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ABSTRACT The ocean has experienced substantial oxygen loss over recent decades, affecting marine ecosystems and fisheries. Investigating ocean deoxygenation during hyperthermal events, such

as the Paleocene-Eocene Thermal Maximum (PETM), offers insights into its future dynamics. Here, sediment cores from the South Atlantic reveal a pronounced decline in foraminifera-bound δ15N,

concurrent with an increase in marine barite δ34S and enhanced ocean productivity during the PETM. These findings suggest an expansion of oxygen-deficient zones (ODZs) from suboxia to

anoxia in the thermocline, with ammonium and sulfide accumulation. Model simulations indicate “ammonium-type” ODZs were driven by Southern Ocean warming and elevated productivity. Intense

fixed nitrogen loss at the upper boundary of these ODZs, along with increased oceanic phosphorus inventory, likely spurred a compensatory rise in N2 fixation. While the Pacific might

experience different oxygenation conditions during the PETM, parts of the Atlantic thermocline became anoxic, highlighting potential spatial variabilities of ocean deoxygenation under global

warming. SIMILAR CONTENT BEING VIEWED BY OTHERS ENHANCED OCEAN OXYGENATION DURING CENOZOIC WARM PERIODS Article Open access 31 August 2022 VERTICAL DECOUPLING IN LATE ORDOVICIAN ANOXIA DUE

TO REORGANIZATION OF OCEAN CIRCULATION Article 01 November 2021 A WELL-OXYGENATED EASTERN TROPICAL PACIFIC DURING THE WARM MIOCENE Article 28 June 2023 INTRODUCTION Oxygen (O2) is critical

to most forms of life. Due to its low solubility in water, the total amount of dissolved O2 in the ocean is less than 1% of that in the atmosphere. Consequently, O2 can become a limiting

resource in many marine environments. Over the past several decades, dissolved O2 levels in both open-ocean and coastal waters have declined, a trend mainly attributed to climate change and

anthropogenic nutrient input1,2,3. While the ongoing oceanic oxygen loss (i.e., ocean deoxygenation) is expected to persist due to rising global temperatures and increased ocean

stratification4, factors such as ocean ventilation and productivity may introduce complexity to these conditions. For example, climate models have yielded inconsistent predictions regarding

the future of ocean deoxygenation5,6. A pressing concern of ocean deoxygenation is the potential expansion of oxygen-deficient zones (ODZs) under global warming. These zones, with the lowest

O2 concentration, typically occur in the subsurface ocean at depths from 100 to 1000 meters. Changes in ODZs significantly influence the marine cycling of key elements, such as nitrogen,

sulfur, and carbon. Under suboxic conditions – where O2 concentrations drop below 5 μmol/kg4,7 – organic matter is respired via denitrification and anaerobic ammonia oxidation (anammox),

resulting in a net loss of biologically available nitrogen, or fixed nitrogen. In addition, ODZs are hotspots of nitrous oxide (N2O) production, a potent greenhouse gas ~ 300 times more

powerful than carbon dioxide over a 100-year period8. Although the modern ocean ODZs are mainly suboxic, they may have been more severely depleted of oxygen in the past. Under anoxic

conditions, where O2 concentrations fall to 0 μmol/kg and other electron acceptors like nitrate are limited, microbial sulfate reduction becomes the dominant pathway for organic matter

remineralization, leading to the accumulation of toxic hydrogen sulfide9. Such scenarios can lead to nitrate depletion in ODZs, with ammonium becoming the dominant form of fixed nitrogen,

thereby altering the marine nitrogen cycle10,11,12. The Paleocene-Eocene Thermal Maximum (PETM; ~ 56 million years ago), as a potential analog for high anthropogenic carbon emission

scenarios, provides important insights into the future evolution of ODZs. During the PETM, the estimated global mean surface temperature was 34.1 °C, exceeding present-day temperatures by 19

 °C13,14. This temperature difference alone would have significantly reduced oxygen solubility, contributing to ocean deoxygenation. Indeed, evidence from marine barite sulfur isotopes

(δ34S, where δ34S = [(34S/32S)sample/(34S/32S)VCDT – 1] × 1000 [‰]) indicates large-scale ocean deoxygenation during the PETM, with ODZs evolving into anoxic and even sulfidic

conditions15,16. However, foraminifera-bound nitrogen isotopes (foraminifera-bound δ15N, where δ15N = [(15N/14N)sample/(15N/14N)air – 1] × 1000 [‰]) from the tropical North Pacific reveal a

decline during the PETM, which was explained as a rise in the upper ocean oxygen concentration caused by a reduction in biological productivity17. These findings suggest that the response of

ocean deoxygenation to global warming is more complex than previously thought, and the variations in ODZs during the PETM warrant further investigation. In this study, we aim to clarify the

extent of ODZs in the South Atlantic during the PETM by examining coupled records of foraminifera-bound δ15N, marine barite δ34S, and productivity on the same samples from Ocean Drilling

Program (ODP) Site 1263 and Site 1265, located on the southeastern margin of subtropical gyre at water depths above the paleo-carbonate compensation depth (Supplementary Fig. 1). This

multi-proxy reconstruction, in combination with modeling, allows for quantifying the impact of warming and productivity changes on the evolution of ODZs across the PETM. RESULTS AND

