Name: preparation of authigenic pyrite from methane-bearing sediments for in situ sulfur isotope analysis using sims
Uploaded: 2017-08-31T12:30:00
Description: 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

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 l...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+sulfate-driven+anaerobic+oxidation+of+methane+affects+the+sulfur+isotopic+composition+of+pyrite:+A+SIMS+study+from+the+South+China+Sea.">How sulfate-driven anaerobic oxidation of methane affects the sulfur isotopic composition of pyrite: A SIMS study from the South China Sea.</a> Chem Geol. 440, 26-41 (2016).</li><li>J&#248;rgensen, B. B., B&#246;ttcher, M. E., L&#252;schen, H., Neretin, L. N., Volkov, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+methane+oxidation+and+a+deep+H2S+sink+generate+isotopically+heavy+sulfides+in+Black+Sea+sediments.">Anaerobic methane oxidation and a deep H2S sink generate isotopically heavy sulfides in Black Sea sediments.</a> Geochim Cosmochim Ac. 68 (9), 2095-2118 (2004).</li><li>Borowski, W. S., Rodriguez, N. M., Paull, C. K., Ussler, III, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+34S-enriched+authigenic+sulfide+minerals+a+proxy+for+elevated+methane+flux+and+gas+hydrates+in+the+geologic+record?.">Are 34S-enriched authigenic sulfide minerals a proxy for elevated methane flux and gas hydrates in the geologic record?.</a> Mar Petrol Geol. 43, 381-395 (2013).</li><li>Canfield, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope+fractionation+by+natural+populations+of+sulfate-reducing+bacteria.">Isotope fractionation by natural populations of sulfate-reducing bacteria.</a> Geochim Cosmochim Ac. 65 (7), 1117-1124 (2001).</li><li>McKibben, M. A., Eldridge, C. 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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 border="0" cellpadding="0" cellspacing="0" width="835">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">secondary ion mass spectroscopy</td>
			<td style="width:128px;">Cameca</td>
			<td style="width:179px;">&#160;IMS-1280</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;thermal field emission scanning electron microscopy</td>
			<td>Quanta</td>
			<td>Quanta 400F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">elemental analyser - isotope ratio mass spectrometry</td>
			<td>ThermoFinnigan</td>
			<td colspan="2">ThermoFinnigan Delta Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">binocular microscope</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">reflected light microscope</td>
			<td>Carl Zeiss</td>
			<td>3519001617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polishing machicine</td>
			<td>Struers</td>
			<td>60210535</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cutting machicine</td>
			<td>Struers</td>
			<td>50110202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">carbon/gold coating machicine</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetic acid&#160;</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">zinc acetate solution (3%)&#160;&#160;&#160;</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl solution (25%)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1 M CrCl2 solution</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">0.1 M AgNO3 solution</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">V2O5 powder</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pure nitrogen</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">syringe</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">filter(&#60;0.45 &#181;m)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tin cups</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">round bottom flasks</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

