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Okenane

Okenane, the diagenetic end product of okenone, is a biomarker for Chromatiaceae, the purple sulfur bacteria. These anoxygenic phototrophs use light for energy and sulfide as their electron donor and sulfur source. Discovery of okenane in marine sediments implies a past euxinic environment, where water columns were anoxic and sulfidic. This is potentially tremendously important for reconstructing past oceanic conditions, but so far okenane has only been identified in one Paleoproterozoic (1.6 billion years old) rock sample from Northern Australia.

Purple sulfur bacteria produce the pigment molecule okenone, which is then diagenetically altered and preserved as the partially saturated okenane. Discovering okenane in sediments is considered evidence of purple sulfur bacteria, implying an anoxic and sulfidic environment.

Background

In the Paleoproterozoic, the water column possibly became sulfidic and anoxic. Purple and green sulfur bacteria likely thrived in this euxinic environment. Purple sulfur bacteria produce the pigment okenone which, during diagenesis, degrades to okenane. Green sulfur bacteria with green pigments produce chlorobactene, which is altered to chlorobactane during burial. Green sulfur bacteria with brown pigments produce isorenieratene, which is preserved as isorenieratane. Each anoxygenic phototroph occupies a different depth range in the ocean, based on their pigment's light absorption. Biomarkers from these species may teach us about anoxic paleoenvironments.

Okenone is a carotenoid, a class of pigments ubiquitous across photosynthetic organisms. These conjugated molecules act as accessories in the light harvesting complex. Over 600 carotenoids are known, each with a variety of functional groups that alter their absorption spectrum. Okenone appears to be best adapted to the yellow-green transition (520nm) of the visible spectrum, capturing light below marine plankton in the ocean. This depth varies based on the community structure of the water column. A survey of microbial blooms found Chromatiaceae anywhere between 1.5m and 24m depth, but more than 75% occurred above 12 meters. Further planktonic sulfur bacteria occupy other niches: green sulfur bacteria, the Chlorobiaceae, that produce the carotenoid chlorobactene were found in greatest abundance above 6m while green sulfur bacteria that produce isorenieratene were predominantly identified above 17m. Finding any of these carotenoids in ancient rocks could constrain the depth of the oxic to anoxic transition as well as confine past ecology. Okenane and chlorobactane discovered in Australian Paleoproterozoic samples allowed conclusions of a temporarily shallow anoxic transition, likely between 12 and 25m.

Okenone is synthesized in 12 species of Chromatiaceae, spanning eight genera. Other purple sulfur bacteria have acyclic carotenoid pigments like lycopene and rhodopin. However, geochemists largely study okenone because it is structurally unique. It is the only pigment with a 2,3,4 trimethylaryl substitution pattern. In contrast, the green sulfur bacteria produce 2,3,6 trimethylaryl isoprenoids. The synthesis of these structures produce biological specificity that can distinguish the ecology of past environments. Okenone, chlorobactene, and isorenieratene are produced by sulfur bacteria through modification of lycopene. In okenone, the end group of lycopene produces a χ-ring, while chlorobactene has a φ-ring. The first step in biosynthesis of these two pigments is similar, formation of a β-ring by a β-cyclase enzyme. Then the syntheses diverge, with carotene desaturase/methyltransferase enzyme transforming the β-ring end group into a χ-ring. Other reactions complete the synthesis to okenone: elongating the conjugation, adding a methoxy group, and inserting a ketone. However, only the first synthetic steps are well characterized biologically.

Preservation

One diagenetic pathway proposed to saturate okenone to okenane is reductive desulphurization, where hydrogen sulfide adds to a double bond and is then removed. More research is needed on other reactions that remove functional groups before preservation.