DISCUSSION NITROGEN-ISOTOPE AND PRODUCTIVITY RECONSTRUCTIONS The nitrogen isotope composition of foraminifera-bound organic matter (foraminifera-bound δ15N) is a reliable proxy for

reconstructing the past marine nitrogen cycling18. In today’s ODZs, water-column denitrification preferentially removes 14N, with isotope fractionation of 15 to 25‰19,20. This process

enriches the remaining nitrate in 15N, which then mixes with water outside the ODZs, raising the δ15N of nitrate in surrounding regions21,22. In areas where surface nitrate is fully

consumed, subsurface nitrate supplied to the euphotic zone is completely assimilated by phytoplankton and converted into organic matter. Foraminifera feeds on this organic matter, causing

foraminifera-bound δ15N to reflect the δ15N of subsurface nitrate in regions with complete surface nitrate consumption18. Consequently, foraminifera-bound δ15N records from ODZs and adjacent

regions have been used to reconstruct the historical expansion and contraction of ODZs23,24,25. Compared to bulk sedimentary δ15N, organic matter trapped within the mineral matrix of

planktic foraminifer shells is less vulnerable to isotopic alteration after burial, making foraminifera-bound δ15N an ideal proxy for preserving the original marine δ15N signature23. Our

foraminifera-bound δ15N record based on mixed species shows values of approximately 10‰ before the PETM (Fig. 1), consistent with the previously reported foraminifera-bound δ15N values of

the early Cenozoic from the Pacific and Atlantic Ocean23. The foraminifera-bound δ15N drops from 10‰ to ~ 4‰ during the PETM, followed by a rapid recovery to the near pre-excursion value of

9‰. It should be noted that the single genus foraminifera δ15N record (based on _Morozovella_, _Acarinina_, and _Subbotina_) was recently published from Site 1263, although their record did

not span the whole PETM period due to changes in the foraminifera genus17. When plotted together, our mixed species foraminifera-bound δ15N are consistent with the published symbiont-bearing

single genus _Morozovella_ and _Acarinina_ δ15N, substantiating the decline of δ15N at the study sites. One datum point in our record showed an anomalously low δ15N of 0.4‰ at 85 kyr

relative to the onset of PETM. While the previous work did not observe a δ15N value close to 0‰, we keep this minimum datum as its nitrogen content falls within the reasonable range (2.5–4.5

 μmol/g; Supplementary Table 1). The δ15N decline reconstructed from the South Atlantic also agrees with the near-shore δ15N record from the Tethys Ocean26 (Supplementary Fig. 2), implying

similar driving mechanisms between coastal and open-ocean settings. We also present the reactive phosphorus accumulation rate (reactive PAR) from the same sites as a measure of phosphorus

supply to the euphotic zone alongside the marine barite accumulation rate (barite AR), which serves as an indicator of export productivity27,28. Reactive PAR shows a four- to nine-fold

increase between 0 and 100 kyrs compared to the pre-PETM levels, peaking at 85 kyrs. This peak coincides with the foraminifera-bound δ15N minimum and the marine barite AR maximum. Notably,

the reactive phosphorus concentration is also higher between 0 and 100 kyrs, indicating that the increase in reactive PAR is not an artifact of mass accumulation rate (Supplementary Table 

2). PRESENCE OF “AMMONIUM-TYPE” ODZS IN THE SOUTH ATLANTIC DURING THE PETM In the modern ocean, nitrate is the dominant form of fixed nitrogen in ODZs (“nitrate-type” ODZs; Fig. 2a). In

these ODZs, nitrate consumption through water-column denitrification is incomplete, resulting in large isotope fractionation of 15 to 25‰19,20. In contrast, denitrification in ocean

sediments is typically complete, removing fixed nitrogen with minimal isotope fractionation (Fig. 2c). Thus, lower rates of water-column denitrification relative to sedimentary

denitrification are expected to decrease mean ocean nitrate δ15N, leading to lower average foraminifera-bound δ15N values globally. The pronounced decline observed in most foraminifera-bound

δ15N records during the PETM, if interpreted at face value, could generally suggest a contraction of nitrate-type ODZs or a reduction in global water-column denitrification relative to

sedimentary denitrification. In the tropical North Pacific, this decline has been previously attributed to reduced ocean suboxia, driven by decreased productivity17. However, this

interpretation is unlikely to apply to the South Atlantic. First, reactive PAR and marine barite AR in the South Atlantic indicate an increase in productivity, not a decline (Fig. 2 and

Supplementary Table 3). Second, independent evidence from foraminiferal Cr-isotope and Ce anomalies, reconstructed from sites at intermediate paleo-water depths in the Atlantic, points to

the presence of oxygen-free water during this period29,30. Third, the concurrent increase in mean ocean sulfate δ34S and δ18O preserved in marine barite indicates the accumulation of

hydrogen sulfide within some ODZs15,16. Although the exact locations of these sulfide-rich ODZs remain unclear due to sulfate’s long residence time in the ocean, microbial sulfate reduction

only occurs after nitrate’s depletion, owing to thermodynamic constraints9. Consequently, the dominant form of fixed nitrogen in sulfide-rich ODZs must be ammonium, not nitrate

(“ammonium-type” ODZs; Fig. 2b). Unlike nitrate-type ODZs, ammonium-type ODZs follow different fixed nitrogen loss pathways and may express weaker isotope effects, which could lead to lower

δ15N values, similar to those observed during the Cretaceous oceanic anoxic events (OAEs)11,12,31,32. We propose that the decline in foraminifera-bound δ15N in the South Atlantic during the

PETM is a consequence of further expansion of suboxia to anoxia in the thermocline water, developing ammonium-type ODZs, which also hosts the previously identified hydrogen sulfide. In these

