Name: experimental study of the relationship between particle size and methane sorption capacity in shale
Uploaded: 2018-08-02T13:30:00
Description: Watch this Scientific Journal Video about Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale at JoVE.com

A lot of assistance was provided by Engineer Gang Chen and Tao Zhang. This work was financially supported by the Major State Research Development Program of China (Grant No.2016YFC0600202) and the China Geological Survey (CGS, Grant No. DD20160183). We thank anonymous reviewers for their constructive comments that greatly improved this paper.

1. Sample Preparation
<ol>
	<li>Sample characterization
	<ol>
		<li>Measure the total organic carbon (TOC) using a TOC apparatus (see Table of Materials) at a temperature of 20 &#176;C and a relative humidity of 65% (per standard GB/T 19145-2003).</li>
		<li>Perform a vitrinite reflectance measurement on polished sections of the shale using a photometer microscope (see Table of Materials).</li>
	</ol>
	</li>
	<li>Sample cleaning and crushing 
	NOT...

<ol>
	<li>Strapoc, D., Mastalerz, M., Schimmelmann, A., Drobniak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geochemical+constraints+on+the+origin+and+volume+of+gas+in+the+New+Albany+Shale+(Devonian-Mississippian),+eastern+Illinois+Basin.">Geochemical constraints on the origin and volume of gas in the New Albany Shale (Devonian-Mississippian), eastern Illinois Basin.</a> AAPG Bulletin. 94, 1713-1740 (2010).</li><li>Loucks, R. G., Ruppel, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mississippian+Barnett+Shale,+Fort+Worth+basin,+north-central+Texas:+Gas-shale+play+with+multitrillion+cubic+foot+potential.">Mississippian Barnett Shale, Fort Worth basin, north-central Texas: Gas-shale play with multitrillion cubic foot potential.</a> AAPG Bulletin. 89, 155-175 (2005).</li><li>Jia, C. Z., Zheng, M., Zhang, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unconventional+hydrocarbon+resources+in+China+and+the+prospect+of+explora+exploration+and+development.">Unconventional hydrocarbon resources in China and the prospect of explora exploration and development.</a> Petroleum Exploration and Development. 39 (2), 129-136 (2012).</li><li>Hou, Y. G., 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=Effect+of+pore+structure+on+methane+sorption+potential+of+shales.">Effect of pore structure on methane sorption potential of shales.</a> Petroleum Exploration and Development. 41 (2), 272-281 (2014).</li><li>Chalmers, G. R., Bustin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organic+matter+distribution+and+methane+capacity+of+the+lower+cretaceous+strata+of+northeastern+British+Columbia,+Canada.">The organic matter distribution and methane capacity of the lower cretaceous strata of northeastern British Columbia, Canada.</a> International Journal of Coal Geology. 70 (1-3), 223-239 (2007).</li><li>Kang, Y. L., 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=Effect+of+particle+size+on+methane+sorption+capacity+of+shales.">Effect of particle size on methane sorption capacity of shales.</a> Natural Gas Geoscience. 28 (2), 272-279 (2017).</li><li>Wang, R., 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=Adsorption+influence+factors+and+characteristics+of+adsorption+isotherm+for+shale+to+methane.">Adsorption influence factors and characteristics of adsorption isotherm for shale to methane.