This research was jointly funded and supported by the Natural Science Foundation of China (No. 91128101, 41273054, and 41373007), the China Geological Survey Project for South China Sea Gas Hydrate Resource Exploration (No. DD20160211), Fundamental Research Funds for the Central Universities (No. 16lgjc11), and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (No. 2011). Zhiyong Lin acknowledges the financial support provided by the China Scholarship Council (No. 201506380046). Yang Lu thanks the Guangzhou Elite Project (No. JY201223) and the China Postdoctoral Science Foundation (No. 2016M592565). We are grateful to Dr. Shengxiong Yang, Guangxue Zhang, and Dr. Jinqiang Liang of the Guangzhou Marine Geological Survey for providing samples and valuable suggestions. We thank Dr. Xianhua Li and Dr. Lei Chen of the Institute of Geology and Geophysics (Beijing), Chinese Academy of Sciences, for help with the SIMS analysis. Dr. Xiaoping Xia is thanked for making available the SIMS Lab of the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, for the filming of this article. The manuscript benefited from comments from Dr. Alisha Dsouza, review editor of JoVE, and two anonymous referees.

1. Collection of Samples from a Sediment Core
Note: The core HS148 was obtained from a site near the gas hydrate drilling zone in the Shenhu area, South China Sea, during a cruise of the R/V Haiyang Sihao in 2006.
<ol>
	<li>Cut the piston core (here, HS148) into sections at intervals of 0.7 m from the top to the bottom (onboard the vessel) and transfer the sections to a cold room (4 &#176;C) for storage after retrieval.</li>
	<li>Transfer the core sections to a cold room (4 &#176;C) in the land-based lab for storage after the cruise. Take the sections out of the cold room and use a saw to cut them into halves lengthwise.</li>
	<li>Clean the surface of the sediment and collect a set of sediment samples (15 cm in length; 1/4 of the sediment core) across the entire length using a knife. Pack the wet samples individually in zippered plastic bags and label them using a marker.</li>
	<li>Place the wet sediment samples (~30 g) in pre-cleaned beakers and dry them at 40 &#176;C in a drying oven for 24 h. After drying, separate the sediments into two aliquots: one for the collection of pyrite aggregates (i.e., authigenic pyrite), and the other for bulk sulfur extraction (see step 3).</li>
	<li>Put one aliquot of dry sediment into beakers and add distilled water to soften the sediment for 2 h. Transfer the slurry (including the sediments and water in the beaker) to a 0.063-mm sieve washed with distilled water.
	<ol>
		<li>Sift the sediment with distilled water so that all fine grains (&#60;0.063 mm) are washed through. Collect the coarse fraction (e.g., quartz grains, fossil shells, and authigenic minerals) in beakers and dry them at 40 &#176;C in a drying oven for 24 h.</li>
	</ol>
	</li>
	<li>Place some of the coarse segment fractions on a glass slide under a binocular microscope (20X magnification). Identify the pyrite aggregates from the coarse fraction. Handpick such pyrite aggregates using a needle and pack them individually into zippered plastic bags. 
	NOTE: Most of the pyrite aggregates are black in color and tubular in shape.</li>
	<li>Pulverize a second aliquot of dry sediment sample into a fine powder (&#60;0.074 mm) using an agate mortar for further bulk sulfur extraction (see step 3).</li>
</ol>
2. Observation of Variable Morphologies
<ol>
	<li>Select some representative pyrite tubes from the handpicked pyrite aggregates under a binocular microscope (20X magnification) for thick section preparation to examine the morphological and textural features of the pyrite aggregates.</li>
	<li>Stick double-sided tape on a slide and place the selected pyrite tubes on the tape. Put a mounting tube (25 mm in diameter) on the slide to cover all pyrite aggregates. Mix 10 mL of epoxy resin with 1.3 mL of hardener at room temperature and pour the mixing liquid into the mounting tube.
	<ol>
		<li>Place the slide and the mounting tube into a vacuum chamber. Pump the air out of the chamber until the pressure in the chamber is below 0.2 bar, so that all the pore spaces of the samples are filled with epoxy. Move the slide and the mounting tube out of the chamber and let the epoxy cure at room temperature for 12 h.</li>
		<li>After the epoxy has cured, hand-grind the pyrite tubes on a fixed, 9-&#181;m diamond mesh pad until the pyrite grains are exposed. Hand-polish the pyrite grains to produce a smooth and flat surface, using 5-, 3-, and 1-&#181;m diamonds successively.</li>
	</ol>
	</li>
	<li>Observe the morphology and the texture of the pyrite under a reflected light microscope at 200X magnification, with a ~3-mm working distance.</li>
	<li>Perform petrographic observation under a reflected light microscope6 and then coat the thick sections with a 25-nm layer of carbon. Examine their morphological and textural features using a thermal field emission scanning electron microscope with secondary electron imaging and backscattered electron modes6,19. 
	NOTE: This step was performed at the School of Earth Science and Geological Engineering, Sun Yat-sen University.</li>
</ol>
3. Bulk Sulfur Isotope Analyses