ODZs, when ammonium is introduced into the euphotic zone through mixing, nitrification-coupled anammox/denitrification occurs at the upper boundary of the chemocline. This process

represents a different pathway of fixed nitrogen loss, which is not present in today’s nitrate-type ODZs33 (Fig. 2d). In the modern Black Sea, ammonium-type ODZs exist below the euphotic

zone (Fig. 2b), where ammonium concentrations are > 20 μM with δ15N of 4‰34,35. Due to salinity stratification in the upper Black Sea, the upward transport of ammonium is slow, resulting

in the majority of ammonium being removed through nitrification-coupled anammox/denitrification in the anoxic-suboxic transition zone. Consequently, the residual nitrate concentration at the

top of the suboxic zone is up to 4 μM, with δ15N of 10 to 14‰34,35. Simple mass-balance calculation suggests a net isotope fractionation effect as low as 2‰ associated with fixed nitrogen

loss in the water column, much weaker than nitrate-type ODZs19,20,21,22. The weakly expressed isotope effect is similar to that of sedimentary denitrification; although the organism-level

isotope effect is identical to that seen in water-column denitrification, the quantitative consumption of nitrate in sediments leaves behind a minimal isotope fingerprint on the seawater

above. Due to the high sea-surface temperature (SST) during the PETM13, the South Atlantic subtropical gyre likely experienced stronger stratification, limiting upward ammonium transport

into the euphotic zone, similar to the conditions observed in the modern Black Sea. If ammonium-type ODZs were prominent during the PETM, this would have increased the proportion of fixed

nitrogen loss through pathways with weak isotope fractionation (i.e., nitrification-coupled anammox/denitrification at the upper boundary of ammonium-type ODZs and/or sedimentary

denitrification) in the global ocean, resulting in a reduction in the mean ocean fixed nitrogen δ15N compared to the pre-PETM condition. In addition, it has been suggested that nitrate-type

ODZs were contracted in the tropical North Pacific due to reduced upwelling during the PETM17. The combined effect of smaller nitrate-type ODZs and expanded ammonium-type ODZs would lower

the mean ocean fixed nitrogen δ15N without necessitating changes in sedimentary denitrification rates. We use a simple one-box model to quantify the effect of ammonium-type ODZs on the mean

ocean fixed nitrogen δ15N during the PETM, and compare it with all existing foraminifera-bound δ15N data17,32,36 (Fig. 3a). In this model, N2 fixation input is assumed to be balanced by

fixed nitrogen loss through sedimentary denitrification, water-column denitrification in nitrate-type ODZs, and nitrification-coupled anammox/denitrification in ammonium-type ODZs

(“Methods”). Compared to the modern ocean, the mean ocean fixed nitrogen δ15N is higher in the pre-PETM (11.3‰ vs. 5‰), indicating more extensive suboxia and an absence of ammonium-type ODZs

(Fig. 3b). The mean ocean fixed nitrogen δ15N shifts towards a lower value of 7.8‰ during the PETM. Assuming no change in sedimentary denitrification, this decline could be explained as a

result of increased nitrogen loss at the upper boundary of ammonium-type ODZs, which constitutes, on average, ~ 30% of water-column fixed nitrogen loss. The same framework might also explain

the particularly low δ15N during the Cretaceous OAE232, where most water-column fixed nitrogen loss occurs in ammonium-type ODZs. One consequence of the intense fixed nitrogen loss at the

upper boundary of ammonium-type ODZs in the South Atlantic is nitrogen limitation in the euphotic zone. Despite this limitation, we observe a rise in productivity during the PETM (Fig. 1),

suggesting that additional fixed nitrogen was supplied to the euphotic zone, most likely through increased N2 fixation. Modern ocean observations indicate that N2-fixing microbes thrive in

water with low N:P ratios37. During the PTEM, it has been suggested that intense weathering led to an increase in the oceanic phosphorus inventory38,39. The phosphorus increase, coupled with

the significant nitrogen loss at the upper boundary of the ammonium-type ODZs, is expected to drive a decrease in the N:P ratio of surface waters, leading to a substantial rise in N2

fixation and thus boosting productivity in the South Atlantic during the PETM. To better constrain the timing and magnitude changes in marine nitrogen cycling in the South Atlantic across

the PETM, we develop a multi-box isotope-enabled model that specifies fixed nitrogen species in different water layers (see “Methods”, Supplementary Fig. 3 and Supplementary Table 4).

Assuming that 1) the oceanic nitrogen inventory is always in a steady state, and 2) the sedimentary denitrification rate is stable at 4 Tmol N/yr, approximately 92% of ammonification occurs

in the subsurface while the rest in the deep ocean, similar to the modern ocean37. We use constraints from the reconstructed foraminifera-bound δ15N, marine sulfate δ34S, and

nutrient/productivity records to calculate the rates of water-column nitrogen loss in ODZs and N2 fixation across the PETM. Our simulation captures the transition in the predominant form of

fixed nitrogen, shifting from nitrate to ammonium in the subsurface ocean in response to ODZs’ expansion in the South Atlantic during the PETM. Prior to the onset of the PETM, suboxia had

developed in the subsurface ocean, where water-column denitrification in nitrate-type ODZs is responsible for 60% of total fixed nitrogen loss (Fig. 4a, b). Assuming sustained anoxia within

ODZs for 100 kyrs, as constrained by marine sulfate isotope data15,16, water-column denitrification must have increased, with a gradual loss of nitrate from the subsurface ocean from 20 μM

to 4.7 μM at 81 kyrs from the onset of the PETM (Fig. 4c). Due to significant consumption of subsurface nitrate, nitrate-type ODZ nitrogen loss briefly decreases until the nitrate

concentration begins to recover. Simultaneously, ammonium accumulates and reaches a maximum concentration of 16.3 μM. As the ammonium is transported to the surface ocean,

nitrification-coupled anammox/denitrification becomes the dominant pathway (90%) of water-column nitrogen loss in the model, with a net isotope fractionation effect of 2‰ (sensitivity tests

in Supplementary Fig. 4). To compensate for the intense fixed nitrogen loss, N2 fixation has to exceed the pre-PETM level by a factor of 4.7 (Fig. 4b). Elevated N2 fixation rates would

stimulate biological productivity, consistent with the reactive PAR and marine barite AR records during the PETM27,28. When dissolved oxygen resumes in ODZs, the marine nitrogen cycle

returns to the pre-PETM structure. This model exercise demonstrates that the δ15N decline in the South Atlantic during the PETM is a combined effect of 1) the development of ammonium-type

ODZs, which results in a decrease in mean ocean fixed nitrogen δ15N, and 2) increased N2 fixation rates above ammonium-type ODZs, (Fig. 4d and sensitivity tests in Supplementary Fig. 4).

