Daniel A. Stolper

The stable carbon and hydrogen isotopic composition of methane is widely used to determine its sources. Methanogenic growth on methanol generates methane with significantly lower ¹³ C/ ¹² C ratios relative to other substrates, which is used as a marker for this metabolism in environmental samples. The biochemical basis for the large carbon isotope effect associated with methanol growth is currently unknown. Here, we measure the change in the carbon and hydrogen stable isotopic compositions of the methane produced by Methanosarcina acetivorans during growth on methanol. We couple these results with an inverse modeling approach to calculate the kinetic isotopic effects (KIEs) of the rate-limiting step, catalyzed by the methanol-specific methyltransferase complex (MTA). Through this process, we estimate the carbon KIE of MTA ( ¹³ ε _MTA ) to be −66‰ and the hydrogen KIE of MTA ( ² ε _MTA ) to be −56‰. Our results indicate that the ¹³ ε _MTA contributes substantially to the large carbon isotope effect observed on methanol. We also show that mutant strains that express only a single copy of the MTA complex (MtaC1B1A1, MtaC2B2A1, or MtaC3B3A1) have ¹³ ε _MTA and ² ε _MTA that are indistinguishable from the wild-type strain. Finally, we propose that methanol activation by MTA will remain rate-limiting, even at low environmental methanol concentrations, and the large ¹³ ε _MTA would be expressed in situ as well. IMPORTANCE: Mapping the biological sources of methane is vital for accurately modeling global emissions of this potent greenhouse gas. Methanogenic growth on methanol produces methane with a uniquely depleted carbon isotope signature, which is commonly used as an environmental indicator of this metabolism. The biochemical mechanisms driving this isotopic signature have remained unclear. In this study, we identify the methanol-specific methyltransferase (MTA) complex as the primary driver of these large carbon isotope effects in the model methanogen, Methanosarcina acetivorans . By utilizing M. acetivorans mutants that express a single MTA complex, we demonstrate that these effects are consistent across MTA isozymes that are expressed during different growth phases. Our results suggest these isotopic signatures are intrinsic to the enzyme complex and persist in natural environments. These findings provide a critical biochemical foundation for using stable isotopes to track methane production from methanol in diverse natural ecosystems.

The stable hydrogen and carbon isotopic composition of methane is widely used to determine its sources. Methanogenic growth on methanol generates methane with significantly lower ¹³ C/ ¹² C ratios relative to other substrates, which is often used as a marker for this metabolism in environmental samples. The biochemical basis for the unusual isotope effect associated with methanol growth is currently unknown. Here, we grew Methanosarcina acetivorans on methanol and measured the change in the carbon and hydrogen stable isotopic compositions of the methane. We coupled these results with an inverse modeling approach to calculate the kinetic isotopic effects (KIEs) of the rate-limiting step, catalyzed by the methanol-specific methyltransferase complex (MTA). Through this process, we estimate the carbon KIE of MTA ( ¹³ ε _MTA ) as −65.5 ‰ and the hydrogen KIE of MTA ( ² ε _MTA ) as −56 ‰. Next, we show that the ¹³ ε _MTA contributes substantially to the large isotopic effect observed for methylotrophic methanogenesis on methanol. We also show that mutant strains that express only a single copy of the MTA complex (either MtaC1B1A1, MtaC2B2A1, or MtaC3B3A1) have ¹³ ε _MTA and ² ε _MTA that are indistinguishable from the wild-type strain. Finally, based on a thermodynamic analysis, we propose that methanol activation by MTA will remain rate-limiting, even at low environmental methanol concentrations, and the large ¹³ ε _MTA would be expressed in situ as well. Importance: Methane is a potent greenhouse gas, and distinguishing between its biological sources is vital for modeling global carbon cycles. Methylotrophic methanogenesis produces methane with a uniquely depleted carbon isotope signature. However, the biochemical mechanisms driving this fractionation have remained unclear. In this study, we identify the methanol-specific methyltransferase (MTA) complex as the primary driver of these large carbon isotope effects. By utilizing Methanosarcina acetivorans mutants, we demonstrate that these effects are consistent across different MTA isozymes. Our results suggest these signatures are intrinsic to the enzyme complex and persist at low substrate concentrations. These findings provide a critical biochemical foundation for using stable isotopes to track microbial methane production in diverse natural ecosystems.