Note: The total sulfur (as sulfide) was extracted as hydrogen sulfide via wet chemical sequential extraction26,27 at the Institut f&#252;r Geologie und Pal&#228;ontologie, Westf&#228;lische Wilhelms-Universit&#228;t M&#252;nster.
<ol>
	<li>Place 4 g of the dried sample powder or 10 mg of pyrite aggregates per flask into round-bottom flasks and add 10 mL of ethanol into each flask as a catalyst.
	<ol>
		<li>Prepare zinc acetate (3%) acetic acid solution in a 500-mL glass flask to trap hydrogen sulfide. Connect the zinc acetate-containing flask to the sample-containing flask. Check the connections of the flasks and flush nitrogen into the flasks to remove the air.</li>
	</ol>
	</li>
	<li>Inject 20 mL of HCl solution (25%) into the round-bottom flasks using a syringe to liberate the acid-volatile (mono)sulfides (AVS) from the sample; allow the samples to react for 1 h at room temperature. 
	NOTE: Here, the analysis revealed that no AVS was present in the studied samples.</li>
	<li>Inject 30 mL of 1 M CrCl2 solution into the round-bottom flasks when the above reaction is completed; allow the samples to react for 2 h at 85 &#176;C. 
	NOTE: Chromium reducible sulfur (CRS, pyrite) reduces to hydrogen sulfide (H2S) after the reaction and precipitates as zinc sulfide in the zinc acetate trap.</li>
	<li>Transfer all the solutions containing zinc sulfide precipitates to beakers and convert the zinc sulfide precipitates to silver sulfide (Ag2S) by adding a 0.1 M AgNO3 solution to the beakers. Place the beakers on the heating plate and heat them to 90 &#176;C so that the finely disseminated Ag2S better coagulates.
	<ol>
		<li>Collect the Ag2S precipitates by filtration (&#60;0.45 &#181;m) after the solution has cooled down to room temperature and dry the filtrate overnight at 40 &#176;C.</li>
	</ol>
	</li>
	<li>Weigh 200 &#181;g of Ag2S precipitate and mix it with an equal amount of V2O5 in tin cups. Have the sulfur composition analyzed on SO2 molecules via combustion using a mass spectrometer connected to an elemental analyzer (EA-IRMS)6. 
	NOTE: The above step was performed at the Institut f&#252;r Geologie und Pal&#228;ontologie, Westf&#228;lische Wilhelms-Universit&#228;t M&#252;nster.</li>
</ol>
4. In Situ SIMS Analysis
<ol>
	<li>Select representative pyrite aggregates with characterized crystal habits (e.g., framboids, overgrows, and euhedral crystals) from different sediment samples after petrographic study. Stick the selected pyrite aggregates and pieces of Sonora pyrite standard to double-sided tape. Mold them within 5 mm of the center of a 25-mm epoxy mount. 
	NOTE: The epoxy disc production process is the same as in step 2.2.
	<ol>
		<li>After the epoxy has cured, hand-grind the disc on a fixed 9-&#181;m diamond mesh pad to the desired level so that pyrite grains are exposed. Hand-polish the epoxy discs to produce a smooth, flat surface, successively using 5-, 3-, and 1-&#181;m diamonds, which is required for high-precision isotope ratio analysis by SIMS28.</li>
	</ol>
	</li>
	<li>Clean the surface of the epoxy disc with deionized water and ethanol. Place the epoxy disc in a gold-coating machine and coat the dry surface sections with a 25-nm layer of gold.
	<ol>
		<li>Observe the sample again under a scanning electron microscope at 1,000X magnification, with a 9-mm working distance, to select spots characterized with different crystal habits (e.g., framboids, overgrows, and euhedral crystals) for SIMS analysis. 
		NOTE: High-spatial resolution SIMS sulfur isotope analysis was applied to reveal the sulfur isotope variability of the different pyrite types.</li>
	</ol>
	</li>
	<li>Perform SIMS analysis15,16. 
	NOTE: Performed at the SIMS lab of the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences.
	<ol>
		<li>Use a Cs+ primary ion beam to measure the sulfur isotope ratios (34S/32S) of pyrite. Focus the Cs+ primary ion beam onto a 15 &#181;m &#215; 10 &#181;m spot at an energy of 10 kV, with 2.5-nA current. Use three off-axis Faraday cups for the simultaneous measurement of 32S, 33S, and 34S in multi-collector mode, with an entrance slit width of 60 &#181;m and an exit slit width of 500 &#181;m on each of the three Faraday cup detectors.</li>
	</ol>
	</li>
	<li>Carry out sulfur isotope analyses in automated sequences, with each analysis consisting of 30 s of pre-sputtering, 60 s of secondary ion automated centering, and 160 s of data acquisition and sulfur isotope signal integration (40 cycles &#215; 4 s).
	<ol>
		<li>Analyze Sonora pyrite as a standard at regular intervals, every 5 - 6 sample analyses. 
		NOTE: See Chen et al.19 for more detailed analytical methods and instrument parameters.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The sulfur isotope analysis of pyrite is a useful approach and can help in identifying the biogeochemical processes that impact pyritization. However, if bulk sulfur isotope analysis is applied, the obtained sulfur isotope signatures commonly represent mixed signals, as sedimentary pyrite aggregates typically consist of multiple, closely interfingering generations. Here, we present a method (i.e., SIMS analysis) for analyzing the in situ sulfur isotopic compositions of various pyrite generations on the ...

