–) −170.9 (–) 6.2 34.7

1.00 1.04 0.95 1.00 1.02 0.98 0.98 0.99 1.02 0.82

−1.5 37.9 −49.9 −3.8 16.9 −16.3 −18.3 −11.9 17.8 −176.0

0.97 1.01 0.92 0.97 0.99 0.96 0.95 0.96 0.99 0.80

−29.0 9.3 −76.1 −31.2 −11.1 −43.4 −45.4 −39.1 −10.2 −198.7

a Fatty acids are designated by the total number of carbon atoms:number of double bonds. a = anteiso; cy = cyclopropane. b Standard deviation of three replicate analyses. c Fractionation factor between fatty acid and water. d Hydrogen isotope fractionation between fatty acid and growth water, defined as εa/b = 1000[(δa + 1000) / (δb + 1000) − 1] (Sessions et al., 1999). e Fractionation factor between fatty acid and substrate (glucose). f Hydrogen isotope fractionation between fatty acid and substrate (glucose). g Not applicable (one analysis available). h Composite glucose as described in Materials and methods.

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4. Discussion

Fig. 1. Total ion chromatogram of phospholipids isolated from piezophilic bacterium Moritella japonica DSK1 grown at a pressure of 30 MPa. See Table 1 for fatty acid identification.

There are significant differences in δD of short-chain (N−44‰) and long-chain polyunsaturated fatty acids (− 171‰) synthesized by piezophilic bacterium M. japonica DSk1. Furthermore, hydrogen isotope fractionation was much less between SCFA and growth water and between SCFA and growth substrate than that observed in surface heterotrophic bacteria grown at atmospheric pressure conditions (e.g., Sessions et al., 1999; Zhang et al., 2009). The observed contrasting difference in δD of fatty acids of M. japonica DSK1 is likely a result of two factors: (1) the effects of high hydrostatic pressure on the kinetics of hydride (H−) and deuteride (D+) transfer from NADPH to fatty acids during lipid biosynthesis; and (2) the operation of two independently functioning fatty acid biosynthetic pathways in the piezophilic bacterium: the FAS- and PKS-based biosynthetic systems (Metz et al., 2001; Napier, 2002).

4.1. Hydrogen isotope fractionation in biosynthesis of short-chain fatty acids via the FAS pathway of lipids previously observed in piezophilic bacteria (Kato et al., 1995; Fang and Kato, 2007). The δD values of fatty acids are given in Table 1. Fractionation of hydrogen isotopes in lipid biosynthesis between fatty acids and growth water and between fatty acids and glucose is illustrated in Fig. 2. Variations in δD can be summarized as follows. First, fatty acids exhibited widely varying δD, from + 44.4 to − 179.5‰. All saturated fatty acids had positive δD values with an average of +3‰, and were enriched in D relative to their corresponding unsaturated counterparts by as much as 36‰. However, there was no consistent relationship between δD and chain length of fatty acids. Second, all saturated fatty acids (except C14:0) were enriched in D relative to water, but depleted in D relative to glucose, albeit to a very small extent. Third, hydrogen isotope fractionations between FA and growth water (εFA/w) (+38 to −50‰) and between FA and substrate (glucose) (εFA/s) (+ 9 to − 76‰) are significantly less than that observed in heterotrophic bacteria grown at atmospheric conditions (−150‰; Sessions et al., 1999). Finally, polyunsaturated fatty acid DHA had the most negative hydrogen isotope ratio (− 171‰) and was much more depleted in D than all the other fatty acids (εPUFA/s = −199, εPUFA/w = −176; Table 1).

50

0

δD

-50

-100

22:6w3

18:0

18:1w9t

18:1w9c

Cy 17:0

16:0

16:1w7c

a15:0

14:0

-200

15:1

SCFA LCFA

-150

Fatty acid Fig. 2. Hydrogen isotope ratios (δD) of fatty acids isolated from piezophilic bacterium Moritella japonica DSK1 grown at 30 MPa (megapascal) pressure. Error bar shows standard deviation of measured δD values of fatty acids (n = 3).