Another potential mechanism for the δ15N decline is greater isotope effects (up to 7‰) associated with alternative (V- and Fe-based) nitrogenases40. However, the deep ocean probably remains

largely oxygenated during the PETM, which would not cause further Mo removal from seawater into sediments41. Thus, it is unlikely that the global oceanic Mo inventory during the PETM is low

enough to favor alternative nitrogenases in triggering the δ15N decline. MECHANISMS FOR EXPANDED SUBSURFACE ANOXIA IN THE ATLANTIC The expansion of ODZs to the extent of anoxia in the South

Atlantic during the PETM is most likely driven by a combination of global warming and enhanced biological productivity. The oxygenation of the subsurface ocean in the South Atlantic is

strongly influenced by O2 supply at high latitudes, particularly from the Southern Ocean surface water. Therefore, SST in the Southern Ocean plays a crucial role in controlling the extent of

ODZs by affecting O2 solubility. Before the PETM, the SST in the Southern Ocean was already ~ 13 °C higher than today (Supplementary Fig. 5). The temperature effect alone would have reduced

O2 solubility by 90 μmol/kg42, preconditioning the ODZs for further expansion. This is consistent with extensive suboxic conditions in the pre-PETM ocean characterized by the high δ15N.

When we further consider an increase in global mean surface temperature by 5.6 °C in the PETM, attributed to nearly doubling atmospheric CO2 concentration, the entire ocean would have been,

on average, 19 °C warmer than today, with the SST in the Southern Ocean higher than 25 °C13. The cumulative temperature effect on O2 solubility is equivalent to a 30% reduction in the

dissolved O2 inventory. Furthermore, intensified warming at high latitudes, through reducing the meridional SST gradient, weakens the ventilation of intermediate and deep waters primarily

originating from the Southern Ocean30. Benthic foraminiferal δ13C and Nd-isotope evidence support a less vigorous ocean overturning during the PETM43,44. Using the Earth system model cGENIE,

we simulate the impact of temperature change on the oceanic O2 distribution (see “Methods”). Compared to today’s ODZs, the ODZs during the PETM have expanded to cover approximately

one-third of the global ocean area in the subsurface ocean, mainly in the Atlantic, eastern tropical Pacific, and Tethys Ocean (Fig. 5). In these areas, the minimum O2 concentration could

have reached zero. We also notice the simulated O2 concentration in some regions of the Pacific remains > 5 μmol/kg, indicating that the response of ODZs to global warming is more complex

in the Pacific compared to the Atlantic, which may be associated with paleogeography restriction and/or local productivity. In addition to global warming, enhanced biological productivity

in the South Atlantic during the PETM could have further intensified the expansion of ODZs by increasing O2 consumption in the thermocline water. While a more stratified ocean would limit

the vertical supply of nutrients to fuel productivity45, it has been suggested that terrestrial weathering has increased globally by 50–60% over 100 kyrs, bringing more phosphorus into the

ocean and promoting productivity during the PETM39,46. The increase in carbon export from the euphotic zone enhances the remineralization of sinking particulate organic matter in the

twilight zone27. As higher seawater temperatures accelerate microbial metabolic activities47, oxygen demand in the interior of the ocean could have become dramatically higher, contributing

to widespread ODZs during the PETM. Despite different residence times of nitrogen and sulfur in the ocean (103 vs. 106 years), we find coupled perturbations to both cycles during the

transient ocean deoxygenation event in the early Cenozoic hypothermal. With widespread suboxia present in the pre-PETM ocean, ODZs are preconditioned to evolve toward anoxia in the South

Atlantic, where ammonium becomes the dominant form of fixed nitrogen. The short-lived accumulation of ammonium in the thermocline water aligns with the coeval sulfide accumulation15,16,

underscoring that significant portions of ODZs have reached anoxic conditions. Ammonium-type ODZs and a compensatory increase in N2 fixation above them offer a plausible explanation of δ15N

decline during the PETM, which might apply to the Cretaceous OAEs and yet to be observed in other Cenozoic warm periods. Our findings suggest that the Paleocene-Eocene warming, alongside

productivity changes, could have led to complex and regionally variable impacts on ocean deoxygenation. While the tropical North Pacific may have undergone less severe deoxygenation or even

an increase in upper ocean oxygen17, the Atlantic Ocean likely experienced intense deoxygenation, marked by the expansion of ODZs enriched with ammonium and hydrogen sulfide. These results

highlight the spatial variability in ocean deoxygenation under extreme climate warming scenarios. In addition, to accurately predict future ocean change in response to anthropogenic warming,

it is crucial to improve our understanding of the future trajectory of the biological carbon pump, a topic currently lacking consensus in Earth System models48. METHODS SITE LOCATION AND

AGE MODEL Sediment samples were collected from ODP Leg 208 Site 1263 Hole C and Hole D (28°31.98′S, 2°46.78′E) drilled at 2717 m water depth and Site 1265 Hole A (28°50.10′S, 2°38.36′E)

drilled at 3060 m water depth on Walvis Ridge. These sites were located on the southeastern margin of the subtropical gyre in the South Atlantic during the PETM (35 °S, 10 °E; Supplementary

Fig. 1). The paleo-water depths of Site 1263 and Site 1265 were approximately 1500 m and 2000 m, respectively49. Composite depth (mcd) allows for stratigraphic correlations among holes43,50.