Methane emissions from sediments are common along continental margins1,2. However, most of the methane in areas of diffusive seepage is oxidized at the expense of sulfate within the sediments, a process known as SO4-AOM (Equation 1)3,4. The production of sulfide during this process commonly results in the precipitation of pyrite. Also, OSR also drives the formation of pyrite by releasing sulfide (Equation 2)5.
CH4 + SO42&#8211; &#8594; HS&#8211; + HCO3&#8211; + H2O (1)
2CH2O + SO42&#8211; &#8594; H2S + 2HCO3&#8211; (2)
It has been found that authigenic sulfide in the sulfate-methane transition zone (SMTZ) reveals high &#948;34S values, which was suggested to be caused by enhanced SO4-AOM in areas of seepage6,7,8. In contrast, pyrite induced by OSR commonly displays lower &#948;34S values9. However, it is challenging to identify different pyrite generations induced by these processes (i.e., OSR and SO4-AOM) if only a bulk sulfur isotope measurement is used, since the successively formed interfingering pyrite generations are characterized by different isotopic compositions. Therefore, microscale in situ sulfur isotope analysis is required to improve our understanding of the actual mineralizing processes10,11,12. As a versatile technique for in situ isotope analysis, SIMS requires only a few nanograms of sample, which sparked its designation as a nondestructive technique. A primary ion beam sputters the target, causing the emission of secondary ions that are subsequently transported to a mass spectrometer for measuring13. In an early in situ sulfur isotope analysis application of SIMS, Pimminger et al. successfully analyzed the &#948;34S values in galena by using a 10 - 30 &#181;m-diameter beam14. This approach has been increasingly applied to the microanalysis of sulfur isotopic compositions in sulfides, with significant improvements in both measurement precision and resolution11,12,13,14,15,16,17,18,19,20. Pyrite with various morphologic attributes and distinct sulfur stable isotope patterns has been reported from seep and non-seep environments21,22,23,24. However, to the best of our knowledge, prior to our recent SIMS study6, only one study used the in situ sulfur isotope analysis of pyrite from seep environments and revealed large sulfur isotope variability in biogenic pyrite25.
In this study, we applied SIMS to analyze the &#948;34S values of different generations of authigenic pyrite from a seepage site in the South China Sea, which allowed for microscale discrimination of OSR- and SO4-AOM-derived pyrite.

