The author wishes to acknowledge Alan Kruger, Todd Boman, Shannon Ostdiek, Elaine Berry, and Ferouz Ayadi who assisted with data collection using the simulated bedded packs. The author also recognizes Tami Brown-Brandl and Dale Janssen for their assistance maintaining the environmental chambers.

The study is designed to be conducted over 42 days with weekly data collection. All animal procedures were reviewed and approved by the US Meat Animal Research Center Institutional Animal Care and Use Committee.
1. Constructing Simulated Bedded Packs
<ol>
	<li>Begin with plastic cylinder containers that are 0.42 m high with a 0.38 m diameter. 
	NOTE: In this study, one particular 10-gallon commercial trash container was used (see Table of Materials), but other similar-sized plastic containers would be suitable.</li>
	<li>Drill six 1-cm holes equally spaced around the circumference of the plastic container into each plastic container approximately 5 cm the top of the plastic container. Remove any plastic remnants from the container.</li>
	<li>Tare the plastic container and record the mass on the side of the plastic container. Weigh 320 g of selected bedding material into weigh pan using a balance and add bedding material to the plastic container. 
	NOTE: Any bedding material deemed suitable for use in livestock facilities can be used9,10,11,12,13,14,15. For modeling deep bedded cattle facilities in the Upper Great Plains, corn stover is considered the most common bedding material1 but soybean stover, wheat straw, and wood chips have also been used1. If using this system to model deep-bedded swine or dairy facilities, wheat straw, barley straw, oat straw, hay, wood shavings, wood chips, sawdust, newspaper, corn cobs, soybean stubble, rice hulls, or sand may be more suitable16,17,18.</li>
	<li>Weigh 320 g of fresh cattle feces on a plastic plate using balance and add to the plastic container. 
	NOTE: Urine and feces are collected and maintained as previously described11.</li>
	<li>Measure 320 mL of fresh cattle urine in 1000-mL graduated cylinder. Empty contents into the plastic container. Using a stirring rod (5.08-cm circumference), mix the bedding material mixture slightly for 30 s. 
	NOTE: In this case, a hollow steel rod with a plastic cover on the end was used. Alternatively, any type of rod could be used.</li>
	<li>Clean the end of the stirring rod between each bedded pack using an antiseptic disposal wipe to prevent cross-contamination of microbes. 
	NOTE: A bucket of warm soapy water can also be used to clean the stirring rod. A plastic sandwich bag can also be secured with a rubber band to the end of the rod and replaced after each bedded pack to prevent cross contamination.</li>
	<li>Weigh and record the final mass of the bedding mixture. Place the plastic container in the environmental chamber19 set to an ambient temperature of 18 - 20 &#176;C with a dew point of 12&#160;&#176;C.</li>
</ol>
2. Maintaining the Simulated Bedded Packs
<ol>
	<li>Forty-eight hours before adding feces and urine, remove frozen feces and urine from freezer and allow to thaw at room temperature (20 - 25&#160;&#730;C).</li>
	<li>Less than an hour before adding urine to bedded pack, measure the pH of the urine.</li>
	<li>Put on appropriate personal protective equipment (gloves, safety glasses) necessary for handling 6 M NaOH.</li>
	<li>Pour 25 mL of 6 M sodium hydroxide (NaOH) into the graduated cylinder. Stir the mixture, then test the pH using a pH probe. Repeat until the urine reaches pH 7.4, physiological pH20.</li>
	<li>Once the pH of the urine is adjusted, replace cap on the urine container when not in use to prevent volatilization of nitrogen from urine.</li>
	<li>Weigh and record the mass of the bedded pack. If fresh bedding is to be added on this day, weigh 320 g of selected bedding material into aluminum pan using balance and add bedding material to the respective bedded packs. If no bedding is to be added on this day, continue to Step 2.7.</li>
	<li>Weigh 320 g of thawed cattle feces on a plastic plate using balance and add to the bedded pack. 
	NOTE: On Day 21, use fresh feces instead of thawed feces.</li>
	<li>Measure 320 mL of thawed cattle urine in 1000-mL graduated cylinder. Empty contents onto the bedded pack. 
	NOTE: On Day 21, use fresh urine instead of thawed urine.</li>
	<li>Using a stirring rod, stir the bedding pack mixture slightly for 30 seconds. Clean the plastic end of the stirring rod between each bedded pack to prevent cross-contamination of microbes. Weigh and record the final mass of the bedding mixture.</li>
	<li>Return the plastic container in the environmental chamber.</li>
	<li>Repeat steps 2.1 - 2.10 on Monday, Wednesday, and Friday of each week, with bedding material being added (Step 2.6) and air samples collected each Wednesday.</li>
</ol>
3. Collecting Samples from the Simulated Bedded Packs
NOTE: Samples are collected from the simulated bedded packs once weekly, prior to adding feces, urine, and fresh bedding.
<ol>
	<li>Preparing to collect air samples from headspace of each simulated bedded pack.
	<ol>
		<li>Turn on all air sampling equipment and allow to warm up according to manufacturer&#39;s directions, approximately 1 hour. 