The ages of these samples were reported as time relative to the onset of the negative carbon isotope excursion (CIE) using the astronomically tuned timescale based on the carbon isotope

record from ODP Site 69050. Mass accumulation rate (MAR) was calculated as the product of the sedimentation rate and the dry bulk density obtained from ODP reports49. ANALYSIS OF

FORAMINIFERA-BOUND NITROGEN ISOTOPES Examinations under a light microscope and scanning electron microscope showed that foraminifera were overall small throughout the PETM interval. The

dominant genus of planktic foraminifera across the PETM were _Morozovella_ and _Acarinina_. Around 5 mg of size fractions (125–250 μm) of mixed-species foraminifera were carefully weighed

and subsequently crushed into smaller pieces using a glass stick and combusted foil. The foraminifera were then meticulously transferred into 15 ml centrifuge tubes. Next, all samples

underwent a 3-min ultrasonication process in 2% sodium polyphosphate with a pH of 8, followed by three thorough rinses with Milli- Q water to remove any potential external sedimentary

contaminants51. To minimize nitrogen contamination associated with metal oxides, a reductive cleaning method was performed using dithionite citrate reagent52, where the samples were soaked

in the reagent and placed in an 80 °C water bath for 1 h with intermittent shaking. Afterward, the samples were rinsed three times with Milli-Q water. Then, the samples were soaked in a

10–15% sodium hypochlorite solution, thoroughly shaken, and left on an orbital shaker overnight (> 12 h) to remove any remaining organic nitrogen contamination. Following these steps, the

samples were rinsed three times with Milli-Q water and dried overnight in an oven at ~ 60 °C. After cleaning, the foraminifera were dissolved using HCl, and the released nitrogen was

oxidized to nitrate using persulfate oxidation25. At least 2 mg of the cleaned foraminifera samples were carefully weighed using clean weighing paper and then transferred into 4 ml combusted

glass vials. 60 µl of 4 N hydrochloric acid was added to the foraminifera samples. The released organic matter was oxidized to nitrate by a low-N potassium persulfate solution. The samples

were then transferred to an autoclave and subjected to 1 h of treatment at 120 °C to ensure complete oxidation. Two amino acid isotope references, USGS 64 (δ15N = + 1.76‰) and USGS 65 (δ15N 

= + 20.68‰), as well as persulfate solution blanks, were used during the oxidation as standards. Following autoclaving, the samples were centrifuged at 6000 rpm for 10 min, and the resulting

solution containing nitrate was transferred to a new, clean, pre-combusted 4 ml vial. pH adjustments were then carried out to ensure a final neutral pH. The ‘denitrifier method’ was used to

measure the isotopic composition of the converted nitrate, which was quantitatively converted into N2O using denitrifying bacteria _P. chlororaphis_ that lacks N2O reductase53. The

resulting N2O was analyzed for δ15N on a customized Gas Bench coupled to a Delta V Plus isotope ratio mass spectrometer at Boston College. Isotope values were calibrated relative to air54

using two nitrate isotope references: the international nitrate standards IAEA-N3 (δ15N = + 4.7‰) and USGS-34 (δ15N = − 1.8‰)55. An in-house coral standard (CBS-II) was used as a quality

control for the overall protocol25. The precision of the entire protocol, as determined by the coral standard, was better than 0.3‰ (1-σ). The foraminifera-bound nitrogen content remains

stable between 2.5 and 4.5 μmol/g and has no apparent correlation with the foraminifera-bound δ15N (see Supplementary Table 1), indicating negligible diagenetic impact. There is one lowest

data point of 0.4‰ at 85 kyrs relative to the onset of the PETM. Although this data point is not replicated due to limited materials, its nitrogen content falls into the same range as other

samples. ANALYSIS OF SEDIMENTARY PHOSPHORUS FRACTIONS We crushed freeze-dried sediments and passed them through a 150 μm sieve to homogenize particle size. Five components of sedimentary P

(i.e., water-soluble P, oxide-associated P, authigenic P, detrital P, organic P) were extracted from about 0.1 g samples into known volumes of extractants, using the modified sequential P

extraction (SEDEX) procedure56,57. We analyzed P concentrations via the molybdenum blue colorimetric method at low pH conditions58 with an EnSpire® multimode microplate spectrophotometer at

Southern University of Science and Technology. The absorbance was at a wavelength of 850 nm. Results were reported as the means with 1-σ errors of triplicate runs on individual components.

The long-term analytical error of reactive P concentration was ± 1.32 μmol P/g sediment using the same deep-sea sample as the in-house standard, similar to those from other sedimentary P

studies59. The reactive P concentration was calculated as the sum of water-soluble P, oxide-associated P, authigenic P, and organic P concentrations. The reactive P accumulation rate

(reactive PAR) was calculated as the product of the concentration of reactive P and MAR (Table 2). ONE-BOX MODEL FOR MEAN OCEAN FIXED NITROGEN Δ15N We build a steady-state one-box model to

explore how the development of ammonium-type ODZs affects mean ocean fixed nitrogen δ15N values. In a steady-state, nitrogen input through N2 fixation (_J__fix_) is balanced by the total

fixed nitrogen loss through sedimentary denitrification (_J__sed_), water-column denitrification (_J__wc_) in nitrate-type ODZs, and water-column nitrification-coupled

anammox/denitrification (_J__N/D_) in ammonium-type ODZs (Eq. 1). $${J}_{{fix}}={J}_{{sed}}+{J}_{{wc}}+{J}_{N/D}$$ (1) We define _f__sed_ as the fraction of sedimentary denitrification to

the total nitrogen loss, and _f__N/D_ as the fraction of nitrification-coupled anammox/denitrification to the water-column nitrogen loss. Assuming the newly fixed nitrogen δ15N value