Pigments and other biomarkers produced by organisms can evade microbial and chemical degradation and persist in sedimentary rocks. Under conditions of preservation, the environment is often anoxic and reducing, leading to chemical loss of functional groups like double bonds and hydroxyl groups. The exact reactions during diagenesis are poorly understood, although some have proposed reductive desulphurization as a mechanism for saturation of okenone to okenane. There is always the possibility that okenane is created by abiotic reactions, possibly from methyl shifts in β-carotene. If this reaction was occurring, okenane would have multiple precursors and the biological specificity of the biomarker would be diminished. However, it is unlikely that isomer specific rearrangements of two methyl groups are occurring without enzymatic activity. The majority of studies conclude that okenane is a true biomarker of purple sulfur bacteria. However, other biological arguments against this interpretation hold merit. Past organisms that synthesized okenone may not be modern analogues of purple sulfur bacteria. There may also be other okenone producing photosynthesizers in today’s ocean that are uncharacterized. A further complication is horizontal gene transfer. If Chromatiaceae gained the ability to create okenone more recently that the Paleoproterozoic, then the okenane does not track purple sulfur bacteria, but rather the original gene donor. These ambiguities indicate that interpretation of biomarkers in billion-year-old rocks will be limited by understanding of ancient metabolisms.

Measurement techniques

GC/MS

Prior to analysis, sedimentary rocks are extracted for organic matter. Typically, only less than one percent is extractable due to the thermal maturity of the source rock. The organic content is often separated into saturates, aromatics, and polars. Gas chromatography can be coupled to mass spectrometry to analyze the extracted aromatic fraction. Compounds elute from the column based on their mass to charge ratio (M/Z) and are displayed based on relative intensity. Peaks are assigned to compounds based on library searches, standards, and relative retention times. Some molecules have characteristic peaks that allow easy searches at particular mass to charge ratios. For the trimethylaryl isoprenoid okenane this characteristic peak occurs at M/Z of 134.

Isotope ratios

Carbon isotope ratios of purple and green sulfur bacteria are significantly different that other photosynthesizing organisms. The biomass of the purple sulfur bacteria, Chromatiaceae is often depleted in δ¹³C compared to typical oxygenic phototrophs while the green sulfur bacteria, Chlorobiaceae, are often enriched. This offers an additional discrimination to determine ecological communities preserved in sedimentary rocks. For the biomarker okenane, the δ¹³C could be determined by an Isotope Ratio Mass Spectrometer.

Case study: Northern Australia

In modern environments, purple sulfur bacteria thrive in meromictic (permanently stratified) lakes and silled fjords and are seen in few marine ecosystems. Hypersaline waters like the Black Sea are exceptions. However, billions of years ago, when the oceans were anoxic and sulfidic, phototrophic sulfur bacteria had more habitable space. Researchers at the Australian National University and the Massachusetts Institute of Technology investigated 1.6-billion-year-old rocks to examine the chemical conditions of the Paleoproterozoic ocean. Many believe that this time had deeply penetrating oxic water columns because of the disappearance of banded iron formations roughly 1.8 billion years ago. Others, spearheaded by Donald Canfield’s 1998 Nature paper, believe that waters were euxinic. Examining rocks from the time uncovered biomarkers of both purple and green sulfur bacteria, adding evidence to support the Canfield Ocean hypothesis. The sedimentary outcrop analyzed was the Barney Creek Formation from the McArthur group in northern Australia. Sample analysis identified both the 2,3,6 trimethylarl isoprenoids (chlorobactane) of Chlorobiaceae and the 2,3,4 trimethylaryl isoprenoids (okenane) of Chromatiaceae. Both chlorobactane and okenane indicate a euxinic ocean, with sulfidic and anoxic surface conditions below 12-25m. The authors concluded that although oxygen was in the atmosphere, the Paleoproterozoic oceans were not completely oxygenated.

History of Hydrogen Isotopes

Earliest Work

The study of hydrogen stable isotopes began with the discovery of deuterium by chemist Harold Urey of the famous Urey and Miller experiment. Even though the neutron was not realized until 1932, Urey began searching for “heavy hydrogen” in 1931. Urey and his colleague George Murphy calculated the redshift of heavy hydrogen from the Balmer series and observed very faint lines on a spectrographic study. To intensify the spectroscopic lines for publishable data, Murphy and Urey paired with Ferdinand Brickwedde and distilled a more concentrated pool of heavy hydrogen, known today as deuterium. This work on hydrogen isotopes won Urey the 1934 Nobel Prize in Chemistry.

Harold Urey, whose pioneering work on hydrogen isotopes won him the 1934 Nobel Prize in Chemistry.