		NOTE: See Table of Materials for ammonia (NH3), hydrogen sulfide (H2S), methane (CH4), nitrous oxide (N2O) and carbon dioxide (CO2) gas analyzers used in this study.</li>
		<li>Measure the distance from the top of the simulated bedded pack to the top of the plastic container holding the simulated bedded pack using a ruler.</li>
		<li>Calculate the volume of the headspace area using the following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/57332/57332eq1.jpg" /> 
		where r = radius of the plastic container, 
		h = distance from the top of the bedded pack to the top of the plastic container, and 
		Vflux chamber = volume of the flux chamber located on top of the plastic container. 
		NOTE: The flux chambers used in this study had an internal volume of 0.007m3 with a surface area of 0.064 m21,22.</li>
		<li>Push a metal stake approximately 5 cm into surface of the bedded pack at the approximate center of the pack. Thread 0.64-cm inert tubing through one of the 1-cm holes at the top of each simulated bedded pack container and secure on a 12.5-cm metal stake 1.3 cm above the surface of the bedding pack. Place stainless steel hemispherical static flux chambers21,22 with rubber skirts on top of each simulated bedded pack (Figure 1). 
		NOTE: Rubber skirts are 61-cm squares made of soft, elastic rubber with 22.9-cm diameter holes cut in the center. The hole fits over the flux chamber and the skirts form a seal on the top of the plastic container when placed on the container.</li>
		<li>Attach 0.64-cm inert tubing to the flux chambers using inert compression fittings. 
		NOTE: The inert tubing is attached to the gas sampling manifold which feeds into the air sampling equipment. The gas sampling system is controlled by a 24-volt Programmable Logic Relay (see Table of Materials) which signals multi-positional 3-way solenoids to open and close one of eight air inlet lines on the gas sampling manifold. One line is opened at a time to allow for individual air sampling from each bedded pack.</li>
		<li>Begin flushing ambient air from the room through the tubing at a rate of 5 L min-1 for 30 minutes. 
		NOTE: See Table of Materials for pump used to flush the air through the sample lines.</li>
	</ol>
	</li>
	<li>Measure concentration of ammonia, carbon dioxide, methane, and hydrogen sulfide in headspace of simulated bedded packs.
	<ol>
		<li>After adequately flushing the simulated bedded packs, open stopcock on sample line to draw ambient air from the room into inert sample lines connected to gas sampling manifold.</li>
		<li>Activate the programmable logic relay to begin pulling air into the air sampling equipment. Record measurements from ambient air for 20 minutes to determine concentration of measured gases in ambient air. This will be used as a background air concentration. When finished collecting ambient air concentration, close the stopcock on the sample line.</li>
		<li>Activate the programmable logic relay to begin sampling air from the inert sample lines attached to each flux chamber. Record measurements from each sample line for 20 minutes to determine concentrations of measured gases in the headspace of each bedded pack.</li>
		<li>Results can be reported as the average concentration of the gas (NH3, CO2, N2O, CH4, H2S) in the air samples (mg kg-1 or ppm), or the flux density (emission rate) of the gas can be calculated on a mass per unit area per unit time basis using the following equation: 
		<img alt="Equation 2" src="/files/ftp_upload/57332/57332eq2.jpg" /> 
		where J = the flux in &#181;g m-2 min-1, 
		A = the area of the source (m2) inside the chamber, 
		Q = the sweep air flow rate m3 min-1, and 
		Cair = the VOC concentration leaving the chamber (&#181;g m-3)23.</li>
	</ol>
	</li>
	<li>Measure concentration of odorous volatile organic compounds in the headspace of simulated bedded packs.
	<ol>
		<li>Put on latex or nitrile disposable gloves.</li>
		<li>After adequately flushing the simulated bedded packs, remove brass storage caps from preconditioned stainless steel sorbent tubes. 
		NOTE: The sorbent tubes used in this study were 89 mm &#215; 6.4 mm OD filled with Tenax TA sorbent (see Table of Materials). Brass caps have polythtrafluorethylene (PTFE) ferrules.</li>
		<li>Attach the scored end of the sorbent tube to the inlet port on the flux chamber using flexible rubber tubing, and the other end of the sorbent tube to a vacuum pump. 