(_δ__fix_) is constant at − 1‰ (60), and the globally mean N-isotope fractionations associated with _J__sed_ and _J__wc_ are 0 and 20‰, respectively19,20, we achieve isotope balance as in

Eq. 2. $$-1={\delta }_{{DIN}}\,{f}_{{sed}}+(1-{f}_{{sed}})[{({\delta }_{{DIN}}-20)1-f}_{(N/D)}+({\delta }_{{DIN}}-{\varepsilon }_{N/D})\,{f}_{N/D}]$$ (2) where _δ__DIN_ denotes the mean

ocean fixed nitrogen δ15N; _ε__D/N_ denotes the N-isotope fractionation associated with nitrification-coupled anammox/denitrification. In the modern ocean, where _J__N/D_ = 0 and δ15NDIN = 

5‰, _f__N/D_ is 0% and _f__sed_ is 70%. Solving for _δ__DIN_: $${\delta }_{{DIN}}=19-(20-{\varepsilon }_{N/D})\,{f}_{N/D}-20{f}_{{sed}}+(20-{\varepsilon }_{N/D})\,{f}_{s{ed}}\,{f}_{N/D}$$

(3) Based on Eq. 3, we generate the contour plots of mean ocean-fixed nitrogen δ15N in Fig. 3b for _ε__N/D_ of 2‰ and 10‰. We also calculate the mean sedimentary δ15N or foraminifera-bound

δ15N values from the compiled modern, pre-PETM, PETM, and OAE2 data, and add them to the contour plot. This simple exercise indicates that ammonium-type ODZs have been more widespread in the

OAE2 than the PETM, but have not occurred in the pre-PETM and modern. THE PETM NITROGEN CYCLE MODEL To further reconstruct the marine nitrogen cycle in the South Atlantic across the PETM,

we establish a multi-box isotope-enabled model by dividing the ocean into four nitrogen reservoirs (i.e., surface fixed nitrogen, subsurface nitrate, subsurface ammonium, and deep nitrate

reservoirs) and simultaneously monitor changes in the nitrogen concentrations and δ15N compositions (see model topology in Supplementary Fig. 3 and Supplementary Table 4). Before exploring

perturbations in nitrogen cycling, we assume an initial steady state for the pre-PETM ocean, where all the reservoirs are mass and isotope balanced (Supplementary Fig. 3a). Similar to the

mean ocean δ15N model above, _J__fix_ in the surface ocean is equal to the sum of _J__sed_ in the deep ocean and _J__wc_ and _J__N/D_ in the subsurface ocean (Eq. 4). In ammonium-type ODZs,

a part of ammonium supplied toward the surface ocean gets nitrified and subsequently undergoes anammox/denitrification in the water column. _J__N/D_ can be calculated as a function of the

fraction of nitrification-coupled anammox/denitrification to total ammonium supply from the subsurface (_k_), the subsurface ammonium concentration (_C__subNH4_), and the mixing rate at the

upper boundary of the subsurface ocean (_v__upper_). In nitrate-type ODZs, _J__N/D_ is negligible as _C__subNH4_ is close to 0.

$${J}_{{fix}}={J}_{{sed}}+{J}_{{wc}}+k{{C}_{{subNH}4}v}_{{upper}}$$ (4) where _k_ remains unchanged at 0.8, indicating that 80% of the ammonium mixed up from the subsurface is lost through

nitrification-coupled anammox/denitrification; _v__upper_ is set to be 3 × 1015 m3/yr61. In an oligotrophic ocean like the South Atlantic, biological assimilation ( _J__assi_) will take up

all the fixed nitrogen in the surface ocean supplied from (i) N2 fixation and (ii) the subsurface nitrate and ammonium (if it exists) reservoirs through vertical mixing (Eq. 5). Thus, we

assume no N-isotope fractionation during biological assimilation of nitrate in an oligotrophic ocean (Eq. 6). $${J}_{{fix}}+[{C}_{{subNO}3}+(1-k){{C}_{{subNH}4}}]v_{{upper}}={J}_{{assi}}$$

(5) $${\delta }_{{fix}}\,{J}_{{fix}} + \left[\right.{\delta }_{{subNO}3}{C}_{{subNO}3}+{\delta }_{{subNH}4}{C}_{{subNH}4} \\ -({\delta }_{{subNH}4}-{\varepsilon

}_{N/D})(1-k){{C}_{{subNH}4}\left]\right.v}_{{upper}}={\delta }_{{bio}}\,{J}_{{assi}}$$ (6) where _C__subNO3_ denotes the subsurface nitrate concentration; _δ__subNO3_, _δ__subNH4_, and

_δ__bio_ denote the δ15N of subsurface nitrate, subsurface ammonium, and biomass, respectively. We set _δ__fix_ = − 1‰ (60) and _ε__N/D_ = 2‰ (taken from the Black Sea) throughout the model

run. In the subsurface ocean, ammonium sourced from biomass degradation (i.e., ammonification) will be subsequently (i) oxidized to nitrogen oxides through nitrification (_J__nitr_) and (ii)

supplied to the surface ocean (Eq. 7). The latter does not exist until ammonium starts to accumulate in ODZs. These processes have no isotope fractionation (Eq. 8).