Also in 1934, scientists Ernest Rutherford, Mark Oliphant, and Paul Harteck, produced the radioactive isotope tritium by hitting deuterium with high energy nuclei. The deuterium used in the experiment was a generous gift of heavy water from the Berkeley physicist Gilbert N Lewis. Interestingly, bombarding deuterium produced two previously undetected isotopes, helium-3 and hydrogen-3. Rutherford and his colleagues successfully created tritium, but incorrectly assumed that helium-3 was the radioactive component. The work of Luis Walter Alvarez and Robert Cornog first isolated tritium and reversed Rutherford's incorrect notion. Alvarez reasoned that tritium was radioactive, but did not measure the half life, although calculations at the time suggested over ten years. At the end of World War II, the physical chemist Willard Libby detected the residual radioactivity of a tritium sample with a Geiger counter, providing a more accurate understanding of the half life, now accepted at 12.3 years.

Impact on Physical Chemistry

The discovery of hydrogen isotopes also impacted the field of physics in the 1940's, as Nuclear Magnetic Resonance (NMR) spectroscopy was first invented. Today, organic chemists utilize NMR for mapping protein interactions or identifying small compounds, but NMR was first a passion project of physicists. All three isotopes of hydrogen were found to have magnetic properties suitable for NMR spectroscopy. The first chemist to fully express an application of NMR was George Pake, who measured gypsum (CaSO₄.2H₂O) as a crystal and powder. The signal observed, called the Pake doublet, was from the magnetically active hydrogens in water. Pake then calculated the proton-proton bond distance. NMR measurements were further revolutionized when commercial machines became available in the 1960's. Before this, NMR experiments involved constructing massive projects, locating large magnets, and hand wiring miles of copper coil. Proton NMR remained the most popular technique throughout advancements in following decades, but deuterium and tritium were used in other flavors of NMR spectroscopy. Deuterium has a different magnetic moment and spin than protium, but generally a much smaller signal. Historically, deuterium NMR is a poor alternative to proton NMR, but has been used to study the behavior of lipids on membranes. Recently, a variation of deuterium NMR called ²H-SNIF has shown potential for understating position specific isotope compositions and comprehending biosynthetic pathways. Tritium is also used in NMR, as it is the only nucleus more sensitive than protium, generating very large signals. However, tritium's radioactivity discouraged many studies of T-NMR.

While tritium radioactivity discourages use in spectroscopy, the energy from decay is essential for nuclear weapons. Scientists began understanding nuclear energy as early as the 1800's, but large advancements were made in studies of the atomic bomb in the early 1940's. War time research, especially the Manhattan project, greatly accelerated scientific understanding of radioactivity. Tritium is a byproduct in reactors, a result of hitting lithium-6 with neutrons, producing almost 5 MeV of energy.

${}^{6}\mathrm {Li} +n\longrightarrow {}^{4}\mathrm {He} +{}^{3}\mathrm {T} +5\ \mathrm {MeV}$

A photograph from the Greenhouse Project in 1952, where the first fission boosted nuclear weapon was tested.

In boosted-fission nuclear weapons a mixture of deuterium and tritium are heated until there is thermonuclear fission to produce helium and release free neutrons. The flurry of fast neutron particles would then excite further fission reactions with uranium, creating a "boosted" nuclear bomb. In 1951, during Operation Greenhouse, a prototype named George, successfully validated the proof of concept for such a weapon. However, the first true boosted fission nuclear device, Greenhouse Item, was successfully tested in 1952, generating 45.5 kilotons of explosive yield, nearly double the value of an un-boosted system. The United States stopped producing tritium in nuclear reactors in 1988, but nuclear weapons testing in the 1950's added large spikes of radioactive elements to the atmosphere, especially radiocarbon and tritium. This complicated measurements for geologists using radiometric dating of carbon. However, some oceanographers benefited from the tritium increase, utilizing the signal in the water to trace physical mixing of water masses.