</a> Natural Gas Geoscience. 26 (3), 580-591 (2015).</li><li>Ruppel, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+methane+on+dry+coal+at+elevated+pressure.">Adsorption of methane on dry coal at elevated pressure.</a> Fuel. 53, 152-162 (1974).</li><li>Zhang, G. D., Han, Y. K., He, Z. P., Gao, X. Y., Chen, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sample+particle+size+on+the+adsorption+capacity+of+the+experimental+study+on+the+effect+of+shale+gas.">The sample particle size on the adsorption capacity of the experimental study on the effect of shale gas.</a> Exploration and Development. 6, 110-116 (2016).</li><li>Ross, D. J., Bustin, R. 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X., Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotherm+adsorption+of+coal+samples+with+different+grain+size.">Isotherm adsorption of coal samples with different grain size.</a> Journal of China University of Mining and Technology. 34, 427-432 (2005).</li><li>Wang, Z. X., 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=A+discussion+on+the+structural+deformation+and+oil/gas+traps+on+the+western+side+of+the+Xuefeng+Mountain.">A discussion on the structural deformation and oil/gas traps on the western side of the Xuefeng Mountain.</a> Geological Bulletin of China. 31 (11), 1812-1825 (2012).</li><li>Xu, Z. Y., Liang, X., Wang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+factors+for+shale+gas+sweet+spots+distribution+in+the+Upper+Yangtze+region:+a+case+study+of+the+Upper+Ordovician+Wufeng+Fm-Lower+Silurian+Longmaxi+Fm,+Sichuan+Basin.">Controlling factors for shale gas sweet spots distribution in the Upper Yangtze region: a case study of the Upper Ordovician Wufeng Fm-Lower Silurian Longmaxi Fm, Sichuan Basin.</a> Natural Gas Industry. 36 (9), 35-43 (2016).</li><li>Chalmers, G. L., Bustin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+cretaceous+gas+shales+in+northeastern+British+Columbia,+Part+I:+geological+controls+on+methane+sorption+capacity.">Lower cretaceous gas shales in northeastern British Columbia, Part I: geological controls on methane sorption capacity.</a> Bulletin of Canadian Petroleum Geology. 56 (1), 1-21 (2008).</li><li>Tan, J. Q., 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=Shale+gas+potential+of+the+major+marine+shale+formations+in+the+Upper+Yangtze+Platform,+South+China,+Part+II:+methane+sorption+capacity.">Shale gas potential of the major marine shale formations in the Upper Yangtze Platform, South China, Part II: methane sorption capacity.</a> Fuel. 129 (4), 204-218 (2014).</li><li>Zhao, Y. J., 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=Study+of+impact+factors+on+shale+gas+adsorption+and+desorption.">Study of impact factors on shale gas adsorption and desorption.</a> Natural Gas Geoscience. 25 (6), 940-946 (2014).</li><li>Xue, H. 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The materials used in this experiment are shown in the Table of Materials. Before the sample pool is removed, it must be confirmed that the temperature and pressure in the sample pool are at normal pressure and normal temperature; otherwise, there is a danger of injury. If the temperature is too high, wait for the temperature to drop and then removethe sample pool. If the pressure is too high or too low, manually set the air pressure on the software and use an inert gas13,</s...

