Author: Andrea Chen
Mentor: Dr. Selva Marroquin
Editor: Ann Zhu

Abstract

Marine sediment chemistry has been employed as a geochemical tool to provide us insights into the ocean’s redox state through time, helping to develop a framework for understanding Earth’s history. Sulfur isotopes (δ³⁴S), commonly buried in its reduced form as pyrite (FeS₂), is hypothesized to be a source of atmospheric oxygen accumulation. However, the behavior of pyrite accumulation and burial depends on the environment and redox conditions at that locality. During microbial sulfate reduction (MSR), organisms preferentially utilize ³²S during the production of sulfide, leaving the residual sulfate reservoir enriched in ³⁴S. Scientists found that pyrite (FeS₂) can capture this fractionation, however, studies overlook sulfur bound into organic molecules which constitutes a second archive of sulfur. Therefore, to analyze pyrite burial as a potential modern analogue we investigate the utility of adding coeval δ³⁴S measurements from sulfur bound into kerogen, to track environmental conditions recorded in the δ³⁴S of both pyrite and organic sulfur. Paired OM and pyrite δ³⁴S profiles make it possible to deconvolve the environmental controls on S-isotopes in the rock record.

To explore the relationship between water column redox and sulfur isotopes, we measured the kerogen and pyrite δ³⁴S, and total organic carbon (TOC) of International Ocean Discovery Program (IODP) ocean cores from the Agulhas Current. We did a sequential sulfur extraction on a single sediment aliquot to separate the different sulfur phases, which are then measured using an Elemental Analyzer and Isotope Ratio Mass Spectrometer. We predict sulfurization of organic matter in an anoxic water column leads to the formation of δ³⁴S-depleted kerogen and a smaller kerogen-pyrite offset. Understanding the δ³⁴S offset for pyrite and kerogen in the modern era will give us insights on using sulfur records to reconstruct the biogeochemical response of the ocean to past climatic events and varying oxygen levels of the water column. 

Introduction

Changes to our climate can no longer be debated. Driven by increased human emissions of greenhouse gasses, glaciers and ice sheets are shrinking, plant and animal geographic ranges are shifting, and plants and trees are blooming sooner. Any further delay in trying to understand our changing planet will cause us to miss the rapidly closing window to secure a livable future.

By the year 2100, dissolved oxygen in the ocean is predicted to decline by about 7%, depleting marine organisms from a key resource they need to survive. Warming-induced deoxygenation is a key driving factor. As sea level temperatures begin to rise, oxygen becomes less soluble, and the ocean holds less of it. This increases stratification within the ocean and induces less mixing of the deeper waters. A second contributing factor is eutrophication, when the ocean becomes progressively enriched with minerals and nutrients, particularly nitrogen and phosphorus, from fertilizer runoff on farms. An increase in nutrients in the ocean stimulates primary productivity and algal growth, but over time, more microorganisms are required to decompose the organic matter. These “blooms” can lead to loss of subaquatic vegetation and the formation of dead zones (oxygen-depleted waters), damaging the ecosystem. By analyzing marine sediment, we can gain insights into the ocean’s composition through time and develop a framework for understanding climate behaviors and Earth’s history. 

Marine sediment can be used to reconstruct the Earth’s global redox history and help us understand the role of Earth’s biosphere in shaping the long-term redox evolution of the ocean-atmosphere system. The global oxygen cycle depends on the redox cycling of carbon, sulfur, and iron through the Earth system and the availability of organic material in oceanic and terrestrial ecosystems. The ocean’s response to climate perturbations can also be reflected in what is preserved in ocean sediments. Additionally, understanding the processes affecting sedimentary sulfur cycling is necessary to accurately predict the effects of global changes in oxygen levels and temperature on the marine sulfur and carbon cycle. Since we have limited access to porewater samples and records from the past, we rely on modern marine sediment samples to observe what is preserved in the sediment and rock record. Analyzing the chemical and physical processes that get incorporated in our samples will allow us to begin reconstructing past ocean and climate behavior. 

Background

1. Key Redox Reactions 

Throughout this study we will be focusing on a few redox reactions that occur in conjunction between the water column and marine sediment. We begin at the most fundamental process: the formation of organic material in the water column. Commonly referred to as photosynthesis, this is the process by which marine algae use sunlight to synthesize organic material (CH₂O) from carbon dioxide and water, generating oxygen as a byproduct (Equation 2). 

CO₂ + H₂O ←→ CH₂O + O₂ (1)

The opposite of this process is respiration (Equation 2), when microorganisms consume oxygen and organic material to generate energy for survival.