Impact in Biogeochemistry

In biogeochemistry, scientists focused primarily on the stable isotope of deuterium as a tracer for environmental processes, especially the water cycle. The American geochemist Harmon Craig, once a graduate student of Urey, discovered the relationship between rainwater’s hydrogen and oxygen isotope ratios. The linear correlation between the two heavy isotopes is conserved worldwide and referred to as the Global Meteoric Water Line. By the late 1960’s, the focus of hydrogen isotopes shifted away from water and towards organic molecules. Plants use water to form biomass, but a 1967 study by Zebrowski, Ponticorvo, and Rittenberg found that the organic material in plants had less deuterium than the water source. Zebrowski’s research measured the deuterium concentration of fatty acids and amino acids derived from sediments in the Mohole drilling project. Further studies by Bruce Smith and Samuel Epstein in 1970 confirmed the depletion of deuterium in organics compared to environmental water. Another duo in 1970, Schiegl and Vogel, analyzed the composition of hydrogen isotopes as water became biomass, as biomass became coal and oil, and as oil became natural gas. In each step they found deuterium further depleted. A landmark paper in 1980 by Marilyn Epstep, now M. Fogel, and Thomas Hoering titled "Biogeochemistry of the stable hydrogen isotopes" refined the links between organic materials and sources.

In this early stage of hydrogen stable isotope study, most isotope compositions or fractionations were reported as bulk measurements of all organic material or all inorganic material. Some exceptions include cellulose and methane, as these compounds are easily separated. Another advantage of methane for compound specific measurements is the lack of hydrogen exchange. Cellulose has exchangeable hydrogen, but chemical derivatization can prevent swapping of cellulose hydrogen with water or mineral hydrogen sources. Cellulose and methane studies in the 1970’s and 1980’s set the standard for modern hydrogen isotope geochemistry.

Measurements of individual compounds was made possible in the late 1990’s and early 2000’s with advancements in mass spectrometry. The Thermo Delta+XL transformed measurements as the first instrument capable of compound specific isotope analysis. It was then possible to look at smaller samples with more precision. Hydrogen isotope applications quickly emerged in petroleum geochemistry by measuring oil, paleoclimatology by observing lipid biomarkers, and ecology by constructing trophic dynamics. Modern advances are currently underway in the clumped isotope composition of methane after development of the carbonate thermometer. Precise measurements are also enabling focus on microbial biosynthetic pathways involving hydrogen. Ecologists studying trophic levels are especially interested in compound specific measurements for construction of past diets and tracing predator-prey relationships. Highly advanced machines are now promising position specific hydrogen isotope analysis of biomolecules and natural gases.

Physical Chemistry of Hydrogen Isotopes

Hydrogen Isotope Formation

Protium or hydrogen-1, with one proton and no neutrons, is the most abundant element in the solar system, formed in the earliest rounds of stellar explosions after the Big Bang. After the universe exploded into life, the hot and dense cloud of particles began to cool, first forming subatomic particles like quarks and electrons, which then condensed to form protons and neutrons. Elements larger than hydrogen and helium were produced with successive stars, forming from the energy released during supernovae.

Deuterium or hydrogen-2, with one proton and one neutron, is also known to have cosmic origin. Like protium, deuterium was produced very early in the universe’s history during the Big Bang nucleosynthesis. As protons and neutrons combined, helium-4 was produced with a deuterium intermediate. Alpha reactions with helium-4 produce many of the larger elements that dominate today’s solar system. However, before the universe cooled, high-energy photons destroyed any deuterium, preventing larger element formation. This is referred to as the deuterium bottleneck, a restriction on the timeline for nucleosynthesis. All of today’s deuterium originated from this proton-proton fusion after sufficient cooling.

Tritium, or hydrogen-3, with one proton and two neutrons, was produced by proton and neutron collisions in the early universe as well, but it has since radioactively decayed to helium-3. Modern tritium cannot be explained by big bang nucleosynthesis because of tritium’s short half-life of 12.3 years. Today’s tritium concentration is instead governed by nuclear reactions and cosmic rays. The radioactive beta decay of tritium to helium releases an electron and an antineutrino, with an average energy release of 18.6 MeV. It is important to note that this is classified as a relatively weak beta reaction, so the radioactivity cannot permeate skin. Tritium is thus only hazardous if directly ingested or inhaled.