		NOTE: The vacuum pump used in this study (see Table of Materials) pulled air through the sorbent tubes at a flow rate of 75 mL min-1.</li>
		<li>Allow the pump to pull air into the sorbent tube for 5 min for a sample volume of 0.375 L, then turn off pump and disconnect sorbent tube. Replace the brass storage caps on the ends of the sorbent tubes.</li>
		<li>Repeat Steps 3.3.1 - 3.3.4 to collect one sorbent tube for each bedded pack.</li>
		<li>Store sorbent tubes until analysis by thermal desorption-gas chromatograph-mass-spectrometry (TD-GC-MS). Tubes may be stored at room temperature (20 - 25 &#730;C) for &#60;24 h. If storing &#62;24 h, store in refrigerator.</li>
		<li>Immediately before sample analysis on the TD-GC-MS system, remove brass storage caps from sorbent tubes and replace with PTFE analytical caps23.</li>
		<li>Analyze sorbent tubes for volatile organic compounds24 (acetic acid, butyric acid, propionic acid, isobutyric acid, isovaleric acid, valeric acid, hexanoic acid, heptanoic acid, phenol, p-cresol, indole, skatole, dimethyl disulfide, and dimethyl trisulfide) using TD-GC-MS23,24,25.</li>
		<li>Results can be reported as concentration of the VOC in the air samples (&#181;g m-3), or the flux density (emission rate) of the VOC can be calculated on a mass per unit area per unit time basis using the following equation: 
		<img alt="Equation 2" src="/files/ftp_upload/57332/57332eq2.jpg" /> 
		where J = the flux in &#181;g m-2 min-1, 
		A = the area of the source (m2) inside the chamber, 
		Q = the sweep air flow rate m3 min-1, and 
		Cair= the VOC concentration leaving the chamber (&#181;g m-3)23.</li>
	</ol>
	</li>
	<li>Collect physical and chemical measurements of the simulated bedded packs. 
	NOTE: Temperature, pH, and evaporative water loss are measured each time additional materials were added to the simulated bedded packs. Nutrient composition is determined at Day 0 and Day 42. Free air space is determined at Day 42 only.
	<ol>
		<li>Determine the temperature of the bedded pack by inserting a temperature probe into the center of the bedded pack, approximately 7.6 cm below the surface of the simulated bedded pack. Allow the temperature to stabilize and record.</li>
		<li>Determine estimated evaporative water loss
		<ol>
			<li>Place the plastic container on the balance.</li>
			<li>Measure and record the mass of the simulated bedded pack before and after each addition of feces/urine/bedding to the simulated bedded pack.</li>
			<li>Calculate the estimated evaporative water loss by subtracting the current day&#39;s beginning mass from the previous day&#39;s ending mass. The difference is the estimated mass of water that evaporated from the bedded pack between the days and can be used to compare relative differences between bedded pack, although it does not reflect absolute loss.</li>
		</ol>
		</li>
		<li>Determine pH of simulated bedded pack
		<ol>
			<li>Collect a representative 5 - 10 g sample from each simulated bedded pack from the center of the pack at a depth of approximately 7.6 cm below the surface of the bedded pack. Put the sample in a plastic 50-mL conical tube, cap, and label.</li>
			<li>Calibrate the pH meter with buffers pH 4 and 7 according to manufacturer&#39;s directions.</li>
			<li>Determine the mass of each conical.</li>
			<li>Dilute each sample 1:2 on a mass basis with distilled, deionized water. Shake the conical to mix the water and bedding material. Insert the pH probe into the conical, measure, and record the pH of the sample.</li>
		</ol>
		</li>
		<li>On Days 0 and 42 only, determine nutrient content of the simulated bedded pack.
		<ol>
			<li>Collect a 50 g representative sample from each simulated bedded pack from the center of the pack at a depth of approximately 7.6 cm below the surface of the bedded pack. Place in a paper soil sample bag.</li>
			<li>Transport to a laboratory for nutrient analysis within 24 hours. Store in refrigerator until samples can be transported to a laboratory for nutrient analysis. 
			NOTE: Any macro or micro nutrient can be analyzed. We analyze for total nitrogen26, phosphorus and sulfur analysis27 at a commercial laboratory.</li>
		</ol>
		</li>
		<li>On Day 42 only, determine free air space in simulated bedded pack.
		<ol>
			<li>Place the plastic container on a balance and record the mass. Slowly fill with water until the surface of the water is even with the surface of the simulated bedded pack. Allow water to settle until no more bubbles are coming from the simulated bedded pack, then record the mass of the plastic container</li>
			<li>Determine the percentage of free air space using the following calculation: 
			<img alt="Equation 3" src="/files/ftp_upload/57332/57332eq3.jpg" /></li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After completing all desired data collection steps (Steps 3.1 - 3.4), add feces, urine, and bedding to the simulated bedded packs following Steps 2.1 - 2.10.</li>
</ol>

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B., Gilley, J., Woodbury, B., Kim, K., Galvin, G., Bartelt-Hunt, S. L., Li, X., Snow, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorous+VOC+emission+following+land+application+of+swine+manure+slurry.">Odorous VOC emission following land application of swine manure slurry.</a> Atmos. Environ. 66, 91-100 (2013).</li><li>Parker, D. B., Koziel, J. A., Cai, L., Jacobson, L. D., Akdeniz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+and+odorous+chemical+emissions+from+animal+buildings:+Part+6.+Odor+activity+value.">Odor and odorous chemical emissions from animal buildings: Part 6. Odor activity value.</a> Trans. ASABE. 55, 2357-2368 (2012).</li><li>Watson, M., Wolf, A., Wolf, N., Peters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+nitrogen.">Total nitrogen.</a> Recommended methods of manure analysis. , 18-24 (2003).</li><li>Wolf, A., Watson, M., Wolf, N., Peters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digestion+and+dissolution+methods+for+P,+K,+Ca,+Mg,+and+trace+elements.">Digestion and dissolution methods for P, K, Ca, Mg, and trace elements.</a> Recommended methods of manure analysis. , 30-38 (2003).</li><li>Euken, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+manure+characteristics+from+bedded+confinement+buildings+for+feedlot+beef+productions:+Progress+report.">A survey of manure characteristics from bedded confinement buildings for feedlot beef productions: Progress report.</a> Animal Industry Report. , (2009).</li><li>Li, L., Li, Q. -. F., Wang, K., Bogan, B. W., Ni, J. -. Q., Cortus, E. L., Heber, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+National+Air+Emission+Monitoring+Study's+southeast+layer+site:+Part+I.+Site+characteristics+and+monitoring+methodology.">The National Air Emission Monitoring Study's southeast layer site: Part I. Site characteristics and monitoring methodology.</a> Trans. ASABE. 56, 1157-1171 (2013).</li></ol>

This research was funded by federally appropriated funds to the USDA Agricultural Research Service, Research Project Number 3040-41630-001-00D.