Shale is a clay rock with a thin sheet of bedding structure, which serves as both a shale gas source rock and a reservoir. Shale has a strong anisotropy consisting of nano- and micron-scale pores, and graptolite fossils are commonly recognized1,2,3.
Shale gas is commercially exploited in the Yangtze Plate, Southern China. As an unconventional gas system that serves as both a source rock and a reservoir for methane, shale gas is derived from the organic matter within the shale through biogenic and/or thermogenic processes4,5. Natural gas stores in reservoirs are in one of three forms: free gas in pores and fractures, adsorbed gas on the surface of organic matter or inorganic minerals, and dissolved gas in bitumen and water6,7. Previous studies suggest that adsorbed gas accounts for 20 - 85% of the total gas in shale formations6. Therefore, research on the adsorption capacity of shale and its controlling factors are significant to the exploration and development of shale gas resource.
The methane adsorption ability of shale has been widely recognized as significantly varying with temperature, pressure, humidity, maturity, mineral composition, organic matter, and specific surface area1,4,5,6,7; and previous studies have confirmed a larger and clearer correlation between external factors like temperature, pressure, and humidity and methane adsorption.
However, studies on the correlation between intrinsic factors like particle size and methane adsorption are controversial. Kang and Ji suggest that the methane adsorption capacity of the same shale samples increases with a decrease in particle size8,14, whereas Rupple and Zhang believe the relevance between the particle size and adsorption is limited based on the isothermal adsorption curves9,10,11. In addition, without standards for a shale gas adsorption evaluation protocol, laboratories in China typically apply the coal adsorption evaluation protocols for evaluating shale gas adsorption. To clarify the relationship between particle size and adsorption,as well as investigate a prospective exploration zone, we obtained shale samples from the thick marine shale deposits of the Wuling Sag in the Upper Yangtze Plate. A gravimetric sorption analyzer was applied to conduct the isothermal adsorption experimentand obtain the relationship between the particle size and adsorption.
The volumetric and gravimetric methods are the main methods used to test the isothermal adsorption of shale. Volume is the key parameter of the volumetric method, which is easily affected by temperature and pressure12,13,14. Because of uncertainty in the error analysis, the cumulative propagation in direct measurements using the volumetric method for calculating adsorption amounts leads to a large error in the measurement results, which causes an abnormal adsorption isotherm14,15. Compared with the volumetric method, the gravimetric method requires fewer parameters and results in smaller errors: because the mass is conserved, the weight and mass of the gravimetric method are not affected by the temperature and pressure12. It is considered a more scientific and accurate method for measuring theadsorption amount of adsorption at present.
A gravimetric sorption analyzer is used in this experiment, which has a maximum testing pressure of 70 MPa (700 bar) and temperature of 150 &#176;C. The temperature and pressure generated by older apparatus are too low toaccurately simulate the temperature and pressure of the deep underground formation. The key to using a sorption analysis apparatus is reaching the magnetic suspension balance for accurately weighing the sample material, with an accuracy of 10 &#181;g. The apparatus adopts a circulating oil bath heating mode and the temperature range can be controlled for a long time to within 0.2 &#176;C. The accuracy of an old apparatus is low, and thus the error would be larger than that obtained with newer instruments. The experimental operations are performed with the software provided by the apparatus.The operating system will be updated regularly to ensure the analysis is close to the actual underground conditions12.
A magnetic suspension balance (MSB) is used in the gravimetric method to test the methane isothermal adsorption of shale without direct contact between the sample and the equipment, in normal temperature and pressure. The sample is placed in the measuring pool, in which the weight of the sample can be transmitted to the balance through a non-contact suspension coupling mechanism12,13. Under the balance, there is a suspended magnet, controlled by a specially designed controller that allows the free suspension of the permanent magnet below. The permanent magnet connects the position sensor and the sample container&#160;with the coupling frame. The function of the coupling frame is to couple or decouple the sample container to the permanent magnet suspension rod14,15,16.
Our measured samples are black organic-rich shales deposited in marine facies of the Long Maxi formation, Lower Silurian in the Daozhen, Guizhou province. The research area is in the Wuling Sag, upper Yangtze plate, which is bordered by the Sichuan Basin to the northwest and Xuefeng Mountain tectonic zone to the southwest17. The Wuling Sag is a structural transfer and transition zone between the Sichuan Basin and Xuefeng Mountain tectonic zone, which received shallow-deep sea shelf deposits, and marine black shale was widely developed during the early Silurian; the sag was then strongly superimposed by tectonic events like the Indo-China Movement, Yanshan Movement, and Himalayan Movement, which formed multistage folds, faults, and unconformities18. The marine black shale in the Wuling Sag has been significantly influenced by the complex geological conditions, which formed shale gas reserves. As a structural transfer zone, the sag is the sweet spot for shale gas exploration, which is characterized by a weaker deformation, better shale gas generation and preservation conditions, and a better natural fracture matching of the traps19.
High-pressure sorption measurements are conducted based on a standardized procedure with the guidance of the isothermal adsorption apparatus protocol, which has been comprehensively elaborated on in several publications10,11,12,13,14,15,16. The isothermal adsorption experiments were completed in the Key Laboratory of Shale Oil and Gas Investigation and Evaluation of the Chinese Academy of Geosciences. A gravimetric measurement carried out with a magnetic suspension balance (MSB) is performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and an adsorption and desorption measurement (Figure 1, Figure 2).