CH₂O + O₂ ←→ CO₂ + H₂O (2)

However, if this organic material were to be buried in the sediment, this respiration reaction (Equation 2) would not be able to occur, and a net flux of oxygen would be released into the atmosphere and ocean. 

In the scenario where we are studying samples in an oxygen-limited marine environment and there are anoxic or partial anoxic conditions in the water column, our respiration redox reaction then becomes oxygen limited. Without available oxygen, the organic material can instead be used by microbes to reduce sulfate in the water column into hydrogen sulfide through the process of microbial sulfate reduction (Equation 3). 

CH₂O + SO₄^-2 → H₂S + CO₂ (3)

We consider the ocean to not be limited in sulfate. Therefore, in completely reducing conditions, all sulfates will be reduced to hydrogen sulfide. The hydrogen sulfide in the presence of highly reactive iron (HRE) will react to form pyrite (Fe₂S₂) as shown in Equation 4. 

H₂S + Fe → FeS₂ (pyrite) (4)

The formation and subsequent burial of both organic material and pyrite becomes a source of atmospheric oxygen and a sink of carbon dioxide. This provides valuable insight when highlighting the impact of these proxies to track atmospheric oxygen levels and to offset carbon emissions.  

2. Sulfur Isotopes & Fractionation 

Sulfur isotopes (δ³⁴S) measured in ancient marine sediments from around the world have been analyzed to reconstruct past global marine conditions and estimate historic atmospheric oxygen content. Sulfur is commonly found in the form of ³²S, which constitutes around 94.99% of all sulfur isotopes. The isotopic ratio (δ) refers to the ratio of the atomic abundances of both sulfur isotopes in terms of the less abundant isotope, ³⁴S/³²S. 

In our study, we will be looking at sulfur isotopes in its commonly reduced form as the mineral pyrite (FeS₂). As shown in Figure 1, samples can be categorized as either being more positive or enriched in δ³⁴S, while a more negative sulfur isotope value means it is relatively depleted in δ³⁴S and more enriched in the lighter isotope δ³²S. The δ³⁴S values of diagenetic pyrite depend on many local biological, chemical, and physical processes that make distinguishing isotope signals difficult. During microbial sulfate reduction (MSR), microbes prefer using ³²S to reduce sulfate into hydrogen sulfide since the lighter isotope has weaker chemical bonds to break. The microbes will continue to uptake ³²S until it is completely depleted. If there is a change in local environmental conditions, the microbes could shift to using ³⁴S instead. This choice by microbes to prefer ³²S over ³⁴S is referred to as its fractionation (ε), and it is expressed as a ratio in both the pools of pore water sulfate and hydrogen sulfide. Sulfate found in the water column is reduced to sulfide via MSR, which introduces a significant fractionation of sulfur isotopes that can be recorded when the sulfide reacts with iron to form pyrite. Pyrite is our solid phase archive we can use to study the fractionation present in the formation of hydrogen sulfide. 

In Figure 2, the processes described above are measured in the marine sediment below the water column. As MSR occurs, the sulfate is consumed to form hydrogen sulfide, changing their respective concentrations. During this reaction, the hydrogen sulfide gradually becomes enriched in ³⁴S as ³²S is consumed. The timing and location of pyrite formation in the sediment dictates the isotope fraction that is captured within our samples. The timing of pyrite formation all depends on conditions such as availability of oxygen and highly reactive iron content.  

There are a couple of factors that influence what portion of sulfur isotopes end up in the sediment record. Organic material is a key requirement for MSR and hydrogen sulfide formation to occur. The amount of highly reactive iron present in the sediment can influence how much and where in the sediment the pyrite forms. Additionally, in the presence of kerogen, the sulfur can also be bound into the organic material as kerogen-bound sulfur through the process of organic material sulfurization. This resulting organic material becomes more resistant to degradation, enhancing its preservation potential and further promoting the burial of carbon.

Here, we investigate the advantages of adding coeval δ³⁴S measurements from sulfur bound into kerogen to our pyrite proxy to track environmental conditions recorded in δ³⁴S. Many new findings also point to sulfur playing an important role in enhancing the preservation potential of organic carbon burial. Sulfurization, the process in which sulfur from sulfide is incorporated into the structure of organic matter, increases its likelihood of being buried and thus makes it less bioavailable. This sulfurization occurs most rapidly in waters that contain no oxygen, further increasing the amount of organic matter that gets buried during oceanic anoxic events. We are working to develop a pyrite-kerogen sulfur isotope proxy to better understand how biogeochemical shifts in our modern oceans, due to variations in temperature and oxygen levels, are reflected in modern isotopic signatures. By collecting and interpreting this data, we hope to contribute to sediment diagenesis models to better reconstruct the interaction between sedimentary and biogeochemical processes. 