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.
USDA is an equal opportunity provider and employer.

The frequent addition of urine and feces to the bedded packs is a critical step. We experimented with adding urine and feces just once weekly, but found that the bedded pack developed a crust, which trapped gases inside the pack and was not representative of commercial facilities. The use of fresh feces at the beginning of the study ensures that the bedded packs is inoculated with common bacterial populations found in cattle facilities. It is also important, when adding the urine, to remember to adjust the pH to physiological pH prior to adding to the bedded packs. On one occasion, an error was made and low pH urine was added to the bedded packs. This killed the methanogenic bacteria population. When setting up the gas sampling system, all fittings need to be secure to prevent leakage that may compromise the quality of the gas measurements.
The protocol has been adapted since it was first developed. Adapting the static flux chamber to be dynamic flux chambers allows researchers to calculate emissions instead of just concentrations in headspace gases. The use of new dynamic gas sampling systems also allows the sampling to be completed by one person instead of needing two to three people to manage all the data collection.
Adaptations could be made to use the simulated bedded packs to evaluate bedding materials or odor amendments used in swine or dairy facilities. Adjustments would need to be made to determine appropriate bedding:manure ratios typical in swine or dairy facilities. Published literature should provide dry matter and nutrient composition expectations from commercial swine or dairy facilities that would help estimate the amount of bedding, feces, and urine that would need to be needed to adjust the simulated bedded pack protocol to represent a swine or dairy facility. The protocol has never been used to measure an inorganic bedding material, such as sand, which is often used in dairy facilities. While there is no reason to believe it would not successfully measure gas emissions from a bedded pack containing inorganic bedding material, this would require additional testing.
There may be additional gases that could be sampled that we have not evaluated. Any gas sampling instrument that can be attached to an inert gas sampling line should, theoretically, be able to be used with this system.
The model could also be adjusted to explore different bedding:manure ratios if a researcher chose to do so. Perhaps a researcher was interested in determining the maximum amount of manure or urine that could be added to a bedded pack before significant odors were detected. Or a researcher wanted to examine different temperature and humidity effects on air quality. The model could also be adjusted to examine these factors.
The protocol was developed to measure air quality and nutrient composition from lab-scaled bedded packs in a controlled environment and has been used to effectively evaluate many different bedding materials, environmental variables (temperature, humidity), and potential mitigation treatments that can improve air quality in commercial deep-bedded mono-slope facilities. The model is dynamic and allows researchers to easily collect many chemical and physical measurements from the bedded pack, including NH3, CH4, N2O, CO2, H2S, VOC, temperature, pH, nutrient composition, free air space, and potentially others that have not yet been measured. Weekly measurements collected over the course of six to seven weeks allows sufficient time to see changes in air quality measurements over time as the bedded pack mature. The data collected from the simulated bedded packs is within the range of concentrations previously measured in commercial deep-bedded mono-slope facilities. Past studies have demonstrated that that 8 - 10 experimental units per treatment are sufficient to detect statistical differences among the simulated bedded packs9,10,11,12,13,14,15. The bedded packs are easy to maintain, requiring less than 10 minutes of labor per bedded pack per week to add urine, feces, and bedding. Sample collection using the gas sampling system requires 20 - 30 minutes per bedded pack, depending on the measurements that are being collected. In the past, as many as 20 bedded packs have been analyzed by one person in a normal 8-hour workday. The use of lab-scaled bedded packs allows the researcher to control variables such as temperature, humidity, and bedding source that are difficult or impossible to control in a research or commercial facility. Several lab-scale studies can be conducted to eliminate possible treatments before trying them in a research or commercial-sized facility.
The primary limitation of the model is that it is not a perfect simulation of &#34;real-world&#34; conditions. It is difficult to perfectly simulate commercial conditions such as continual additional of urine and feces which occurs in a livestock facility. Based on the dry matter content and nutrient composition of the simulated bedded packs compared to commercial facilities, and the labor available in our laboratory, we have determined thrice weekly additions of urine and feces to be sufficient. However, if a modification could be developed to periodically add fresh urine and feces multiple times daily, that would better simulate the commercial environment.