<table border="0" cellpadding="0" cellspacing="0" style="width:1587px;" width="1586">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">XRF D8 DISCOVER X-Ray diffractometer</td>
			<td style="width:427px;">Brook,Germany</td>
			<td style="width:140px;">204458</td>
			<td style="width:608px;">For mineralogy X-ray diffraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EBSD three element integration system with spectrum&#160;</td>
			<td>EDAX,USA</td>
			<td>Trident XM4</td>
			<td>For nanoscale imaging (SEM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mercury injection capillary pressure (MICP)</td>
			<td>USA micromeritics Instrument company</td>
			<td>AutoPore IV 9520</td>
			<td>For the immersion method to measure macropores(Porosity)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen gas adsorption at low temperature</td>
			<td>USA micromeritics Instrument company</td>
			<td>ASAP2460/2020</td>
			<td>For the low pressure nitrogen gas adsorption to measure mesopores and micropores(BET)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Finnigan MAT-252 mass spectrometer</td>
			<td>ThermoFinnigan,USA</td>
			<td>TRQ/Y2008-004</td>
			<td>For C isotope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LECO CS-230 analyzer&#160;</td>
			<td>Research Institute of Petroleum Exploration and Development</td>
			<td>617-100-800</td>
			<td>TOC apparatus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3Y-Leica MPV-SP photometer microphotometric system&#160;</td>
			<td>Leica,Germany</td>
			<td>M090063016</td>
			<td>Ro apparatus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Suspension Balance Isothermal adsorption analyzer</td>
			<td>Rubotherm,Germany</td>
			<td>2015-1974CHN</td>
			<td>For methane adsorption tests</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sieve(20/40/60/80/100/120mesh)</td>
			<td>Sinopharm Chemical Reagent Beijing Co.Ltd</td>
			<td>200*50GB6003.102012</td>
			<td>Used to screen samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent cotton, hammer, tweezers and acetaldehyde</td>
			<td>Sinopharm Chemical Reagent Beijing Co.Ltd</td>
			<td>standard</td>
			<td>Used to clean materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Residual gas tight grinder</td>
			<td>Nantong Huaxing Petroleum Instrument Co., Ltd</td>
			<td>TY2013000237</td>
			<td>Sample smasher</td>
		</tr>
	</tbody>
</table>

