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Introduction

Hydrogen isotope biogeochemistry is the scientific study of biological, geological, and chemical processes in the environment using the distribution and relative abundance of hydrogen isotopes. There are two stable isotopes of hydrogen, ¹H and ²H, which vary in relative abundance on the order of hundreds of permil. The ratio between these two species can be considered the hydrogen isotopic fingerprint of a substance. Understanding isotopic fingerprints and the sources of fractionation that lead to variation between them can be applied to address a diverse array of questions ranging from ecology and hydrology to geochemistry and paleoclimate reconstructions. 

Paleoreconstructions

The isotopic compositions of biomolecules preserved in the sedimentary record can be used as a proxy for paleoenvironment reconstructions. Since water is the primary hydrogen source for photoautotrophs, the hydrogen isotope composition of their biomass can be related to the composition of their growth water and thereby used to gain insight into some properties of ancient environments. Studying hydrogen isotopes can be very valuable, as hydrogen is more directly related to climate than other relevant stable isotope systems. However, hydrogen atoms bonded to oxygen, nitrogen, or sulfur atoms are exchangeable with environmental hydrogen, which makes this system less straightforward [ref to earlier H exchange section]. To study the hydrogen isotope composition of biomolecules, it is preferable to use compounds where the hydrogen is largely bound to carbon, and therefore not exchangeable on experimental timescales. By this criteria, lipids are a much better subject for hydrogen isotope studies than sugars or amino acids.

The net fractionation between source water and lipids is denoted as ε_l/w, and can be represented as

$\epsilon _{l/w}={\frac {R_{l}}{R_{w}}}-1={\frac {\delta D_{l}+1}{\delta D_{w}+1}}-1$  

where w refers to the water, and l refers to the lipids.  

While the δD of source water is the biggest influence on the δD of lipids, discrepancies between fractionation factor values obtained from the slope and from the intercept of the regression suggest that the relationship is more complex than a two-pool fractionation. In other words, there are multiple fractionation steps that must be taken into account in understanding the isotopic composition of lipids.  

Plant leaf waxes

Photograph of a leaf, with sunlight reflecting off of the waxy surface.

Terrestrial plants make leaf waxes to coat the surfaces of their leaves as an adaptation to minimize water loss. These waxes are comprised largely of straight-chain n-alkyl lipids. They are insoluble, non-volatile, chemically inert, and resistant to degradation, making them easily preserved in the sedimentary record, and therefore good targets as biomarkers. 

Soil water and leaf water

The starting δD value of source water is highly variable, and is influenced by temperature, amount, and geography (the continental effect). The main water source for terrestrial plants is soil water, which largely resembles the hydrogen isotope composition of rain water, but varies between environments and with enrichment by precipitation, depletion by evaporation, and exchange with atmospheric water vapor. [ref to Sang's hydrology section]

There can be a significant offset between the δD value of source water and the δD value of leaf water at the site of lipid biosynthesis. No fractionation is associated with water uptake by roots, a process usually driven by capillary tension, with the one exception of xerophytes that burn ATP to pump water in extremely arid environments (with a roughly 10‰ depletion). However, leaf water can be substantially enriched relative to soil water due to transpiration, an evaporative process which is influenced by temperature, humidity, and the composition of surrounding water vapor. The leaf water hydrogen isotope composition can be described with a modified Craig-Gordon model, where ΔD_e is the steady state enrichment of leaf water, ε_eq is the temperature-dependent equilibrium fractionation between liquid water and vapor, ε_k is the kinetic isotope effect from diffusion between leaf internal air space and the atmosphere, ΔD_v is the leaf/air disequilibrium, e_a is atmospheric vapor pressure, and e_i is internal leaf vapor pressure.

$\Delta D_{e}=\epsilon _{eq}+\epsilon _{k}+(\Delta D_{v}-\epsilon _{k})({\frac {e_{a}}{e_{i}}})$

The Péclet effect, which describes the opposing forces of advection and diffusion can be added to the model as

$P={\frac {EL}{CD}}$

where E is the transpiration rate, L is the length scale of transport, C is the concentration of water, and D is the diffusion coefficient.

$\Delta D_{leaf}=\Delta D_{e}{\frac {(1-e^{P})}{P}}$

[ref to Sujung's ecohydrology section]

While the role of rain water δD as the fundamental control on the final δD of lipids is well documented, the importance of fractionation effects from rain water to soil water and leaf water on ε_l/w is appreciated but remains poorly understood.

Biochemical effects

Organic biomolecules are generally depleted relative to the δD of leaf water. However, differences between organisms, biosynthetic pathways, and biological roles of different molecules can lead to huge variability in fractionation; the diversity of lipid biomarkers spans a 600‰ range of δD values .  