It has been shown that two different fatty acid biosynthetic pathways, the FAS- and the PKS-based systems, co-exist in piezophilic bacteria (Metz et al., 2001; Napier, 2002). The FAS-based pathway is one common to surface bacteria which synthesizes the common bacterial fatty acids (i.e., the SCFA). The short-chain fatty acids in strain DSK1 are probably synthesized via the classical bacterial FAS pathway. The FAS is a group of individual enzymes, encoded by discrete genes that catalyze the successive steps in fatty acid synthesis (Schweizer and Hofmann, 2004). Its constituent chemical reactions and the respective component enzymes have been well illustrated, and the chemical mechanisms are strongly conserved across most bacterial and eukaryotic phyla (Campbell and Cronan, 2001; Baillif et al., 2009). In essence, a fatty acid is biosynthesized from primer acetyl-CoA and chain extender malonyl-CoA, with repetitive rounds of four enzymatic reactions on the growing fatty acyl chain: condensation (enzyme: KS), reduction (KR), dehydration (DH), and reduction (ER) (Fig. 3). The sources of hydrogen for microbial biosynthesis of fatty acids are water, acetate and NADPH. Acetate is derived from growth substrate glucose via glycolysis and pentose phosphate pathway (McInnes et al., 1983; Zhang et al., 2009). In heterotrophic bacteria, NADPH is generated in NADP+ reduction in the TCA cycle and the pentose phosphate pathway of glucose metabolism (White, 2000; Baillif et al., 2009). In fatty acid biosynthesis, hydrogens (as hydride, H−, or deuteride, D−) are transferred from NADPH to the nascent fatty acids via two reduction reactions: β-ketoacyl ACP reductase reaction (KR) which transfers H from NADPH to odd-numbered carbon positions of the emerging fatty acid, and enoyl ACP reductase reaction (ER) which transfers H from NADPH to all carbon positions (Baillif et al., 2009). High growth pressure likely changes isotope effects by affecting the kinetics of multi-step enzymatic reactions, as it does affecting carbon isotope fractionation in biosynthesis of fatty acids by piezophilic bacteria (Fang et al., 2006), or by altering the intrinsic isotope effect through hydrogen tunneling (Northrop, 2006). Hydrogen tunneling is sensitive to hydrostatic pressure, and can result in a large change in activation volume of enzymes, e.g., NADPH, before and after hydride transfer, and thus, an increase in reaction rate (Northrop, 2006). Therefore, it would seem reasonable to propose that high growth pressure accelerates the rate of hydrogen transfer from NADPH to fatty acids (Northrop, 2006) and, therefore, entails relatively less hydrogen isotope fractionation in biosynthesis of fatty acids by piezophilic bacteria than by surface bacteria. This is evidenced by the much greater fractionation factors observed, αFA/w: 0.95 to 1.04 and αFA/s: 0.92 to 1.01 (Table 1), which are significantly higher than those observed in surface heterotrophic bacteria (αFA/w = 0.44–0.60) (Zhang et al., 2009). Thus, the short-chain fatty acids synthesized by strain DSK1 via the FAS system under high pressure conditions

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Fig. 3. Piezophilic bacterial synthesis of short-chain fatty acids via the fatty acid synthase (FAS) system (gray area) and of long-chain polyunsaturated fatty acids (DHA, all cis-4,7,10,13,16,19-docosahexaenoic acid is shown) via the polyketide synthase (PKS) system (blue area) (modified from Heath et al., 2001; Ratledge, 2004). Both the FAS and the PKS system initially utilize the same building molecules, acetyl-CoA and malonyl-CoA, to synthesize β-ketoacyl-ACP (green area) which serves as a primer for fatty acid synthesis. Presumably, DHA is synthesized via the sequence 8:1(5) ➔ 10:2(4,7) ➔ 12:3(3,6,9) ➔ 14:3(5,8,11) ➔ 16:4(4,7,10,13) ➔ 18:5(3,6,9,12,15) ➔ 20:5(5,8,11,14,17) ➔ 22:6(4,7,10,13,16,19). Hydrogens shown in bold, italics, and plain type represent those from NADPH, water, and growth substrate, respectively. ACP, acyl carrier protein; Acc, acetyl-CoA carboxylase; FabD, malonyl CoA:ACP transacylase (MCAT); FabB, FabF, and FabH are β-Ketoacyl-ACP synthases I, II, and III, respectively. KS, keto synthase; KR, β-ketoacyl-ACP reductase; DH, dehydratases; ER, enoyl-ACP reductase; 2,3I, 2,3-isomerase.

would retain more positive δD values compared to those synthesized by surface bacteria under atmospheric conditions. 4.2. Hydrogen isotope fractionation in biosynthesis of polyunsaturated fatty acids via the PKS pathway The PKS-based pathway is a fundamentally different biosynthetic pathway which involves polyketide synthases and catalyzes the biosynthesis of long-chain polyunsaturated fatty acids (Metz et al., 2001; Okuyama et al., 2007). Indeed, previous studies have demonstrated the existence and operation of the two co-existing fatty acid biosynthetic pathways in piezophilic bacteria (Metz et al., 2001; Napier, 2002; Okuyama et al., 2007). The PKS pathway operates independently of the FAS in piezophilic bacteria (Wallis et al., 2002). The significant depletion of D in polyunsaturated fatty acid DHA relative to SCFA observed