<table><tbody><tr><td>secondary ion mass spectroscopy</td><td>Cameca</td><td>IMS-1280</td><td></td></tr><tr><td>thermal field emission scanning electron microscopy</td><td>Quanta</td><td>Quanta 400F</td><td></td></tr><tr><td>elemental analyser - isotope ratio mass spectrometry</td><td>ThermoFinnigan</td><td>ThermoFinnigan Delta Plus</td><td></td></tr><tr><td>binocular microscope</td><td>any</td><td>NA</td><td></td></tr><tr><td>reflected light microscope</td><td>Carl Zeiss</td><td>3519001617</td><td></td></tr><tr><td>polishing machicine</td><td>Struers</td><td>60210535</td><td></td></tr><tr><td>cutting machicine</td><td>Struers</td><td>50110202</td><td></td></tr><tr><td>carbon/gold coating machicine</td><td>any</td><td>NA</td><td></td></tr><tr><td>ethanol</td><td>any</td><td>NA</td><td></td></tr><tr><td>acetic acid</td><td>any</td><td>NA</td><td></td></tr><tr><td>zinc acetate solution (3%)</td><td>any</td><td>NA</td><td></td></tr><tr><td>HCl solution (25%)</td><td>any</td><td>NA</td><td></td></tr><tr><td>1 M CrCl&lt;sub&gt;2&lt;/sub&gt; solution</td><td>any</td><td>NA</td><td></td></tr><tr><td>0.1 M AgNO&lt;sub&gt;3&lt;/sub&gt; solution</td><td>any</td><td>NA</td><td></td></tr><tr><td>V&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;5 &lt;/sub&gt;powder</td><td>any</td><td>NA</td><td></td></tr><tr><td>pure nitrogen</td><td>any</td><td>NA</td><td></td></tr><tr><td>syringe</td><td>any</td><td>NA</td><td></td></tr><tr><td>filter(&amp;lt;0.45 &amp;micro;m)</td><td>any</td><td>NA</td><td></td></tr><tr><td>tin cups</td><td>any</td><td>NA</td><td></td></tr><tr><td>round bottom flasks</td><td>any</td><td>NA</td><td></td></tr><tr><td>epoxy</td><td>Struers</td><td>41000004</td><td></td></tr></tbody></table>

preparation of authigenic pyrite from methane-bearing sediments for in situ sulfur isotope analysis using sims

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Data Expression - Bulk Sulfur Isotopes:
The bulk sulfur isotope ratio is expressed in relation to the Vienna Canyon Diablo Troilite (V-CDT) standard, and the analytical precision is better than &#177;0.3&#8240;. The sulfur isotope measurements were calibrated with international reference materials: IAEA-S1 (&#948;34S = -0.30&#8240;), IAEA-S2 (&#948;34S = -21.55&#8240;), IAEA-S3 (&#948;34S = -31.4&#8...

Watch this Scientific Journal Video about Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS at JoVE.com

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

preparation-authigenic-pyrite-from-methane-bearing-sediments-for-situ

Research

JoVE Journal

Environment

7.6K Views.  Sun Yat-sen University. Analyses van de zwavel isotopensamenstelling (δ34van S) van pyriet van methaan-bevattende sedimenten hebben meestal gericht op laboratoriummonsters. Hier, pasten we secundaire ion massaspectrometrie voor het analyseren van de δ34S-waarden van verschillende generaties van pyriet te begrijpen van de diagenetic geschiedenis van pyritization.