Materials & Methods

To explore the relationship between water column redox and sulfur isotopes, we measured the kerogen δ³⁴S, pyrite δ³⁴S, highly reactive iron content, and total organic carbon (TOC), from International Ocean Discovery Program (IODP) ocean cores representing different depositional environments. Since the isotopic signatures of kerogen and pyrite are preserved for over a decade, even under their oxic storage conditions, we can still confidently work with IODP cores that were collected years prior. Reactive organic matter competes with iron for any available sulfide in the water column. By measuring residual total organic carbon (TOC) in sediments, we can estimate the fraction of organic matter that was remineralized by sulfate reduction. This will help determine if the exported organic matter is returned to the ocean system as CO₂ or removed from the surface through the carbon cycle. 

1. Agulhas Basin Sediment Samples

The samples we have been working with are from IODP Expedition 361, which drilled six sites on the southeast African margin and in the Indian-Atlantic Ocean gateway from January to March of 2016. The cruise was targeted to reconstruct the history of the greater Agulhas Current system over the past 5 million years. The Agulhas Basin is an oxic closed system that consists of a warm water current running along the southern boundary of Africa (Figure 3). The current has been predicted to control the strength of the Atlantic Meridional Overturning Circulation (AMOC) during the Late Pleistocene. The introduction of the saline Agulhas water into the South Atlantic stimulates buoyancy anomalies that act as control mechanisms on the AMOC, impacting convective activity in the North Atlantic and global climate conditions. Additionally, the variability of the current has effects on present-day terrestrial climates, rainfall patterns, and river runoff. 

Site	Water Column Depth (m)	Sedimentation Rate (cm/kyr)
1474B	3045	3.7
1475D	2669	2.8
1478B	488	9

We selected 25 sediment samples from 3 different sites at varying depths within the sediment core to ensure we got an even spread of data points throughout the sediment. The sites that we selected are U1474, U1475, and U1478. For each of the sites we have data on their water column depths, sedimentation rate, and geographical location (Figure 4).

Looking at the preliminary information on the sites, we immediately notice that site U1474 and U1475 have similar water column depths around 3000m and similar sedimentation rates of about 3 cm/kyr. However, site U1478 has a couple of key differences including its proximity to the continental boundary, therefore suggesting that it could have high iron contents. Additionally, it has the shallowest water column depth so we predict it may have high total organic carbon (TOC) levels since primary productivity is most abundant in nutrient-rich shallower waters. With a quick sedimentation rate of about 9 cm/kyr, we believe that this might influence the isotope offset since the microbes could have less time to select the lighter, ³²S isotope. 

2. Sequential Sulfur Extraction

In order to measure the 𝛿³⁴S within pyrite and kerogen at our three sites, we first must extract the necessary forms of sulfur from our samples. We performed a sequential sulfur extraction on our samples to ensure separation of the various forms of sulfur isotopes for analysis (Figure 5). We took about 0.5 g splits of each of the samples and the first step of the extraction involved rinsing them with degassed H2O to remove any sulfide that may be trapped within the pore water. Then we placed the samples in MeOH on a shaker table to mix for 12 hours to remove the elemental sulfur and bitchimin. The MeOH was then removed and kept for analysis and the samples were transferred to a round bottom flask. The next step was a 1-hour acidic volatile sulfur (AVS) extraction, shown on the right, that involved mixing 10 mL of 6N HCl into the samples on stir plates. Nitrogen gas was bubbled through the HCl solution and any FeS was trapped in our zinc acetate solution for analysis. The last step was a 2-hour extraction with a mix of 20mL of 1M chromium (III) chloride and 5 mL of 12N HCl. The solutions were brought to a boil in heating nests (shown on left) and any hydrogen sulfide present was collected in our zinc acetate trap solution in the form of zinc sulfide. After the extraction, the chromium chloride solution was disposed of and the remaining sample was dried down to be used for kerogen-bound sulfur isotope analysis. 

3. Sequential Iron Extraction

Another factor that influences pyrite formation is the presence of highly reactive (HR) iron which is categorized as iron that is reactive towards hydrogen sulfide. The amount of HR iron present at a given locality in the sediment can dictate where and how much pyrite forms, which is important for determining how much of the sulfur isotope offset profile gets captured in the solid phase pyrite. HR iron is composed of a few different forms of iron, including iron in carbonates, oxides, magnetite, and pyrite. 