The overall goal of this procedure is to analyze the In Situ sulfur isotopic compositions of various pyrite generations by secondary ion mass spectroscopy in order to understand the diagenetic history of pyritization in methane bearing sediments. This method can help identify the biogeochemical processes that impact pyritazation, like organiclastic sulfur reduction and sulfur-driven anaerobic oxidation of methane which can now be resolved by a box of isotopic analyzers. The main advantage of this technique is high-resolution and precision, which allowed us to analyze the sulfur isotopic composition of a pyrite on a micro-scale.Demonstrating the SIMS analyzed procedure will be Qing Yang, a technician from the SIMs laboratory at the Guangzhou Institute of Geochemistry. To begin, clean the surface of a sediment core and collect a set of samples across the entire length using a knife. Pack the wet samples individually in zippered plastic bags, and label them using a marker.When ready to continue, transfer the wet sediment samples into pre-cleaned beakers and place them in an oven at 40 degrees Celsius for 24 hours to dry them out. After drying, separate the sediments into two aliquots, one for the collection of pyrite aggregates and the other for bulk sulfur extraction. Add 400 milliliters of distilled water to soften one aliquot of the sediment for two hours.Transfer the slurry into a 063 millimeter civ and sift the sediment with distilled water so that all fine grains are washed through the civ. Collect the coarse fraction in beakers and dry them in an oven at 40 degrees Celsius for 24 hours. Once dry, place some of the coarse segment fractions on a glass slide and set the slide under a binocular microscope.Turn to the 20x objective and identify the pyrite aggregates. Handpick the identified pyrite aggregates using a needle and pack them individually into zippered plastic bags. Pulverize a second aliquot of dry sediment sample into a fine powder using an agate mortar for further bulk sulfur extraction.Store the powder in zippered plastic bags. Select some representative pyrite tubes from the selected aggregates under a binocular microscope to examine the morphological and textural features of the pyrite aggregates. Stick double-sided tape on a slide, and place the selected pyrite tubes on the tape.Then, place a 25 millimeter diameter mounting tube on the slide to cover all of the pyrite aggregates. Mix 10 milliliters of epoxy resin with 1.3 milliliters of hardener at room temperature and pour the mixing liquid into the mounting tube. Then, 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. Next, move the slide and the mounting tube out of the chamber and let the epoxy cure at room temperature for 12 hours. Once the epoxy has cured, hand grind the pyrite tubes on a fixed nine micrometer diamond mesh pad until the pyrite grains are exposed.Then, hand polish the pyrite grains to produce a smooth and flat surface. Observe the morphology and the texture of the pyrite under a reflected light microscope at 200x magnification using a three millimeter working distance. Select representative pyrite aggregates with characterized crystal habits from different sediment samples after the petrographic study.And then stick them to double-sided tape. Then, position the samples within five millimeters of the center of a 25 millimeter mount, and add epoxy. After the epoxy has cured, hand grind the disc on a fixed nine micrometer diamond mesh pad to the desired level so that the pyrite grains are exposed.As before, hand polish the epoxy discs to produce a smooth, flat surface successively using five, three, and one micrometer diamond pads. Next, clean the surface of the epoxy disc with deionized water, followed by ethanol. Observe the sample under a reflective light microscope at 200x magnification, and a three millimeter working distance.For greater magnification, gold coat the sample and image using an electron microscope. For SIMS analysis, use a cesium primary ion beam to measure the sulfur isotope ratios of pyrite. Focus the cesium primary ion beam onto a 15 micrometer by 10 micrometer spot at an energy of 10 kilobolts, with a 2.5 nanoamp current.The identification of pyrite aggregates should be large enough to avoid missing a optimum pyrogenetic faces. Besides, it's better to analyze a sufficient number of spots to ensure the obtained isotopes in nature are representative. Use three off-axis faraday cups for the simultaneous measurement of sulfur-32, sulfur-33, and sulfur-34 in multi-collector mode with an entrance slit width of 60 micrometers and an exit slip width of 500 micrometers on each the three faraday cup detectors.Carry out the sulfur isotope analyses and automated sequences with each analysis consisting of 30 seconds of pre-sputtering, 60 seconds of secondary ion automated centering, and 160 seconds of data acquisition and sulfur isotope signal integration. Analyze Sonora pyrite as a standard at regular intervals between every five to six samples. Most pyrite aggregates handpicked from the sediment are black in color and tubular in shape varying from three to eight millimeters in length, and 0.2 to 0.6 millimeters in diameter.These longitudinal cross-sections of the pyrite tubes show typical hollow interiors and different wall thicknesses. When analyzed by secondary ion mass spectrometry, the pyrite found in shallow zones is mainly framboidal and depleted in sulfur-34. Meanwhile, overgrowths and euhedral crystals enriched in sulfure-34 are abundant in deeper zones.The amount of chromium reducible sulfur content ranges from zero weight percent to 0.98 weight percent in the sample shown here. Below 50 centimeters below sea-floor, the sample shows minor fluctuations around the main value of 0.44 weight percent. And two distinct peaks of 0.98 weight percent at 490 centimeters below sea-floor and 0.78 weight percent that 590 centimeters below sea-floor are present.The sulfur isotopic composition of three types of pyrite in the delta sulfur-34 values of chromium reducible sulfur content and handpicked pyrite aggregates are shown here. The shaded portion of the graph refers to the area affected by the sulfate-driven anaerobic oxidation of methane. The dashed line separates a zone to the left, suggested to be dominated by organiclastic sulfate reduction and the zone to the right suggested to be dominated by the sulfate driven anaerobic oxidation of methane.After watching this video, you may have a good understanding of how to prepare orthogenic pyrites from a thin bearing sediments for institute sulfur isotope analysis using SIMS. Following this procedure, only a few pyrite material samples are needed for sulfur isotope analysis, and you can obtain the results in a few minutes, which is much more effective than the traditional bulk sulfur isotope analysis. This approach can serve as a sensitive tool for reconstructing the pyrite elation sequences that develop during diagenesis in modern marine sediments.What's more, it should also target ancient sedimentary sequences aiming to result of different biogeochemical processes on mineral formation when is the data is lacking.

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Research

JoVE Journal

Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

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 ...

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A multidimensional gas chromatography method for the analysis of dissolved hydrogen sulfide in liquid crude oil samples is presented. A Deans switch is used to heart-cut light sulfur gases for separation on a secondary column and detection on a sulfur chemiluminescence detector.

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...