Another recognized limitation is the use of frozen and thawed feces and urine. While every effort is made to quickly freeze the urine and feces to prevent volatilization of the nitrogen and any bacterial growth, urine and feces collected from a balance study are only collected once daily. It takes an hour or longer to collect, weigh, reset the collection containers, and partition the urine and feces. It also requires several hours for a 20-L carboy of urine to completely freeze, even after being placed in a -4 &#730;C freezer. During this time, volatilization and bacterial growth may occur. To compensate for this time delay between collection and freezing, the urine is acidified to pH 4 immediately after removing the container from the collection apparatus to prevent bacterial growth and nitrogen volatilization. The urine is restored to pH 7 once it is thawed, but this may not be exactly the same as adding fresh urine. However, as there have been no observed increases in NH3 volatilization following the addition of fresh urine to the bedded packs compared to frozen urine, we believe we have minimized this limitation. Bacterial populations are killed or decreased when feces is frozen. This is a recognized limitation of the protocol that we have attempted to minimize by adding fresh feces on Day 0 and Day 21.
The use of a steel rod to gentle mix the freshly added feces and urine with the bedding material may not perfectly simulate the weight of the cattle in a commercial facility, thus resulting in somewhat different compaction and water holding capacity. To account for the porosity of the bedded backs, and as an indication of the free air space that may be present in the bedded pack, water was poured into the bedded packs at the end of each study to determine a percentage of free air space present in each bedded pack9,10,11,12,13,14,15. Free air space stayed generally uniform from one study to another, but has not been compared to the free air space that is present in a commercial facility.
The protocol has not been tested with other livestock species or facility types, such as swine deep-bedded hoops or Swedish deep-bedded farrowing facilities, dairy compost barns or other dairy bedded facilities, or any type of poultry facility using bedding. While it seems that the model would have potential to be used as a model for other livestock facilities, adjustments to the protocol may be necessary to adequately represent any facility beyond a beef cattle deep-bedded facility.
While the model is not a perfect simulation of commercial facilities, it can offer a starting point when evaluating factors such as bedding, temperature, humidity, or amendments that can be added to a bedded pack in a livestock facility. It allows a researcher to evaluate treatment differences in a controlled environment and eliminate potentially less effective treatment options before spending money on the resources necessary for a full-scale commercial-size operation.

Beef cattle confinement facilities are a popular housing option in the Midwest and Upper Great Plains. Confinement facilities are more common in this region than the Southern Plains because the region receives more annual precipitation, which creates more feedlot runoff that must be contained. Many producers chose to build mono-slope barns for beef cattle. The primary reasons cited by producers for selecting a mono-slope facility was the ability to schedule labor and manure removal, and improved performance compared to open lot feedlots1. A majority of cattle producers (72.2%) using mono-slope barns maintain a bedded pack for one turn of cattle or longer, using a deep-bedding management system for bedding and waste1. The most common bedding material used is corn stover, although producers report using soybean stubble, wheat straw, corn cobs, and sawdust1. Because of the regional demand for corn stover bedding, many producers were interested in alternative bedding materials that could be used in mono-slope facilities. In addition to economics and animal comfort, producers questioned how the bedding material would impact the environment of the facility, including the production of odorous gases, nutrient composition of the resulting manure/bedding, and presence of pathogens.
Few studies have been conducted to measure air quality resulting from different bedding materials used in livestock housing, with most focusing only on ammonia. Most of the previous evaluations of air quality include on-farm data collection with one or two experimental units per treatments being analyzed at once2,3,4,5. Having limited numbers of experimental units requires the study to be repeated multiple times, thus adding additional variables such as weather conditions, age or stage of production of animals, and perhaps bedding materials produced in different growing seasons.
With no known lab-scaled model to study factors affecting air quality and nutrient composition of the manure/bedding mixture resulting from beef deep-bedded mono-slope facilities, researchers first attempted to utilize commercial cattle facilities using a deep-bedded system6,7,8. Static flux chambers were used to measure NH3 concentrations on the surface of mono-slope deep bedded cattle facilities over an 18 month period6. Two pens in each of two barns were measured. Chopped corn stalks were the preferred bedding material, but wheat straw and soybean stalks were also used for bedding during brief periods of this project. Bedding use ranged from 1.95 - 3.37 kg per animal per day and pen density ranged from 3.22 - 6.13 m2 per animal. Subsequent studies measured ammonia and hydrogen sulfide emissions from the barn7, and particulate matter concentrations outside the barn8. These studies were conducted over a 2 year period using two to four barn locations. The challenge with on-farm data collection is the lack of control that the research has over the system. Producers change cattle diets, move animals from pen to pen, use bedding materials from different sources, and clean and re-bed pens as their production and labor force allows, thus confounding many variables. On-farm research also involves travel expenses and large quantities of experimental treatments (such as bedding material). The objective of this project was to develop a lab-scale model that could be used to study factors affecting air quality and nutrient management in cattle deep-bedded mono-slope facilities.