 Fe_HR = Fe_carb  + Fe_oxides +  Fe_magnetite + Fe_pyrite (5)

The extraction process takes place in four different solutions which are shaken on a shaker table to ensure all the sediment is in contact with the solution for optimal extraction. After taking another 0.5 g split of each sample, they were placed in an ascorbic acid solution at a pH of 7.5 for 24 hours to remove any carbonate Fe. 100 L of the solution was saved for analysis and the sample was then mixed in sodium dithionite at a pH of 4.8 for another 2 hours to remove any Fe oxides, goethite and hematite specifically, and the same process was repeated to extract magnetite using ammonium oxalate at a pH of 3.2 for 6 hours. A separate 0.5 g split of each sample was mixed with sodium acetate at a pH of 4.5 for 24 hours to remove the carbonate irons, siderite, and ankerite. 

The four extraction solutions were then pipetted into trays and ferrozine reagent was added to each one (Figure 6). Depending on the amount of iron present, the ferrozine reagent changes the solution color into a deep purple color. The solutions are then analyzed on a spectrophotometer, with a deeper purple color indicating a higher content of iron, to determine the concentrations of each specific Fe pool.  

4. Hydrochloric Acid Digest

Since organic material is utilized by the microbes to reduce sulfate into hydrogen sulfide, it is helpful to know how much organic material is present in the sample to see if it can be a potential limiting reagent in pyrite formation. However, we must determine the carbonate weight percent of each sample since the carbonate is not included in the TOC measurement. To do this, we reacted the sediment with hydrochloric acid since any carbonate present would be released as carbon dioxide. Using the mass difference between the original sample and de-carbonated sample, we can calculate its carbonate weight percent. 

5. EA-IRMS (Elemental Analyzer & Isotope Ratio Mass Spectrometry)

Now that we have extracted our various forms of sulfur and organic carbon, we can now weigh splits of each sulfur extraction and the decarbonated samples into tins for analysis. The tins are combusted in the EA-IRMS where the abundances and isotope values of both carbon and sulfur are determined. 

Results

For each of the 25 samples we were able to gather the following information: 

Preliminary Information	Sample depth within the sediment (m) Pore water sulfate concentration (ppm)
Sequential Sulfur Extraction	Sulfur in pyrite weight percent Organic S weight percent
Sequential Iron Extraction	Highly reactive Fe weight percent Fraction of HR Fe that is in the form of pyrite
HCl Digest	Carbonate weight percent
EA-IRMS	δ34S isotopes in pyrite δ34S isotopes in kerogen Resulting offset δ34Sk-p Total organic carbon weight percent

Utilizing all the data we collected we can plot the trends with depth at each of the three sites. 

We first took a closer look at the sulfur isotope offsets between the three different sites and summarized the average δ³⁴S for both pyrite and organic sulfur (Figure 7). 

Site	Average δ34Spyrite	Average δ34SOrg S	Average Offset
U1474	-43.26	-11.22	31.72
U1475	-41.58	-16.94	24.63
U1478	-33.31	-15.41	17.90

Comparing the three sites we can see that there is the greatest average offset at site U1474 and the smallest offset at site U1478. The large offset at U1474 is characterized by the most depleted δ³⁴S_pyrite and most enriched δ³⁴S_{Org S}. The exact opposite is happening at site U1478 with the most enriched δ³⁴S_pyrite and more depleted δ³⁴S_{Org S}. So, is the offset in δ³⁴S driven primarily by the δ³⁴S_pyrite or δ³⁴S_{Org S}?

Site	𝜺k-p  (‰ avg)	𝛿34Spyrite  (‰ avg)	𝛿34SOrgS  (‰ avg)	HR Fe (wt% avg)	Pyr/HR Fe (% avg)	Sedimentation (cm/kyr)
U1474	31.72	-43.26	-11.22	0.49	34	3.7
U1475	24.63	-41.58	-16.94	0.26	73	2.8
U1478	17.90	-33.31	-15.41	0.96	34	9

Summarizing the average results from the three sites, we can use the preliminary site information and iron content to provide a possible explanation for the varying offsets. 