The chemical compositions of island arc basalts (IAB) reflect contributions from the mantle as well as fluids and melts from the subducting slab. Addition of radiogenic seawater Sr to oceanic crust through hydrothermal alteration and subsequent subduction is often invoked to explain elevated ⁸⁷ Sr/ ⁸⁶ Sr signatures in modern IAB. However, changes in the ⁸⁷ Sr/ ⁸⁶ Sr of island arc magmatic rocks through time has not been investigated, limiting our understanding of the factors influencing the Sr budgets of arcs throughout Earth’s history. To address this, we compiled ⁸⁷ Sr/ ⁸⁶ Sr values from island arc magmatic rocks ranging in age from modern to Paleoproterozoic, only including data from island arc localities that best preserve initial magmatic ⁸⁷ Sr/ ⁸⁶ Sr. Median initial ⁸⁷ Sr/ ⁸⁶ Sr values are consistently elevated compared to depleted mantle ⁸⁷ Sr/ ⁸⁶ Sr over this period, indicating persistent enrichment in radiogenic Sr in island arcs. Moreover, the elevation in island arc ⁸⁷ Sr/ ⁸⁶ Sr relative to the depleted mantle is variable. A notable rise in island arc ⁸⁷ Sr/ ⁸⁶ Sr during the late Neoproterozoic coincides with a steep increase in seawater ⁸⁷ Sr/ ⁸⁶ Sr and Sr concentration. To investigate this potential connectivity, we modeled the ⁸⁷ Sr/ ⁸⁶ Sr of island arc magmas between 0 and 830 Ma with inputs of depleted mantle ⁸⁷ Sr/ ⁸⁶ Sr, seawater ⁸⁷ Sr/ ⁸⁶ Sr, and seawater Sr concentration. The model reproduces the overall trajectory of the compiled data. We interpret the observed temporal variation in island arc ⁸⁷ Sr/ ⁸⁶ Sr values and its close association with fluctuations in seawater chemistry as evidence that changes in marine geochemistry have strongly influenced the Sr isotopic record of island arc magmas over time.

Photorespiration can limit gross primary productivity in terrestrial plants. The rate of photorespiration relative to carbon fixation increases with temperature and decreases with atmospheric [CO ₂ ]. However, the extent to which this rate varies in the environment is unclear. Here, we introduce a proxy for relative photorespiration rate based on the clumped isotopic composition of methoxyl groups ( R –O–CH ₃ ) in wood. Most methoxyl C–H bonds are formed either during photorespiration or the Calvin cycle and thus their isotopic composition may be sensitive to the mixing ratio of these pathways. In water-replete growing conditions, we find that the abundance of the clumped isotopologue ¹³ CH ₂ D correlates with temperature (18–28 °C) and atmospheric [CO ₂ ] (280–1000 ppm), consistent with a common dependence on relative photorespiration rate. When applied to a global dataset of wood, we observe global trends of isotopic clumping with climate and water availability. Clumped isotopic compositions are similar across environments with temperatures below ~18 °C. Above ~18 °C, clumped isotopic compositions in water-limited and water-replete trees increasingly diverge. We propose that trees from hotter climates photorespire substantially more than trees from cooler climates. How increased photorespiration is managed depends on water availability: water-replete trees export more photorespiratory metabolites to lignin whereas water-limited trees either export fewer overall or direct more to other sinks that mitigate water stress. These disparate trends indicate contrasting responses of photorespiration rate (and thus gross primary productivity) to a future high-[CO ₂ ] world. This work enables reconstructing photorespiration rates in the geologic past using fossil wood.

Carbonate mud represents one of the most important geochemical archives for reconstructing ancient climatic, environmental, and evolutionary change from the rock record. Mud also represents a major sink in the global carbon cycle. Yet, there remains no consensus about how and where carbonate mud is formed. Here, we present stable isotope and trace-element data from carbonate constituents in the Bahamas, including ooids, corals, foraminifera, and algae. We use geochemical fingerprinting to demonstrate that carbonate mud cannot be sourced from the abrasion and mixture of any combination of these macroscopic grains. Instead, an inverse Bayesian mixing model requires the presence of an additional aragonite source. We posit that this source represents a direct seawater precipitate. We use geological and geochemical data to show that “whitings” are unlikely to be the dominant source of this precipitate and, instead, present a model for mud precipitation on the bank margins that can explain the geographical distribution, clumped-isotope thermometry, and stable isotope signature of carbonate mud. Next, we address the enigma of why mud and ooids are so abundant in the Bahamas, yet so rare in the rest of the world: Mediterranean outflow feeds the Bahamas with the most alkaline waters in the modern ocean (>99.7th-percentile). Such high alkalinity appears to be a prerequisite for the nonskeletal carbonate factory because, when Mediterranean outflow was reduced in the Miocene, Bahamian carbonate export ceased for 3-million-years. Finally, we show how shutting off and turning on the shallow carbonate factory can send ripples through the global climate system.