experimental study of the relationship between particle size and methane sorption capacity in shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57705/57705fig1.jpg" /> 
Figure 1: Experimental set-up for gravimetric gas adsorption at high temperatures and pressures. This figure shows the set-up for the isothermal adsorption experiment: (a) the oil bath heating device for the fluid bath; (b) the electrical heating device for the electrical heating; (c) the magnetic suspens...

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Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

This method can help answer key questions in the shale gas field about the relationship between particle size and the adsorption capacity of shale. The main advantage of this technique is that the gravimetric method requires fewer parameters and produces better results in terms of accuracy and simulating actual underground structure. First, turn on the magnetic suspension balance computer, and start the magnetic suspension coupling controller.Open the gas two helium cylinder, and roughly adjust it to the appropriate output pressure with the control valve. Next, confirm that the magnetic suspension coupling controller knob is in the Off position. Then, turn on the instrument temperature controller, the oil bath heater, and the vacuum pump.Switch the coupling controller to the Zero Point position, wait one to two minutes, and confirm that the readings are normal and stable. Check the Measuring Point positions in the same way. Then, set the controller to the Zero Point position.Start the System Control Software. Select the Blank Measurement program, and open the configuration window. Name the measurement, select gas two to use helium gas, select other fluid, and select fluid bath.Set the maximum pressure to 70 bars with seven pressure steps, the pressure ramp to two bars per minute, and the oil bath temperature to 50 degrees Celsius. Run the program, and calculate the mass and volume of the empty sample container. After the blank test has finished, set the gas two helium pressure setpoint to one bar.Start gas dosing to fill the sample pool with helium until it is close to atmospheric pressure. Then, stop gas dosing. Next, pull down the temperature sensor.Disassemble the insulating cotton jacket and the double-walled thermostat. Support the double-walled thermostat when the last screw is removed. Unload the double-walled thermostat, being careful not to knock the sensor traverse tube with the wrench.Then, disassemble the sample pool by removing the six screws symmetrically. Carefully open the sample pool. Next, check whether the instrument balance frame is level.To adjust the balance frame, turn the magnetic coupling controller knob to Off, and carefully raise or lower the feet and support screws. Once the system is balanced, take out the sample container, and load a clean, crushed shale rock sample into the sample container. Install the sample container, and then set the coupling to the appropriate position.Then, reinstall the sample pool, being careful to keep the edges within the flange. Install the screws symmetrically, and turn the screws with gentle, uniform force. After installing the sample pool, try installing the double-walled thermostat to ensure that it can be installed smoothly in the later buoyancy measurement.Once this has been confirmed, remove the double-walled thermostat. Next, turn the coupling controller knob to each position to check the balance. Insert the temperature sensor, and install the electrical heating sheath and insulation cover.Connect the electrical heating power supply, and turn on the power. Then, select the Pretreatment program in the instrument software, and open the configuration menu. Name the measurement, choose the vacuum source, and select electrical heating.Set the sample temperature to 105 degrees Celsius, the coupling temperature to 20 degrees Celsius, and the duration to 600 minutes. Then, run the Pretreatment program. Afterwards, turn off the electrical heating, and disconnect the power supply.Remove the electric heating sheath, insert the temperature sensor, and install the double-walled thermostat. Wrap the thermostat with an adhesive insulation cover. Next, in the software, select the Buoyancy Measurement program, and open the configuration window.Set the oil bath temperature to 50 degrees Celsius. Set the maximum pressure to 70 bar, the pressure ramp to two bars per minute, and the number of pressure steps to seven. Then, run the process, which usually takes five to eight hours.After that, select the Sorption Measurement program. If desorption will be evaluated, maintain the oil bath at 50 degrees Celsius, and set 19 pressure points, stepping up from zero bars to 250 bars and back to zero bars. Then, choose gas one instead of gas two for the experimental gas, and run the program.Use a pressurized pump if needed to manually increase the experimental gas pressure to the target value. Let the process finish before removing any components from the instrument. Two shale specimens were crushed and divided by particle size.For all samples, the absolute methane adsorption capacity linearly increased with pressure between zero and 60 bars. After that, the absolute adsorption capacity gradually plateaued. The excess adsorption capacity followed a similar trend at lower pressures, reached a maximum at about 80 to 100 bars, and then decreased with increasing pressure.Shale samples with particle sizes of about 250 micrometers generally had the highest maximum absolute and excess adsorption capacity. This was attributed to the rock being crushed into smaller particles and to micropores and mesopores connecting to form mesopores and macropores, decreasing the total specific surface area of the shale. We first had this idea for this method when we considered how large particles should be used for isothermal adsorption experiments of shale in different regions.Once mastered, this technique can be done in 24 hours if it is performed properly.

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Environment

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Protocol for Measuring the Thermal Properties of a Supercooled Synthetic Sand-water-gas-methane Hydrate Sample

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future energy resources and energy storage media. To develop a method for gas production from natural MH-bearing sediments and hydrate-based technologies, it is imperative to understand the thermal properties of gas hydrates.
The thermal properties' measurements of samples comprising sand, water, methane, and MH are difficult because the melting heat of MH may affect the measurements. To solve this problem, we performed thermal properties' measurements at supercooled conditions during MH formation. The measurement protocol, calculation method of the saturation change, and tips for thermal constants' analysis of the sample using transient plane source techniques are described here.
The effect of the formation heat of MH on measurement is very small because the gas hydrate formation rate is very slow. This measurement method can be applied to the thermal properties of the gas hydrate-water-guest gas system, which contains hydrogen, CO2, and ozone hydrates, because the characteristic low formation rate of gas hydrate is not unique to MH. The key point of this method is the low rate of phase transition of the target material. Hence, this method may be applied to other materials having low phase-transition rates.