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in strain DSK1 suggests that the PKS system has much greater hydrogen isotope fractionation than the FAS system (Fig. 3). The distinctly different δD values between short-chain FA and long-chain PUFA DHA provide the first evidence in isotope geochemistry (apart from genomic sequencing evidence) that suggests that the two groups of fatty acids in piezophilic bacterium M. japonica DSK1 were likely biosynthesized by two different biosynthetic pathways, the former by the FAS system and the latter by the PKS-based biosynthetic pathway, as described below (Fig. 3). The biosynthetic sequence of polyunsaturated fatty acids via the PKS system starts with the formation of the nascent fatty acyl chain using acetyl-CoA and malonyl-CoA as the building blocks, via the condensing enzyme KS, reductive enzyme KR, and the dehydration enzyme DH (Fig. 3). However, different from the FAS system, the unsaturated fatty acyl chain synthesized via the PKS system is not reduced into a saturated one as in the FAS system. Instead, double bonds introduced by DH are retained during the ensuing successive reaction cycles of condensation, ketoreduction, and dehydration/ isomerization (Fig. 3). During each cycle, an additional double bond with trans configuration in the acyl chain via the dehydration reaction is introduced while the acyl chain is elongated. Subsequently, a precise double-bond isomerization takes place, namely, the original trans configuration at the 2,3-position is converted into the cis configuration and simultaneously moved to the 3,4-position in the fatty acyl chain (Fig. 3) (Wallis et al., 2002; Ratledge, 2004). The synthesis of DHA would follow the following sequence: 6:1(3) ➔ 8:1(5) ➔ 10:2(4,7) ➔ 12:3(3,6,9) ➔ 14:3(5,8,11) ➔ 16:4(4,7,10,13) ➔ 18:5(3,6,9,12,15) ➔ 20:5(5,8,11,14,17) ➔ 22:6(4,7,10,13,16,19,all cis) (Fig. 3). The source of hydrogens for each of the enzymatic steps is illustrated in Fig. 3. Thus, we can conclude that the PKS mechanism of PUFA synthesis is different from that of the FAS system in that (1) double bonds of the synthesized fatty acid are formed by multiple cycles of double bond insertions via the abbreviated reduction reactions (KS ➔ KR ➔ DH); (2) the D/H signature of the NADPH hydrogens incorporated into the fatty acid is conserved in the polyunsaturated fatty acids; and (3) less hydrogen is received directly fromcellular water (roughly 13% vs. 25% in surface bacteria; Sessions et al., 2002) (Fig. 3). Therefore, the isotope signature of hydrogens derived from NADPH is more pronounced, and is preserved in the fatty acids synthesized via the PKS pathway. Given that NADPH has the most depleted δD values (e.g.,−250‰) (Hayes, 2001; Schmidt et al., 2003), and that hydrogens are transferred directly by the flavin-free nucleotidedependent reductases from NADPH to fatty acids (McMurry and Begley, 2005), rather than by hydride transfer via flavoproteins which may cause isotope exchange with water (and would cause D-enrichment in fatty acids), PUFAs synthesized via the PKS system would have much more depleted δD, as observed in this study, than unsaturated fatty acids synthesized by the FAS system also possessed by the surface bacteria. 5. Conclusions and implications Results from this study showed that there is a mechanistic link between hydrogen isotope fractionation and biosynthetic pathway of fatty acids. The short-chain and long-chain polyunsaturated fatty acids of piezophilic bacteria were likely synthesized by two co-existing, biosynthetic pathways in the bacterium, the FAS and the PKS systems, which produce distinctly different δD in fatty acids. Thus, the lipid biosynthetic pathways can exert first-order control on the hydrogen isotope signature of bacterial membrane lipids. Our findings have important implications in marine biogeochemistry. The deep ocean probably represents the largest biotope of the Earth (Bartlett, 1999). Given that PUFAs synthesized by piezophilic bacteria retain much more negative δ13C (Fang et al., 2006) and δD (this study), the carbon and hydrogen isotope signatures of lipids can be used as tracers to determine the source of organic matter in deep sea. A case in mind is the long-chain PUFAs (e.g., DHA). LC-PUFA produced by marine plankton in surface ocean has more positive δD (Li et al., 2009), whereas PUFAs synthesized by piezophilic bacteria retain much more negative