The deep ocean is generally considered to have changed from anoxic in the Precambrian to oxygenated by the Late Paleozoic (∼420–400 Ma) due to changes in atmospheric oxygen concentrations. When the transition occurred, that is, in the Early Paleozoic or not until the Late Paleozoic, is less well constrained. To address this, we measured Fe³⁺/ΣFe of volcanic rocks, sheeted dykes, gabbros, and ultramafic rocks from the Early Paleozoic (∼485 Ma) Bay of Islands (BOI) ophiolite as a proxy for hydrothermal alteration in the presence or absence of O₂derived from deep marine fluids. Combining this data with previously published data from the BOI indicates that volcanic rocks are oxidized relative to intrusive crustal rocks (0.35 ± 0.02 vs. 0.19 ± 0.01, 1 standard error), which we interpret to indicate that the volcanic section was altered by marine‐derived fluids that contained some dissolved O₂. We compare our results directly to the Macquarie Island and Troodos ophiolites, drilled oceanic crust, previously compiled data for ophiolitic volcanic rocks, and newly compiled data for ophiolitic intrusive rocks. These comparisons show that the BOI volcanic (but not intrusive) rocks are oxidized relative to Precambrian equivalents, but are less oxidized relative to Late Paleozoic to modern equivalents. We interpret these results to indicate that the Early Paleozoic ocean contained dissolved O₂, but at concentrations ∼2.4× lower than for the Late Paleozoic to today.

Solid-earth processes act as both sources and sinks for atmospheric O₂. They act as sinks by introducing reduced minerals and gases to the earth9s surface that can remove O₂ from the atmosphere and ocean. They act as sources by exporting organic carbon and sedimentary pyrite to the mantle via subduction. Here we examine the relative sizes of igneous source and sinks of O₂ for the modern earth to determine their magnitudes and if they are in balance today. We find that igneous sinks for O₂ remove 1.83×10¹² mol O₂/yr (±0.43, 1σ) while subduction indirectly releases 1.56×10¹² mol/O₂ yr (±0.33, 1σ). This indicates that today igneous O₂ sinks are balanced by solid earth sources. We propose this balance is achieved by negative feedbacks associated with either low-temperature hydrothermal sinks for O₂, which are sensitive to deep-ocean O₂ concentrations, or the amount of organic carbon and pyrite buried in sediments and subducted, which are sensitive to dissolved O₂ concentrations. We also explore how igneous sinks for O₂ may have varied in the Neoproterozoic when atmospheric O₂ concentrations are thought to have been lower and the deep ocean anoxic. We find that despite these changes, the igneous O₂ sink was essentially the same as today: 1.78×10¹² (±0.43, 1σ) mol O₂/yr. We explore how this sink would change as the deep ocean accumulated sulfate, became oxygenated, and began oxidizing oceanic crust such that there was an increase in the subduction flux of oxidants to the subarc mantle. We propose that significant changes to the O₂ cycle, both in terms of positive and negative feedbacks could occur during these transitions. For example, accumulation of sulfate in the deep ocean would increase the oxidation state of high-temperature hydrothermal fluids, decreasing the size of this O₂ sink and thus promoting an increase in atmospheric O₂. In contrast, the oxygenation of the deep ocean would have allowed hydrothermally derived H₂S to react with and consume O₂ instead of being titrated out via reactions with dissolved Fe²⁺. Additionally, deep-ocean oxygenation would have initiated the oxidation of oceanic crust at low temperatures, creating new sinks for O₂. Finally, the oxidation of the subarc mantle via subduction of newly oxidized sediments and altered oceanic crust would have increased the oxygen fugacity of arc volcanic gases, decreasing their overall demand for O₂, allowing yet more O₂ to accumulate in the atmosphere. We place these changes into a conceptual framework and discuss their potential impact on the history of atmospheric and marine O₂ concentrations from the Neoproterozoic to late Paleozoic.

Circulation more than temperature: Changes in continental configuration and sea level affected the ocean's oxygen levels and the rate of denitrification between 70 and 30 million years ago. That finding by Kast et al. shows a fundamental difference from the modern ocean, in which the extent of suboxia is controlled primarily by global temperature. Changes in the nitrogen isotopic composition of marine organic matter correlate with the collision of India and Asia and the circulation changes that occurred as a result. Later, isotopic composition changed further in response to a fall in sea level as global cooling caused ice sheets to grow. Science , this issue p. 386