Elsevier

Organic Geochemistry

Volume 64, November 2013, Pages 105-111
Organic Geochemistry

Hydrogen isotope fractionation during lipid biosynthesis by Tetrahymena thermophila

https://doi.org/10.1016/j.orggeochem.2013.09.007Get rights and content

Highlights

  • δDtetrahymanol and δDfatty acid correlated with δDwater in Tetrahymena cultures.

  • Temperature increase caused D enrichment in some lipids.

  • At 36 °C, relative abundance of tetrahymanol increased markedly.

  • Tetrahymanol was D depleted vs. water at 24 °C and 30 °C and D enriched at 36 °C.

Abstract

Hydrogen isotope ratio values of lipids are increasingly used to reconstruct past variation in hydrological conditions. However, apart from recording the hydrogen isotope composition of ambient water, δD values of lipids also depend on specific biosynthetic pathways and growth conditions. We have evaluated the hydrogen isotope fractionation by the ciliated protozoan, Tetrahymena thermophila, grown in pure culture at three temperatures (24 °C, 30 °C and 36 °C) and in water with a range of hydrogen isotope composition. T. thermophila synthesizes tetrahymanol, a pentacyclic triterpenoid alcohol and the diagenetic precursor of the biomarker gammacerane. We focused our attention on the isotopic controls on tetrahymanol and various fatty acids (FAs).

The δD values of FAs and tetrahymanol correlated linearly with the hydrogen isotope composition of water, but growth temperature was also clearly an important factor controlling lipid D/H compositon. Hydrogen isotope fractionation during tetrahymanol biosynthesis changed with higher growth temperatures, resulting in D-depleted signatures relative to water at 24 °C and 30 °C and D-enriched composition at 36 °C. T. thermophila grown at 36 °C – a temperature above opitmum growth conditions – showed a significant change in lipid composition, with the abundance of tetrahymanol increasing relative to total FAs. We suggest the change is a response to temperature stress and a decrease in the stability of the cell membrane. The temperature effect is also presumed to impact δDlipid by altering the hydrogen isotopic composition of NADPH and potentially intracellular water.

Introduction

Hydrogen isotope ratio values of lipids are determined by the hydrogen isotopic composition of ambient water, the redox-active hydrogen in reducing agents (e.g. NADPH) and, in the case of heterotrophic organisms, carbon substrate and metabolic pathways (Sessions et al., 1999, Sessions et al., 2002, Zhang and Sachs, 2007, Zhang et al., 2009a, Dirghangi and Pagani, 2013). Ubiquitous lipids synthesized by a variety of organisms [e.g. palmitic acid (n-C16 fatty acid, FA)] have been used as proxies for water hydrogen isotope composition (Huang et al., 2002; Hou et al., 2006), but the approach is potentially complicated because of differences in biosynthetic D/H fractionation between species responsible for biomarker production, as well as isotopic effects associated with growth conditions. For example, pure cultures of the freshwater green algae Eudorina unicocca and Volvox aureus, and three species of Botryococcus braunii produced n-C16 FA that differed in δD values by ca. 100‰, demonstrating the potential for substantial uncertainty in water D/H reconstruction using lipid compounds (Zhang and Sachs, 2007). On the other hand, lipids synthesized by more specific algal species or families (e.g. botryococcene from Botryococcus algae) can act as more reliable δDwater proxies (Zhang and Sachs, 2007).

Tetrahymanol is the source of the biomarker gammacerane, a diagenetic pentacyclic triterpane first observed in the Green River Shale (Hills et al., 1966). Gammacerane is derived from tetrahymanol via sulfurization and consequent carbon–sulfur cleavage (Sinnighe Damsté et al., 1995), as opposed to the initially proposed mechanism involving dehydration and hydrogenation (ten Haven et al., 1989).

The first reported source of tetraymanol was Tetrahymena pyriformis (Mallory et al., 1963), a ciliated Protozoan belonging to the class Oligohymenophorea (subclass Hymenostomatia). However, a land fern and a purple non-sulfur bacterium (Rhodopseudomonas palustris) are also known to synthesize it under certain conditions (Kleemann et al., 1990, Rashby et al., 2007). Nonetheless, ciliates are clearly the dominant source of tetrahymanol in sediments (ten Haven et al., 1989), as confirmed by stable carbon isotopic composition (Sinnighe Damsté et al., 1995).