Lipid biosynthesis is biochemically complex, involving multiple enzyme-dependent steps that can lead to isotope fractionations. There are three major pathways of lipid biosynthesis, known as the mevalonate pathway, the acetogenic pathway, and the 1-deoxyD-xylulose-5-phosphate/2-methylerythroyl-4-phosphate pathway. The acetogenic pathway is responsible for the production of n-alkyl lipids like leaf waxes, and is associated with a smaller δD depletion relative to source water than the other two lipid biosynthesis pathways. While leaf water is the main source of hydrogen in leaf biomolecules, relatively depleted hydrogen from acetate or NADPH is often added during biosynthesis, and contributes to the hydrogen composition of the final molecule. Secondary hydrogen exchange reactions, meaning hydrogenation and dehydrogenation reactions outside of the primary biosynthetic pathway, also contribute substantially to the variability of lipid hydrogen isotope composition.                  

It is important to note that biological differences in fractionation stem not only from biochemical differences between different molecules, but also from physiological differences between different organisms. For example, the δD values of multiple leaf wax molecules are enriched in shrubs (median ~ -90‰) relative to trees (median ~ -135‰), which themselves are enriched relative to both C3 (median ~ -160‰) and C4 grasses (median ~ -140‰). Between indiviudal species, substantial variation of δD values have been documented. Other physiological factors that contribute to variable leaf wax δD values include the seasonal timing of leaf development, response to external stress or environmental variability, and the presence or absence of stomata.                  

It can be difficult to distinguish between physiological factors and environmental factors, when many physiological adaptations are directly related to environment.                  

Environmental effects

Several environmental factors have been shown to contribute to leaf wax δD variability, in addition to environmental effects on the δD of source water. Humidity is known to impact lipid δD values at moderate humidity levels, but not at particularly high (>80%) or low (<40%) humidity levels, and a broad trend of enriched δD values, meaning smaller ε_l/w, is seen in arid regions. Temperature and sunlight intensity, both correlated to geographic latitude, have strong effects on the rates of metabolism and transpiration, and by extension on ε_l/w. Additionally, the average chain length of leaf wax molecules varies with geographic latitude, and ε_l/w has been shown to increase with increasing chain length.

When using biomarkers as a proxy for reconstructing ancient environments, it is important to be aware of the biases inherent in the sedimentary record. Leaf matter incorporated into sediment is largely deposited during the autumn, so seasonal variations in leaf waxes must be considered accordingly. Furthermore, sediments average leaf waxes over lots of different plants in both space and time, making it difficult to calibrate the biological constraints on ε_l/w. Finally, preservation of biomolecules in the geologic record does not faithfully represent whole ecosystems, and there is always the threat of hydrogen exchange, particularly if the sediments are subjected to high temperatures.

Summary of leaf wax δD as a paleoenvironment proxy

The hydrogen isotope relationships between rain water and leaf wax lipids. Adapted from Sachse et al., 2012.

The hydrogen isotope composition of leaf waxes can be summarized as the δD of rain water, with three main fractionation steps- evaporation from soil water, transpiration from leaf water, and lipid biosynthesis, which can be combined and measured as the net fractionation, or ε_l/w. With the application of ever-improving measurement techniques for single molecules, and correlation with other independent proxies in the geological record that can help constrain some variables, investigating the hydrogen isotope composition of leaf waxes can be extremely productive. Leaf wax δD data has been successfully applied to improving our understanding of climate driven changes in terrestrial hydrology, by demonstrating that ocean circulation and surface temperature have a significant effect on continental precipitation. Leaf wax δD values have also been used as records of paleoaltimetry to reconstruct the elevation gradients in ancient mountain ranges based on the effect of altitude on rain water δD.

Alkenones

Scanning electron micrograph of a single Emiliana huxleyi cell.

Another group of molecules frequently used in paleoreconstructions are alkenones, long chain largely unsaturated lipids produced exclusively by coccolithophores. Coccolithophores are marine haptophyte algae, and include the globally iconic species Emiliania huxleyi, one of the main CaCO₃ producers in the ocean. The δD values of alkenones are highly correlated to the δD values of sea water, and therefore can be used to reconstruct paleoenvironmental properties that constrain the isotopic composition of sea water. The most notable reconstruction that alkenone δD values are applied to is the salinity of ancient oceans.

Alkenones as a paleosalinity proxy

Both the δD values of sea water and the fractionations associated with hyptophyte biochemistry (ε_bio) are fairly well understood, so alkenones can be readily used to observe the secondary effect of salinity on δD. There is a well established positive linear correlation between salinity and ε_l/w, on the order of a ~3‰ change in fractionation per salinity unit. Hypothesized mechanisms for this effect include enrichment of D in intracellular water due to reduced exchange with extracellular water at higher salinity, removal of H from intracellular water due to increased production of solutes to maintain osmotic pressure at higher salinity, and lower haptophyte growth rates at higher salinity.

Alkenone δD values have been used successfully to reconstruct past salinity changes in the Mediterranean Sea, Black Sea, Panama Basin, and Mozambique Channel. As an extension of salinity, this data was also used to draw further conclusions about ancient environments, such as ancient freshwater flooding events, and the evolution of plankton in response to environmental changes.