<table><tbody><tr><td>10 gallon plastic cylinder containers</td><td>Rubbermaid</td><td>Model 2610</td><td>Other similar-sized plastic containers are suitable</td></tr><tr><td>Mass balance</td><td>Any</td><td></td><td>Capable of measuring 0.1 gram</td></tr><tr><td>Electric drill with 1 cm bit</td><td>Any</td><td></td><td></td></tr><tr><td>Methane analyzer</td><td>Thermo Fisher Scientific</td><td>Model 55i Methane/Non-methane Analyzer</td><td></td></tr><tr><td>Hydrogen sulfide analyzer</td><td>Thermo Fisher Scientific</td><td>Model 450i</td><td></td></tr><tr><td>Ammonia analyzer</td><td>Thermo Fisher Scientific</td><td>Model 17i</td><td></td></tr><tr><td>Carbon dioxide analyzer</td><td>California Analytical</td><td>Model 1412</td><td></td></tr><tr><td>Nitrous oxide analyzer</td><td>California Analytical</td><td>Model 1412</td><td></td></tr><tr><td>Programmable Logic Relay</td><td>TECO</td><td>Model SG2-020VR-D</td><td></td></tr><tr><td>Stainless steel flux chambers</td><td>Any</td><td></td><td>Constructed using the parts list and directions cited at Woodbury et al., 2006</td></tr><tr><td>Rubber skits</td><td>Any</td><td></td><td>Constructed from flexible rubber material. Cut into squares (61 cm x 61 cm) with 22.9 cm diameter hole in center.&amp;nbsp;</td></tr><tr><td>pH meter</td><td>Spectrum Technologies</td><td>IQ150</td><td></td></tr><tr><td>thermometer</td><td>Spectrum Technologies</td><td>IQ150</td><td></td></tr><tr><td>Ruler or tape measure</td><td>Any</td><td></td><td>Capable of measuring in cm</td></tr><tr><td>Sorbent tubes</td><td>Markes International</td><td>Tenax TA</td><td></td></tr><tr><td>Pocket pumps</td><td>SKC Inc.</td><td>Series 210</td><td></td></tr><tr><td>Inert sampling line</td><td>Teflon</td><td></td><td>0.64 cm diameter</td></tr><tr><td>Pump</td><td>Thomas</td><td>107 series</td><td>Used to flush air through sample lines</td></tr></tbody></table>

lab-scale model to evaluate odor and gas concentrations emitted by deep bedded pack manure

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope facilities. This protocol has been used to effectively evaluate many different bedding materials, environmental variables (temperature, humidity), and potential mitigation treatments that can improve air quality in commercial deep-bedded mono-slope facilities. The model is dynamic and allows researchers to easily collect many chemical and physical measurements from the bedded pack. Weekly measurements, collected over the course of six to seven weeks, allows sufficient time to see changes in air quality measurements over time as the bedded pack matures. The data collected from the simulated bedded packs is within the range of concentrations previously measured in commercial deep-bedded mono-slope facilities. Past studies have demonstrated that 8 - 10 experimental units per treatment are sufficient to detect statistical differences among the simulated bedded packs. The bedded packs are easy to maintain, requiring less than 10 minutes of labor per bedded packs per week to add urine, feces, and bedding. Sample collection using the gas sampling system requires 20 - 30 minutes per bedded pack, depending on the measurements that are being collected. The use of lab-scaled bedded packs allows the researcher to control variables such as temperature, humidity, and bedding source that are difficult or impossible to control in a research or commercial facility. While not a perfect simulation of &#34;real-world&#34; conditions, the simulated bedded packs serve as a good model for researchers to use to examine treatment differences among bedded packs. Several lab-scale studies can be conducted to eliminate possible treatments before trying them in a research or commercial-sized facility.

To date, seven research studies have been published9,10,11,12,13,14,15 using this procedure, with modifications and adjustments made to improve the model and reflect objectives of the specific experiments. This procedure has been used to evaluate the effect of numerous bedding materials and ambient temperature on odor and gas production, as well as amendments that can be added to control ammonia emissions. Chemical and physical properties of the bedded packs have been measured in commercial barns6,28 as well as in the simulated bedded packs (Table 1). This data was used to determine if the protocol was a suitable model to supplement expensive on-farm research trials. Air quality data has been collected from commercial facilities and simulated bedded packs using two different methods (Table 2). The gas sampling system described in this protocol is new technology that has been tested and compared to previously used methods.