$${f}_{{subammo}}\,{J}_{{assi}}={J}_{{nitr}}+{C}_{{subNH}4}{v}_{{upper}}$$ (7) $${\delta }_{{bio}}\,{f}_{{subammo}}\,{J}_{{assi}}={\delta }_{{subNH}4}\,{J}_{{nitr}}+{\delta

}_{{subNH}4}{C}_{{subNH}4}{v}_{{upper}}$$ (8) where _f__subammo_ denotes the constant fraction of ammonification in the subsurface ocean to biological assimilation. Nitrate in the subsurface

ocean is controlled by nitrification, water-column denitrification, and nitrate exchange with the surface and the deep ocean (Eq. 9). Nitrate exchange fluxes are a function of the

subsurface nitrate concentration, the deep nitrate concentrations (_C__deep_), and the mixing rate at the upper and lower boundaries of the subsurface ocean. Water-column denitrification

expresses strong N-isotope fractionation (_ε__wc_) as observations in today’s ocean, which will drive the overall marine δ15N toward higher values (Eq. 10).

$${J}_{{nitr}}+{C}_{{deep}}{v}_{{lower}}={J}_{{wc}}+{C}_{{subNO}3}({v}_{{upper}}+{v}_{{lower}})$$ (9) $${{\delta }_{{subNH}4}\,J}_{{nitr}} +{\delta

}_{{deep}}{C}_{{deep}}{v}_{{lower}}=\left({\delta }_{{subNO}3}-{\varepsilon }_{{wc}}\right){J}_{{wc}} \\ +{\delta }_{{subNO}3}{C}_{{subNO}3}({v}_{{upper}}+{v}_{{lower}})$$ (10) where

_δ__deep_ denotes deep nitrate δ15N; _v__lower_ denotes the mixing rate at the lower boundary of the subsurface ocean, which is set to be 1 × 1014 m3/yr61. In the deep ocean, nitrate remains

the dominant form of fixed nitrogen due to deep-sea ventilation. Ammonification in the deep ocean is balanced by sedimentary denitrification and nitrate exchange between the subsurface and

the deep ocean (Eq. 11). Due to complete nitrate consumption in sediment porewaters, N-isotope fractionation associated with sedimentary denitrification is negligible (Eq. 12).

$${f}_{{deepammo}}\,{J}_{{assi}}+{C}_{{subNO}3}{v}_{{lower}}={C}_{{deep}}{v}_{{lower}}+{J}_{{sed}}$$ (11) $${\delta }_{{bio}}\,{f}_{{deepammo}}\,{J}_{{assi}}+{\delta

}_{{subNO}3}{C}_{{subNO}3}{v}_{{lower}}={\delta }_{{deep}}({C}_{{deep}}{v}_{{lower}}+{J}_{{sed}})$$ (12) where _f__deepammo_ denotes the constant fraction of ammonification in the deep ocean

to biological assimilation, which is equivalent to 1 – _f__subammo_. At the initial steady state, our model achieves equilibrium when approximately 92% of organic matter is recycled through

ammonification in the subsurface ocean (i.e., _f__subammo_ = 0.92) while the rest is recycled in the deep ocean (_f__deepammo_ = 0.08), similar to the present day37. This yields the

N-isotope fractionation of 19.2‰ associated with water-column denitrification in nitrate-type ODZs, which corresponds to the deep nitrate δ15N of 9.7‰ and the biomass δ15N of 9.3‰,

consistent with the foraminifera-bound δ15N values in the pre-PETM. To further model the response of nitrogen cycling to ocean deoxygenation, we use constraints from the foraminifera-bound

δ15N, marine sulfate δ34S, and productivity records. To reproduce the observed N-isotope decline, we make the following assumptions: * i. The oceanic nitrogen inventory remains constant

throughout the model run. Nitrogen input through N2 fixation is set to always compensate for nitrogen output. The ocean δ15N changes with these processes. * ii. The timing of anoxia within

ODZs is between 0 and 100 kyrs relative to the onset of the PETM, as constrained by the positive sulfur and oxygen isotope excursions of marine sulfate15,16. Once ODZs become oxygen-free,

ammonium sourced from biomass degradation can no longer be substantially oxidized to nitrogen oxides (Supplementary Fig. 3b). Thus, we force the ratio of nitrification to ammonification to

decrease at the onset, cease within the time interval (as an extreme case), and recover at 100 kyrs when the oxygen concentration returns to the pre-PETM level. * iii. Water-column nitrogen

loss fluxes are a function of subsurface fixed nitrogen concentrations. With intensified ocean deoxygenation, water-column denitrification in nitrate-type ODZs increases until subsurface

nitrate is progressively depleted. The increasing fixed nitrogen loss through water-column denitrification in nitrate-type ODZs and nitrification-coupled anammox/denitrification in

ammonium-type ODZs will be compensated by increased N2 fixation, which further influences biological assimilation to reflect changes in productivity. With these assumptions, our model

reconstructs the evolution of marine nitrogen cycling in response to a transient expansion and recovery of ODZs during the PETM. When dissolved oxygen resumes in ODZs, elevating

nitrification reduces the ammonium concentration and, subsequently, ammonium-type ODZ nitrogen loss and assimilation. In addition, we test the sensitivity of the model to N2 fixation. In the

sensitivity test, we force a constant N2 fixation flux, while keeping the rest parameters and forcing same as above (Supplementary Fig. 4b). In this scenario, N2 fixation cannot compensate

nitrogen loss during the expansion of ODZs. The oceanic nitrogen inventory is reduced by 12% during the PETM. The deficiency of nitrogen in the ocean will cause a reduction in biological

assimilation by ~ 70%, which is opposed to productivity records27,28. With a constant N2 fixation flux, the drawdown of δ15N could only be achieved with a close-to-zero isotope fractionation

effect associated with nitrification-coupled anammox/denitrification in ammonium-type ODZs. However, this scenario overall fits foraminifera-bound δ15N poorer compared to that of a constant

oceanic nitrogen inventory. INTERMEDIATE-COMPLEXITY EARTH SYSTEM MODEL CGENIE We utilize the late-Paleogene configuration of the carbon-centric Grid Enabled Integrated Earth system model