Tetrahymena is predominantly a freshwater organism, although it has been reported from thermal springs and soils (Corliss, 1973). Tetrahymanol was also found to be the dominant neutral lipid in eight strains of marine ciliates, indicating that its synthesis is not restricted to Tetrahymena alone, but is common across the class Oligohymenophorea (Harvey and McManus, 1991). Thus, although tetrahymanol is not as source-specific as botryococcene, it derives from a more specific source than many sterols and hopanols.

The presence of gammacerane in petroleum is commonly used as a marker for a stratified water column during source-rock deposition under hypersaline conditions (Peters et al., 2005a, Peters et al., 2005b, Rullkötter et al., 1984, Rullkötter et al., 1985, Fowler and McAlpine, 1995, Terken and Frewin, 2000) and tetrahymanol has been reported in ancient and modern hypersaline deposits (ten Haven et al., 1989, Barbe et al., 1990, Romero-Viana et al., 2013). However, tetrahymanol is synthesized predominantly by freshwater and marine bacteriovorous ciliates that are not strictly restricted to hypersaline environments. Tetrahymanol-producing ciliates are bacteriovores, feeding on bacteria at the oxic-anoxic interface and associated mainly with water column stratification (Sinnighe Damsté et al., 1995). Hypersaline lakes are often characterized by stratification and are favorable environments for tetrahymenol-producing ciliates, and account for the association of tetrahymanol and gammacerane with hypersaline deposits.

We have studied D/H fractionation of tetrahymanol and FAs by growing a pure culture of Tetrahymena thermophila in water with varying δD values across a range of temperature.

Section snippets

Culture experiments

A T. thermophila strain was obtained from the Tetrahymena Stock Center at Cornell University and grown in batch culture (200 ml in 1 l Erlenmeyer flasks) in a growth medium consisting of 5.5 g/l dextrose, 0.625 g/l bacto peptone, 2.5 g/l yeast extract and 33 μM FeCl3. It was grown in a shaker bath at 24 °C, 30 °C and 36 °C. For each temperature experiment, five isotopically distinct growth media were used. The hydrogen isotope composition of the medium was altered by adding aliquots of D2O (99%; Table 1

Growth rate of T. thermophila

T. thermophila growth rate exhibited only small differences with increasing temperature: 1.35 divisions day−1 at 24 °C, 1.37 divisions day−1 at 30 °C and 1.36 divisions day−1 at 36 °C.

Lipid distribution of T. thermophila

Tetrahymanol was the dominant neutral lipid in all cultures, but abundance varied with temperature. At 30 °C, a variety of FAs were observed, including saturated n-acids (C14, C16, C17 and C18), with n-C16:0 the most abundant and n-C17:0 the least abundant. T. thermophila grown at 24 °C contained lower molecular weight

Consideration of carbon substrate

The medium had dextrose as the primary carbon source, but also contained small amounts of peptone and yeast extract, which are mixtures of various compounds including carbohydrates, proteins and amino acids. Some of them can potentially act as carbon substrates, but δD values of these trace compounds were unknown. Lipids synthesized by T. thermophila were significantly D-enriched, indicating that a D-enriched substrate was consumed. This suggests that dextrose (δDdextrose 29.97‰) was the

Conclusions

T. thermophila was grown in pure culture at three temperatures – 24 °C, 30 °C and 36 °C. Growth rate was highest at 30 °C and lower at 24 °C and 36 °C. Tetrahymanol and FAs of various chain length (n-C12:0, n-C14:0, n-C16:0, n-C17:0 and n-C18:0) were synthesized in varying amounts. The δD values of tetrahymanol and FAs showed near perfect correlation with δDwater, although temperature exerted a significant influence on the relationship. Tetrahymanol, in particular, was characterized by a reversal of D

Acknowledgements

We would like to thank the Tetrahymena Stock Center at Cornell University for providing us with the T. thermophila strain. We would also like to thank A.L. Sessions and an anonymous reviewer for comments that were helpful for the improvement of the manuscript.

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