At site U1474, we see the greatest average offset between kerogen and pyrite, but the pyrite is the most depleted in ³⁴S. A possible explanation for this is that the pyrite formed rather quickly sampling only the lighter ³²S isotopes. At site U1475, we see that it has the smallest average highly reactive iron weight percent, suggesting that the sediments could be iron limited. With less HR Fe available, less pyrite can form and therefore the sulfur could become bound into kerogen. The lighter isotopes, instead of being incorporated into the pyrite, are incorporated in the kerogen as organic sulfur, represented in the highly depleted δ³⁴S_pyrite. At our last site, U1478, we see the smallest offset and most enriched δ³⁴S_pyrite. The small offset could be explained by the quick sedimentation rate since even though it is not iron limited there is still only 34% of the iron being incorporated into pyrite. With the quick burial of organic material, the microbes could have less time to choose the lighter ³²S isotope, possibly contributing to the most enriched δ³⁴S_pyrite. 

Conclusion

At our three sites we can come to preliminary conclusions regarding explanations for the varying δ³⁴S offset between kerogen-bound sulfur and pyrite. At our first site, U1474, we believe that pyrite is driving our offset and that the pyrite formed quickly and early, therefore only profiling the lighter sulfur isotopes. At our second locality, we instead believe that organic sulfur is dictating the offset since the site has low HR Fe availability, which causes OM sulfurization to occur over pyrite formation. The lighter isotope of sulfur is bound into the organic matter causing the δ³⁴S_{Org S} to be highly depleted, resulting in a smaller offset. At our last site, we believe that pyrite is dedicating our offset with influence by sedimentation rates. Even with an abundance of highly reactive iron available, there is still a small portion that is found in the form of pyrite. We believe that pyrite quickly sampled a large portion of the H₂S isotope profile and became enriched in ³⁴S. Additionally, with such quick burial and sedimentation rates, the microbes could have less selectivity and time when choosing between ³²S and ³⁴S. 

Global climate change has significant impacts on biogeochemical responses of the oceans since it involves differences in nutrient regimes and primary production. Each of these sites represents common past and present marine environmental conditions that can provide insight into the atmosphere-ocean conditions at that time. Organic material sulfurization can promote carbon burial and could be a potential offset to rising carbon emissions. Since iron levels can be dictated by seasonality and rainfall it could give clues to the climate conditions at the time of pyrite formation. This work will begin to help us understand the link between sulfur cycling and organic remineralization, allowing for better predictions to be made about water column redox and nutrient input. Additionally, our results reinforce the benefits of pairing kerogen and pyrite as a sulfur isotope proxy to capture a complete picture regarding environmental conditions at our study sites. Similarly, being able to identify future ocean dead zones more quickly will have great impacts on fisheries and marine life and give us insight on how marine sediment responds to natural perturbations in the water column. Additionally, the connection between nutrient input and sulfurization processes in promoting carbon burial may allow tracking of feedback processes to improve climate planning and mitigation. Looking to the sediment record will give us key insights to the biogeochemical response of the ocean due to past and future climate perturbations. All results from this study will be shared to the broader scientific community and public through presentations at larger scientific meetings and manuscripts. 

As we analyze more samples and expand our site locations, we can begin to find patterns that will continue to support our findings. The next site we are analyzing is the sub-oxic to anoxic Baltic Sea through sediment cores retrieved with IODP Expedition 347. Comparing the oxic Agulhas Basin and anoxic Baltic Sea samples will allow us to determine the influence of water column redox on the isotope offset between pyrite and organic sulfur. During sub-oxic conditions in the water column, I would expect enhanced rates of microbial sulfate reduction, which preferentially utilizes the lighter isotope, δ³²S, therefore producing sulfide with very low δ³⁴S values. Therefore, I hypothesize that the sulfurization of organic matter in an oxygen deficient water column leads to the formation of ³⁴S-depleted kerogen and a smaller kerogen-pyrite offset. To gain a better understanding of the interactions between the water column chemistry and what gets preserved in the sediment, we are working to develop a localized computer model to map the fluxes at play. By combining computer models and data from sediment samples, we can hopefully begin to reconstruct climate conditions throughout time.

References

Fike, David A., et al. “Rethinking the Ancient Sulfur Cycle.” Annual Review of Earth and Planetary Sciences, vol. 43, no. 1, 2015, pp. 593–622. Annual Reviews,  https://doi.org/10.1146/annurev-earth-060313-054802.

Raiswell, Rob, et al. “The Determination of Labile Fe in Ferrihydrite by Ascorbic Acid  Extraction: Methodology, Dissolution Kinetics and Loss of Solubility with Age and de-Watering.” Chemical Geology, vol. 278, no. 1, Nov. 2010, pp. 70–79. ScienceDirect, https://doi.org/10.1016/j.chemgeo.2010.09.002.