${}^{3}\mathrm {T} \longrightarrow {}^{3}\mathrm {He} +{e}^{-}+18.6\ \mathrm {MeV}$

Quantum Properties

Protium is a spin 1/2 subatomic particle and is therefore a fermion. Other fermions include neutrons, electrons, and the radioactive isotope tritium. Fermions are governed by Pauli's exclusion principle, where no two particles can have the same quantum number. However, bosons like deuterium and photons, are not bound by exclusion and multiple particles can occupy the same energy state. This fundamental difference in ¹H and ²H manifests in many physical properties. Integer spin particles like deuterium follow Bose-Einstein statistics while fermions with half integer spins follow Fermi-Dirac statistics. Wave functions that describe multiple fermions must be antisymmetric with respect to swapping particles, while boson wave functions are symmetric. Because bosons are indistinguishable and can occupy the same state, collections of bosons behave very differently than fermions at colder temperatures. As bosons are cooled and relaxed to the lowest energy state, phenomena like superfluidity and superconductivity occur.

Kinetic and Equilibrium Isotope Effects

  Isotopes differ according to their number of neutrons, which directly impacts physical properties based on mass and size. Typical hydrogen is called hydrogen-1 or protium and has no neutrons. Deuterium or hydrogen-2 has one neutron and tritium or hydrogen-3 has two neutrons. These additional neutrons significantly impact the mass of the element, leading to different chemical physical properties. This effect is especially prevalent in hydrogen isotopes, since addition of a neutron doubles the mass from protium to deuterium. For higher order elements like carbon, oxygen, nitrogen, or sulfur, the mass difference is diluted.

Physical chemists often model bonding with the quantum harmonic oscillator, simplifying a hydrogen-hydrogen bond as two balls connected by a spring. The quantum harmonic oscillator is itself based on Hooke’s Law and acts as a good approximation of the Morse potential that accurately describes bonding. Modeling hydrogen and deuterium in a chemical reaction demonstrates the energy distributions of isotopes in products and reactants. Lower energy levels for the heavier isotope deuterium can be explained mathematically by the harmonic oscillator's dependence on the inverse of the reduced mass, denoted μ. Thus, a larger reduced mass is a larger denominator and thus a smaller zero point energy and a lower energy state in the quantum well.

A simplified model of a chemical reaction with pathways for H and D isotopes of hydrogen. The positions on the energy wells are based on the quantum harmonic oscillator. Note the lower energy state of the heavier isotope and the higher energy state of the lighter isotope. Under equilibrium conditions, the heavy isotope is favored in the products as it is more stable. Under kinetic conditions, like an enzymatic reaction, the lighter isotope is favored because of a lower activation energy.

Calculating the reduced mass of a hydrogen-hydrogen bond versus a deuterium-deuterium bond gives:

$\mu _{H}={\cfrac {m_{H}m_{H}}{m_{H}+m_{H}}}={\cfrac {1}{2}}.\!\,$

$\mu _{D}={\cfrac {m_{D}m_{D}}{m_{D}+m_{D}}}={\cfrac {4}{4}}=1.\!\,$

The quantum harmonic oscillator has energy levels of the following form, where k is the spring constant and h is plank's constant.

$E_{n}=\hbar {\sqrt {k \over \mu }}\left(n+{1 \over 2}\right).$

The effects of this energy distribution manifest in the kinetic isotope effect and the equilibrium isotope effect. In a reversible reaction, under equilibrium conditions, the reaction will proceed forwards and backwards, distributing the isotopes to minimize thermodynamic free energy. Some time later, at equilibrium, more heavy isotopes will be on the product side. The stability of the lower energy drives the products to be enriched in deuterium relative to reactants. Conversely, under kinetic conditions, reactions are generally irreversible. The limiting step in the reaction is overcoming the activation energy barrier to reach an intermediate state. The lighter isotope has a higher energy state in the quantum well and will thus be preferentially formed into products. Thus under kinetic conditions the product will be relatively depleted in deuterium.

Kinetic isotope effects are common in biological systems and are especially important for hydrogen isotope biogeochemistry. Kinetic effects usually result in larger fractionations than equilibrium reactions. In any isotope system, kinetic effects are stronger for larger mass differences. Light isotopes in most systems also tend to move faster but form weaker bonds. At high temperatures, entropy explains a large signal in isotope composition. However, when temperature decreases isotope effects are more expressed and randomness plays less of a role. These general trends are exposed in further understanding of bond breaking, diffusion or effusion, and condensation or evaporation reactions.