(cGENIE) after Ref. 62 and modify two parameters in the initial set-up for the pre-PETM to reconcile proxy information. First, we assume the pre-PETM atmospheric CO2 concentration is 1120

ppm, exceeding the preindustrial CO2 concentration (280 ppm) by a factor of four. The × 4 CO2 concentration is chosen because (i) it falls within the range of × 2 to × 8 CO2 projected by

previous studies13,29,30,45,63 and (ii) the resulting pre-PETM surface water temperatures and dissolved O2 concentrations match proxy observations (Supplementary Fig. 5 and Table 3). Second,

we change the initial ocean temperature to 13 °C for the pre-PETM. While the default configuration is 10 °C, the latest proxy and data assimilation results suggest warmer bottom water

temperatures at ~ 13 °C in the pre-PETM13,64,65,66,67. Before carbon emission, the model is spun up to achieve long-term equilibrium. The simulated minimum O2 concentration is consistent

with redox proxy records that the development of suboxic and anoxic waters is mainly restricted in the Atlantic and Tethys in the pre-PETM. The atmospheric _p_O2 level could be another

factor affecting oxygen in the ocean. The previous study suggested that after the PETM, the atmospheric _p_O2 level experienced a gradual rise from ~ 17% in the early Eocene to as high as

23% in the Oligocene, followed by a decline toward the present value of 21%68. If true, the initial atmospheric _p_O2 level and, consequently, the dissolved O2 concentration in the ocean

during the PETM would be about 20% lower than the present-day level. However, considering debates on the atmospheric _p_O2 history, our cGENIE simulations stick with the modern-like

atmospheric _p_O2 level of 21% in this study, as most of the previous modeling work did30,62. We test different carbon emission scenarios following the rates and magnitudes of carbon

emissions suggested by previous studies62,63,69,70, ranging from a release of 3000 Pg C (low-emission) to 10,900 Pg C (high-emission) within 5000 years, and investigate the responses of the

oceanic O2 concentration. All the scenarios agree with enhanced ocean deoxygenation, where the minimum O2 concentration in the subsurface Atlantic, Tethys, and Eastern Pacific may have

reached anoxia during the PETM (Supplementary Fig. 6). These results strongly support the “ammonium ocean” mechanism in the subsurface South Atlantic. We also note that compared to the South

Atlantic, the minimum O2 concentration is overall higher in the tropical North Pacific – consistent with proxy data (Supplementary Table 3) – and more variable to different scenarios. While

discussing the Pacific is beyond the scope of this study, we focus on the South Atlantic, where all the simulations agree on subsurface ocean anoxia during the PETM. Our cGENIE results are

also consistent with the previous General Circulation Model (GCM) results for future climate warming (SSP5-8.5)17, where the subsurface O2 concentration mainly increases at low latitudes but

decreases at ~ 30°N and ~ 30°S. In other words, the mechanism of ocean oxygen rise/decline is unlikely to apply to the whole ocean – while some parts of the tropical Pacific undergo

enhanced oxygenation, the subtropical Atlantic is experiencing significant oxygen loss. DATA AVAILABILITY All data generated in this study are provided in the Supplementary Information. CODE

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Palaeocene–Eocene Thermal Maximum. _Nat. Geosci._ 4, 481––4485 (2011). Article  ADS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank A. Paytan for discussion and Y. Hu and J.

Liao for their assistance with sample preparation. This research was supported by the National Natural Science Foundation of China (42376049), National Key R&D Program of China

(2022YFF0802900), Guangdong Natural Science Foundation (2024A1515012537), Science and Technology Innovation Committee of Shenzhen Municipality (20231114150312001) grants to W.Y., and Boston

College Start-up funds to X.T.W. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Ocean Science and Engineering, Southern University of Science and Technology, Shenzhen,

Guangdong, China Weiqi Yao, Ruixiang Zhai, Ruiling Zhang & Yilin Liu * Department of Earth and Environmental Sciences, Boston College, Chestnut Hill, MA, USA Tianshu Kong, Xingchen Tony

Wang & Ruixiang Zhai Authors * Weiqi Yao View author publications You can also search for this author inPubMed Google Scholar * Tianshu Kong View author publications You can also search

for this author inPubMed Google Scholar * Xingchen Tony Wang View author publications You can also search for this author inPubMed Google Scholar * Ruixiang Zhai View author publications You

can also search for this author inPubMed Google Scholar * Ruiling Zhang View author publications You can also search for this author inPubMed Google Scholar * Yilin Liu View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS W.Y. and X.T.W. conceived the project. T.K. performed nitrogen isotope analyses. Y.L. performed

phosphorus analyses. W.Y., R.Zhai and R.Zhang contributed to the computations. W.Y. provided samples and composed the manuscript with contributions from T.K. and X.T.W. W.Y., T.K., X.T.W.,

R.Zhai, R.Zhang and Y.L. contributed to data interpretation and the final manuscript. CORRESPONDING AUTHORS Correspondence to Weiqi Yao or Xingchen Tony Wang. ETHICS DECLARATIONS COMPETING

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the Atlantic during the Paleocene-Eocene Thermal Maximum. _Nat Commun_ 15, 9053 (2024). https://doi.org/10.1038/s41467-024-53423-x Download citation * Received: 26 March 2024 * Accepted: 11

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