The dry matter composition of simulated bedded packs were within the range of published dry matter content of bedded pack material collected from commercial facilities6,28. The first time the protocol was used11, 400 g of bedding were initially added to the bedded packs with subsequent additions of 200 g per week of fresh bedding, and 400 g each of urine and feces added three times weekly. This was set up to simulate commercial barns in which multiple bales of bedding are added initially and only one or two bales added to the pack per week thereafter. The ratio of bedding:livestock waste was estimated using data collected from commercial deep bedded mono-slope facilities1,6. At the end of the first study, the dry matter content of the bedded packs was similar to the dry matter content measured in bedded pack material collected from commercial facilities6,28. However, visual observation of the bedded packs indicated that there was a lot of variability in the water holding capacity of the bedding materials. For example, bedded packs with corn cobs appeared very wet, but had a dry matter content of 27.2 &#177; 1.5%16, while bedded packs with wheat straw bedding appeared relatively dry, but had a dry matter content of 21.2 &#177; 1.1%11. To try to increase the dry matter content of the bedded packs to better represent commercial barns6,28, the protocol was adjusted slightly with 320 g each of bedding, urine, and feces added when the pack was started, three weekly additions of 320 g each of urine and feces, and one weekly addition of 320 g of bedding material added to the packs. This protocol raised the dry matter content of the bedded packs, but was highly dependent upon the bedding material13 used in the experiment and the temperature of the environmental chambers14. Even though it was variable, the dry matter content of the simulated bedded packs were within the range measured in commercial barns so the second protocol has been used for all subsequent studies.
The nutrient composition, pack temperature, and pH of the simulated bedded packs provide further evidence that the simulated bedded packs are a good model to represent manure bedded packs in commercial facilities. Total N, total P, total S, and total K have consistently been within the range of the nutrient content measured from commercial deep-bedded mono-slope facilities6,28. Partial composting occurs in the bedded packs of deep-bedded mono-slope facilities, so it was important to replicate the temperature of commercial facilities in the lab-scaled simulated bedded packs. Temperature of bedded packs in deep-bedded commercial facilities when ambient air temperature was between 0 and 20.6 &#730;C was 19.2 &#177; 0.3 &#730;C 6. The temperature in the environmental chambers was set at 20 &#730;C for most of the studies conducted using this protocol. In these studies, the temperature of the simulated bedded packs has consistently been between 18.3 and 20.1 &#730;C. The exception to this was when temperature was a factor that was tested in a three-way factorial experiment. Two environmental chambers were set at 40 &#730;C and two were set at 10 &#730;C. In that study, the temperature of the simulated bedded packs was 12 - 13 &#730;C in the cold chambers and 32 - 35 &#730;C in the warmer chambers. Once again, this reflected commercial barns, where pack temperatures were 15.4 &#177; 0.4 &#730;C when ambient temperatures were 0 &#730;C or colder, and 29.0 &#177; 0.3 &#730;C when ambient air temperature was greater than 20.6 &#730;C 6. The pH of the bedded packs in commercial barns using corn stover bedding was measured in one study6 and ranged from 7.5 - 8.0. Simulated bedded packs with corn stover bedding have had pH values of 7.1 - 7.311,13. The pH of all simulated bedded packs has ranged from 6.2 to 9.0, which reflects a variety of bedding materials used in the experiments.
The gas sampling system that is used in this protocol was adapted from a series of studies conducted in commercial poultry, swine, and dairy barns as part of the National Air Emissions Monitoring Study29. This system flushes room air through the flux chamber, creating a dynamic flux chamber that measures the concentration of the selected gases that are emitted over a 20-minute period. Previous to using the gas sampling system, the steady-stead concentration of NH3 was determined by collecting air samples from each bedded pack using static flux chambers with acid traps containing 2 mol L-1 sulfuric acid6,22. The air within the chamber was recycled through the acid traps at a rate of 1 L min-1 for 20 minutes. Total reduced sulfides were collected using a hand-held sampler. Air samples were recirculated through the static flux chambers using a small pump at a flow rate of 1 L min for no longer than 4 minutes. A minimum of four consecutive samples were drawn from each simulated bedded pack. Greenhouse gas concentrations (N20, CO2, and CH4) were determined by collecting one 20 mL sample of air from each simulated bedded pack using the septa at the top of each static flux chamber. The samples were later analyzed using a gas chromatograph. The previous method of collecting all these gas samples was very labor intensive and required two or three people to manage all the collection equipment. Use of the gas sampling system is much less labor intensive. One person is able to set up the gas sampling system, initiate the programmable logic relay and return approximately 160 minutes later when the samples have finished collecting gas data from 8 simulated bedded packs.