We present a protocol for measuring the thermal properties of synthetic hydrate-bearing sediment samples comprising sand, water, methane, and methane hydrate.

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future ...

Image-based Lagrangian Particle Tracking in Bed-load Experiments

Image analysis has been increasingly used for the measurement of river flows due to its capabilities to furnish detailed quantitative depictions at a relatively low cost. This manuscript describes an application of particle tracking velocimetry (PTV) to a bed-load experiment with lightweight sediment. The key characteristics of the investigated sediment transport conditions were the presence of a covered flow and of a fixed rough bed above which particles were released in limited number at the flume inlet. Under the applied flow conditions, the motion of the individual bed-load particles was intermittent, with alternating movement and stillness terms. The flow pattern was preliminarily characterized by acoustic measurements of vertical profiles of the stream-wise velocity. During process visualization, a large field of view was obtained using two action-cameras placed at different locations along the flume. The experimental protocol is described in terms of channel calibration, experiment realization, image pre-processing, automatic particle tracking, and post-processing of particle track data from the two cameras. The presented proof-of-concept results include probability distributions of the particle hop length and duration. The achievements of this work are compared to those of existing literature to demonstrate the validity of the protocol.

The manuscript presents a protocol for the conduction of bed-load sediment transport experiments where the moving particles are tracked by image analysis. The experimental facility, the procedures for run realization and data processing, and finally some proof-of-concept results are presented here.

Image analysis has been increasingly used for the measurement of river flows due to its capabilities to furnish detailed quantitative depictions at a ...

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.

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.

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

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

Investigating the Relationship between Sea Surface Chlorophyll and Major Features of the South China Sea with Satellite Information

Satellite observations offer a great approach to investigate the features of major marine parameters, including sea surface chlorophyll (CHL), sea surface temperature (SST), sea surface height (SSH), and factors derived from these parameters (e.g., fronts). This study shows a step-by-step procedure to use satellite observations to describe major parameters and their relationships in seasonal and anomalous fields. This method is illustrated using satellite datasets from 2002&#8211;2017 that were used to describe the surface features of the South China Sea (SCS). Due to cloud coverage, monthly averaged data were used in this study. The empirical orthogonal function (EOF) was applied to describe the spatial distribution and temporal variabilities of different factors. The monsoon wind dominates the variability in the basin. Thus, wind from the reanalysis dataset was used to investigate its driving force on different parameters. The seasonal variability in CHL was prominent and significantly correlated with other factors in a majority of the SCS. In winter, a strong northeast monsoon induces a deep mixed layer and high level of chlorophyll throughout the basin. Significant correlation coefficients were found among factors at the seasonal cycle. In summer, high CHL levels were mostly found in the western SCS. Instead of a seasonal dependence, the region was highly dynamic, and factors correlated significantly in anomalous fields such that unusually high CHL levels were associated with abnormally strong winds and intense frontal activities. The study presents a step-by-step procedure to use satellite observations to describe major parameters and their relationships in seasonal and anomalous fields. The method can be applied to other global oceans and will be helpful for understanding marine dynamics.

Sea surface chlorophyll, temperature, sea level height, wind, and front data obtained or derived from satellite observations offer an effective way to characterize the ocean. Presented is a method for the comprehensive study of these data, including overall average, seasonal cycle, and intercorrelation analyses, to fully understand regional dynamics and&#160;ecosystems.

Satellite observations offer a great approach to investigate the features of major marine parameters, including sea surface chlorophyll (CHL), sea surface ...