Video: Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. 
This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.

Microbial sulfate reduction is a process of great importance in environmental biotechnology. The success of the sulfidogenic reactors depends among other factors on the microbial composition of the sludge. Here, we present a protocol to develop sulfidogenic sludge from hydrothermal vents sediments in a UASB reactor for reductive dechlorination purposes.

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate ...

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is built and can be transported to a temporary work space close to field sampling site(s) to facilitate rapid analysis. Cores are extruded into a portable glove bag, where they are sectioned and each 1-3 cm thick section (depending on core diameter) is sealed into 50 ml centrifuge tubes. Pore waters are separated with centrifugation outside of the glove bag and then returned to the glove bag for separation from the sediment. These extracted pore water samples can be analyzed immediately. Immediate analyses of redox sensitive species, such as sulfide, iron speciation, and arsenic speciation indicate that oxidation of pore waters is minimal; some samples show approximately 100% of the reduced species, e.g. 100% Fe(II) with no detectable Fe(III). Both sediment and pore water samples can be preserved to maintain chemical species for further analysis upon return to the laboratory.

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Metal-silicate Partitioning at High Pressure and Temperature: Experimental Methods and a Protocol to Suppress Highly Siderophile Element Inclusions

Estimates of the primitive upper mantle (PUM) composition reveal a depletion in many of the siderophile (iron-loving) elements, thought to result from their extraction to the core during terrestrial accretion. Experiments to investigate the partitioning of these elements between metal and silicate melts suggest that the PUM composition is best matched if metal-silicate equilibrium occurred at high pressures and temperatures, in a deep magma ocean environment. The behavior of the most highly siderophile elements (HSEs) during this process however, has remained enigmatic. Silicate run-products from HSE solubility experiments are commonly contaminated by dispersed metal inclusions that hinder the measurement of element concentrations in the melt. The resulting uncertainty over the true solubility and metal-silicate partitioning of these elements has made it difficult to predict their expected depletion in PUM. Recently, several studies have employed changes to the experimental design used for high pressure and temperature solubility experiments in order to suppress the formation of metal inclusions. The addition of Au (Re, Os, Ir, Ru experiments) or elemental Si (Pt experiments) to the sample acts to alter either the geometry or rate of sample reduction respectively, in order to avoid transient metal oversaturation of the silicate melt. This contribution outlines procedures for using the piston-cylinder and multi-anvil apparatus to conduct solubility and metal-silicate partitioning experiments respectively. A protocol is also described for the synthesis of uncontaminated run-products from HSE solubility experiments in which the oxygen fugacity is similar to that during terrestrial core-formation. Time-resolved LA-ICP-MS spectra are presented as evidence for the absence of metal-inclusions in run-products from earlier studies, and also confirm that the technique may be extended to investigate Ru. Examples are also given of how these data may be applied.

We present a procedure to determine the metal-silicate partitioning of siderophile elements, emphasizing techniques that suppress the formation of metal inclusions in experiments for the noble metals. The results of these experiments are used to demonstrate the effect of core-formation on the highly siderophile element composition of the mantle.

Estimates of the primitive upper mantle (PUM) composition reveal a depletion in many of the siderophile (iron-loving) elements, thought to result from ...

Chemistry

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

Simulation of Early Earth Hydrothermal Chimneys in a Thermal Gradient Environment

Deep sea hydrothermal vents are self-organizing precipitates generated from geochemical disequilibria and have been proposed as a possible setting for the emergence of life. The growth of hydrothermal chimneys in a thermal gradient environment within an early Earth vent system was successfully simulated by using different hydrothermal simulants, such as sodium sulfide, which were injected into an early Earth ocean simulant containing dissolved ferrous iron. Moreover, an apparatus was developed to sufficiently cool the ocean simulant to near 0 &#176;C in a condenser vessel immersed in a cold water bath while injecting a sulfide solution at hot to room temperatures, effectively creating an artificial chimney structure in a temperature gradient environment over a period of a few hours. Such experiments with different chemistries and variable temperature&#160;gradients resulted in a variety of morphologies in the chimney structure. The use of ocean and hydrothermal fluid simulants at room temperature resulted in vertical chimneys, whereas the combination of a hot hydrothermal fluid and cold ocean simulant inhibited the formation of robust chimney structures. The customizable 3D printed condenser created for this study acts as a jacketed reaction vessel that can be easily modified and used by different researchers. It will allow the careful control of injection rate and chemical composition of vent and ocean simulants, which should help accurately simulate prebiotic reactions in chimney systems with thermal gradients similar to those of natural systems.