Chemistry of Hydrogen Exchange

One of the major complications in studying hydrogen isotopes is the issue of exchangeability. At many time scales, ranging from hours to geological epochs, scientists have to consider if the hydrogen moieties in studied molecules are the original species or if they represent exchange with water or mineral hydrogen near by. Research in this area is still inconclusive in regards to rates of exchange, but it is generally understood that hydrogen exchange complicates the preservation of information in isotope studies.

Rapid exchange

Hydrogen atoms easily separate from electronegative bonds such as hydroxyl bonds (O-H), nitrogen bonds (N-H), and thiol/mercapto bonds (S-H) on hour to day long timescales. This rapid exchange is particularly problematic for measurements of bulk organic material with these functional groups because isotope compositions are more likely to reflect the source water and not the isotope effect. For this reason, records of paleoclimate that are not measuring ancient waters, rely on other isotopic markers. Advancements in the 1990's held promising potential to resolve this problem: samples were equilibrated with two variations of heavy water and compared. Their ratios represent an exchange factor that can calibrate measurements to correct for hydrogen and deuterium swapping..

Carbon bound hydrogen exchange

For some time, researchers believed that large hydrocarbon molecules were impervious to hydrogen exchange, but recent work has identified many reactions that allow isotope reordering. The isotopic exchange becomes relevant at geological time scales and has impacted work of biologists studying lipid biomarkers as well as geologists studying ancient oil. Reactions responsible for exchange include:

The trend of carbocation stability. Note the stabilization effects from adjacent carbons that donate electrons to the positive charge. The opposite trend is seen in carbanion stability. In isotopes, a tertiary bound hydrogen is more likely to be lost because the resulting carbocation is the most stable species.

1. Radical reactions that cleave C-H bonds.
2. Ionic exchange that of tertiary and aromatic hydrogen.
3. Enolizations that activate hydrogens on ketone alpha carbons.
4. Stereochemical exchange that causes sterochemical inversion.
5. Constitutional exchange like methyl shifts, double bond migrations and carbon backbone rearrangements.

Detailed kinetics of these reactions have not been determined. However, it is known that clay minerals catalyze ionic hydrogen exchange faster than other minerals. Thus hydrocarbons formed in clastic environments exchange more than those in carbonate settings. Aromatic and tertiary hydrogen also have greater exchange rates than primary hydrogen. This is due to the increasing stability of associated carbocations. Primary carbocations are considered too unstable to physically exist and have never been isolated in an FT-ICR spectrometer. On the other hand, tertiary carbocations are relatively stable and are often intermediates in organic chemistry reactions. This stability, which increases the likelihood of proton loss, is due to the electron donation of nearby carbon atoms. Resonance and nearby lone pairs can also stabilize carbocations via electron donation. Aromatic carbons are thus relatively easy to exchange.

Many of these reactions have a strong temperature dependence, with higher temperatures typically accelerating exchange. However, different mechanisms may prevail at each temperature window. Ionic exchange, for example, has the most significance at low temperatures. In such low temperature environments, there is potential for preserving the original hydrogen isotope signal over hundreds of millions of years. However, many rocks in geologic time have reached significant thermal maturity. Even by the onset of the oil window it appears that much of the hydrogen has exchanged. Recently, scientists have explored a silver lining: hydrogen exchange is a zero order kinetic reaction (for carbon bound hydrogen at 80-100°C, the half-times are likely 10⁴ - 10⁵ years). Applying the mathematics of rate constants would allow extrapolation to original isotopic compositions. While this solution holds promise, there is too much disagreement in the literature for robust calibrations.

Vapor Isotope Effects

Vapor isotope effects occur for protium, deuterium, and tritium, because each isotope has different thermodynamic properties in the liquid and gaseous phases. For water molecules, the condensed phase is more enriched while the vapor is more depleted. For example, rain condensing from a cloud will be heavier than the vapor starting point. Generally, the large variations in deuterium concentrations of water are from the fractionations between liquid, vapor, and solid reservoirs. In contrast to the fractionation pattern of water, non-polar molecules like oils and lipids, have gaseous counterparts enriched with deuterium relative to the liquid. This is thought to be associated with the polarity from hydrogen bonding in water that does not interfere in long chain hydrocarbons.