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δ13C (Fang et al., 2006) and δD (this study). Given that piezophilic bacteria contribute PUFAs to the deep ocean sediment by two orders of magnitude more than surface ocean plankton (Fang et al., 2006), the carbon and hydrogen isotope signatures of PUFAs can be useful in determining the source, diagenesis, and vertical transport of organic matter in oceanic environments (e.g., Jones et al., 2008; Li et al., 2009, 2011). Finally, extensive water–rock interactions (e.g., serpentinization) can take place in the crustal aquifer where hydrogen and methane are produced (Cardace and Hoehler, 2010). Molecular hydrogen is a favorable electron donor for microorganisms and hydrogen oxidation can be a major metabolic pathway in the high-pressure environment of the crust biosphere (Edwards et al., 2012). Because of the unique δD signature of fatty acids from piezophilic bacteria and the nearly identical lipid/water hydrogen isotopic fractionations in eukaryotes and bacteria (e.g., Sessions et al., 1999; Zhang and Sachs, 2007), the δD of lipids can be a proxy to study microbial metabolism, the interactions between microbes, rock, and water in the subsurface, and the relationship between life in the deep biosphere and the global biogeochemical processes. Molecular hydrogen can also be produced in radiolysis of water (Lin et al., 2006). Thus, given different δD for H2 from different sources, the δD and δ13C of lipids can be useful in quantifying the relative importance of different sources of hydrogen for subsurface life and tracing the carbon flow in the deep biosphere. Acknowledgments We are gratefully to Dr. Michael E. Böttcher and two anonymous reviewers whose constructive comments improved the manuscript. This work was supported by the National Natural Science Foundation of China (No. 41240039) and the Department of Education (No. 20120072110026), by the State Key Laboratory of Marine Geology, Tongji University, and by the Trustees' Scholarly Endeavors Program of Hawaii Pacific University to J. F., and by the 973 Program (2013CB955704) to C. L. References Baillif, V., Robins, R.J., Le Feunteun, S., Lesot, P., Billault, I., 2009. Investigation of fatty acid elongation and desaturation steps in Fusarium lateritium by quantitative twodimensional deuterium NMR spectroscopy in chiral oriented media. J. Biol. Chem. 284, 10783–10792. Bartlett, D.H., 1999. Microbial adaptations to the psychrosphere/piezosphere. J. Mol. Microbiol. Biotechnol. 1, 93–100. Campbell, J.W., Cronan Jr., J.E., 2001. Escherichia coli FadR positively regulates transcription of the fabB fatty acid biosynthetic gene. J. Bacteriol. 183, 5982–5990. Campbell, B.J., Li, C., Sessions, A.L., Valentine, D.L., 2009. Hydrogen isotopic fractionation in lipid biosynthesis by H2-consuming Desulfobacterium autotrophicum. Geochim. Cosmochim. Acta 73, 2744–2757. Cardace, D., Hoehler, T.M., 2010. Extremophiles in serpentinizing systems: implications for life on the Early Earth and Other Planets. In: Harrison, S., Rajakaruna, N. (Eds.), Serpentine: A Model for Evolution and Ecology. University of California Press, pp. 29–48. Chikaraishi, Y., Naraoka, H., 2006. Carbon and hydrogen isotope variation of plant biomarkers in a plant–soil system. Chem. Geol. 231, 190–202. Chikaraishi, Y., Yamada, Y., Naraoka, H., 2005. Carbon and hydrogen isotopic compositions of sterols from riverine and marine sediments. Limnol. Oceanogr. 50, 1763–1770. Dirghangi, S.S., Pagani, M., 2013. Hydrogen isotope fractionation during lipid biosynthesis by Haloarcula marismortui. Geochim. Cosmochim. Acta 119, 381–390. Edwards, K., Fisher, A.T., Wheat, C.G., 2012. The deep subsurface biosphere in igneous ocean crust: frontier habitats for microbiological exploration. Front. Microbiol. 3, 1–11. Englebrecht, A.C., Sachs, J.P., 2005. Determination of sediment provenance at drift sites using hydrogen isotopes and unsaturation ratios in alkenones. Geochim. Cosmochim. Acta 69, 4253–4265. Fang, J., Findlay, R.H., 1996. The use of a classic lipid extraction method for simultaneous recovery of organic pollutants and phospholipids. J. Microbiol. Methods 27, 63–71. Fang, J., Kato, C., 2007. FAS or PKS, lipid biosynthesis and stable carbon isotope fractionation in deep-sea piezophilic bacteria. In: Méndez-Vilas, A. (Ed.), Communicating Current Research and Educational Topics and Trends in Applied Microbiology (2007). The Formatex Microbiology Book Series, Formatex Center, Spain, pp. 190–200. Fang, J., Uhle, M., Billmark, K., Bartlett, D.H., Kato, C., 2006. Fractionation of carbon isotopes in biosynthesis of fatty acids by a piezophilic bacterium Moritella japonica DSK1. Geochim. Cosmochim. Acta 70, 1753–1760. Hayes, J.M., 2001. Fractionation of carbon and hydrogen isotopes in biosynthetic processes. Rev. Mineral. Geochem. 43, 225–277. Heath, R.J., White, S.W., Rock, C.O., 2001. Lipid biosynthesis as a target for antibacterial agents. Prog. Lipid Res. 40, 467–497.

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