The goal of this protocol is to form simulated hydrothermal chimneys via chemical garden injection experiments and introduce a thermal gradient across the inorganic precipitate membrane, using a 3D printable condenser that can be reproduced for educational purposes.

Deep sea hydrothermal vents are self-organizing precipitates generated from geochemical disequilibria and have been proposed as a possible setting for the ...

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

Separation of Uranium and Thorium for 230Th-U Dating of Submarine Hydrothermal Sulfides

The age of a submarine hydrothermal sulfide is a significant index for estimating the size of hydrothermal ore deposits. Uranium and thorium isotopes in the samples can be separated for 230Th-U dating. This article presents a method to purify and separate U and Th isotopes in submarine hydrothermal sulfide samples. Following this technique, the separated U and Th fractions can meet measuring requirements by multi-collector inductively coupled plasma mass spectrometry (MC-ICPMS). The age of the hydrothermal sulfide sample can be calculated by measuring the present-day activity ratios of 230Th/238U and 234U/238U. A super clean room is necessary for this experiment. Cleaned regents and supplies are used to reduce the contamination during the sample processes. Balance, hotplate, and centrifuge are also used. The sulfide sample is powdered for analysis and less than 0.2 g sample is used. Briefly, the sample is weighed, dissolved, added to 229Th-233U-236U double spike solution, Fe co-precipitated, and separated on an anion-exchange resin extraction column. Approximately 50 ng U is consumed for 230Th-U dating of sulfides sample by MC-ICPMS.

Separation of Uranium and Thorium for 230Th-U Dating of Submarine Hydrothermal Sulfides

The protocol describes a method to purify and separate the U and Th nuclide in submarine hydrothermal sulfide sample with Fe co-precipitation and extraction chromatography for 230Th-U disequilibrium dating.

The age of a submarine hydrothermal sulfide is a significant index for estimating the size of hydrothermal ore deposits. Uranium and thorium isotopes in ...

Using Flexible Gold-Titanium Reaction Cells to Simulate Pressure-Dependent Microbial Activity in the Context of Subsurface Biomining

Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging obstacles, including the special environmental conditions that need to be replicated, e.g., high pressure and in some cases acidic solutions. The former requires an experimental setup suitable for pressurization up to 100 bar, while the latter demands a fluid container with high chemical resistance against corrosion and unwanted chemical reactions with the container wall. To meet these conditions for an application in the field of in situ biomining, a special flexible gold-titanium reaction cell inside a rocking high-pressure reactor was used in this study. The described system allowed simulation of in situ biomining through sulfur-driven microbial iron reduction in an anoxic, pressure-controlled, highly chemically inert experimental environment. The flexible gold-titanium reaction cell can accommodate up to 100 mL of sample solution, which can be sampled at any given time point while the system maintains the desired pressure. Experiments can be performed on timescales ranging from hours to months. Assembling the high-pressure reactor system is fairly time consuming. Nevertheless, when complex and challenging (microbiological) processes occurring in the earth&#39;s deep subsurface in chemically aggressive fluids have to be investigated in the laboratory, the advantages of this system outweigh the disadvantages. The results found that even at high pressure the microbial consortium is active, but at significantly lower metabolic rates.

This protocol describes microbial experiments under elevated pressures to study in situ biomining processes. The experimental approach employs a rocking high-pressure reactor equipped with a gold-titanium reaction cell containing a microbial culture in an acidic, iron-rich medium.

Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging ...

Engineering

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

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