Results from the previous, labor-intensive sampling protocol are shown, as well as results from the gas sampling system (Table 2). Because of the amount of labor required to collect the data, not all data was able to be collected from commercial facilities. Ammonia concentration was collected using the acid-trap method from the surface of bedded packs in commercial mono-slope facilities and compared to the simulated bedded packs. Ammonia concentrations measured in the simulated bedded packs were consistently similar to NH3 concentrations measured from bedded packs in commercial cattle facilities. Ammonia concentrations using the new gas sampling system appear to be on the low end of the concentrations present in cattle facilities. That may be caused by the NH3 analyzer or it may be a reflection of the treatments in the experiments that used the new gas sampling system. It could also reflect a higher rate of air flow over the simulated bedded packs compared to the air flow in the commercial barns, which would dilute the concentration of the ammonia samples. One series of experiments tested the use of alum as a surface amendment that can be applied to the bedded packs to lower the pack pH, thereby reducing volatilization of nitrogen as NH3. Carbon dioxide, CH4, and N2O have not been measured at the surface of a bedded pack in commercial facilities. However, the range of concentrations of these gases measured in simulated bedded packs using the previous gas chromatography method and the range of concentrations measured using the gas sampling system are very similar. Somewhat higher concentrations were produced when the simulated bedded packs were housed in a 35 &#730;C environmental chamber compared to 20 &#730;C chamber, which accounts for the variability among experiments. Comparing TRS to hydrogen sulfide is not a direct comparison, since TRS includes more than just hydrogen sulfide. Therefore, it is not surprising that TRS concentrations from simulated bedded packs are slightly higher than H2S concentrations measured using the gas sampling system. This is also a reflection of the studies conducted using the two sampling protocols. Bedded packs that contained green cedar bedding generated very high TRS concentrations12, while those that contain corn stover bedding did not. The samples collected using the gas sampling system have used corn stover, wheat straw, soybean stover, and pine chip bedding materials but no green cedar bedding.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Commercial Barns1-2</td>
			<td colspan="4">Simulated Bedded Packs3-6</td>
		</tr>
		<tr>
			<td>Dry matter,%</td>
			<td>29.99 &#177; 3.15</td>
			<td>16.0&#160; &#8211; 36.6</td>
			<td>20.8 &#8211; 27.2</td>
			<td>22.3 &#8211; 26.1</td>
			<td>24.0 &#8211; 58.0</td>
			<td>20.8 &#8211; 24.9</td>
		</tr>
		<tr>
			<td>Total N,g kg-1</td>
			<td>60.97 &#177; 13.77</td>
			<td>21.2 &#8211; 23.6</td>
			<td>19.4 &#8211; 28.2</td>
			<td>17.8 &#8211; 22.3</td>
			<td>15.6 &#8211; 18.6</td>
			<td>17.8 &#8211; 23.8</td>
		</tr>
		<tr>
			<td>Total P, g kg-1</td>
			<td>14.13 &#177; 3.99</td>
			<td>6.7 &#8211; 7.5</td>
			<td>6.2 &#8211; 9.6</td>
			<td>7.1 &#8211; 9.6</td>
			<td>6.7 &#8211; 8.5</td>
			<td>6.2 &#8211; 9.6</td>
		</tr>
		<tr>
			<td>Total S, g kg-1</td>
			<td>7.88 &#177; 1.48</td>
			<td>5.6 &#8211; 6.7</td>
			<td>3.6 &#8211; 6.5</td>
			<td>4.5 &#8211; 5.3</td>
			<td>---</td>
			<td>3.6 &#8211; 6.5</td>
		</tr>
		<tr>
			<td>Total K, g kg-1&#160;</td>
			<td>32.74 &#177; 8.39</td>
			<td>15.5 &#8211; 21.1</td>
			<td>16.3 &#8211; 23.1</td>
			<td>---</td>
			<td>18.8 &#8211; 25.6</td>
			<td>16.3 &#8211; 25.2</td>
		</tr>
		<tr>
			<td>Lignin, g kg-1</td>
			<td>---</td>
			<td>---</td>
			<td>26.5 &#8211; 139.6</td>
			<td>49.9 &#8211; 136.9</td>
			<td>---</td>
			<td>62.6 &#8211; 139.6</td>
		</tr>
		<tr>
			<td>Ash, g kg-1</td>
			<td>---</td>
			<td>154 - 214</td>
			<td>119.3 &#8211; 200.5</td>
			<td>98.9 &#8211; 223.6</td>
			<td>---</td>
			<td>119.3 &#8211; 200.5</td>
		</tr>
		<tr>
			<td>C:N Ratio</td>
			<td>---</td>
			<td>---</td>
			<td>17.4 &#8211; 28.2</td>
			<td>20.2 &#8211; 29.7</td>
			<td>---</td>
			<td>20.6 &#8211; 27.5</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>---</td>
			<td>7.5 - 8</td>
			<td>6.2 &#8211; 7.2</td>
			<td>6.8 &#8211; 7.6</td>
			<td>8.5 &#8211; 9.0</td>
			<td>7.4 &#8211; 7.7</td>
		</tr>
		<tr>
			<td>Temperature, &#730;C</td>
			<td>---</td>
			<td>15.4 &#8211; 29.0</td>
			<td>18.3 &#8211; 19.9</td>
			<td>18.4 &#8211; 20.0</td>
			<td>12.0 &#8211; 35.0</td>
			<td>19.7 &#8211; 20.1</td>
		</tr>
		<tr>
			<td colspan="7">1Euken, 2009. Standard deviation as reported by Euken, 2009 is shown. Total P and total K were calculated by converting reported P2O5 and K2O composition, respectively.</td>
		</tr>
		<tr>
			<td colspan="7">2Spiehs et al., 2011. Data collected from two pens in each of two barns . Chopped corn stalks were the preferred bedding material, but wheat straw and soybean stalks were also used for bedding during brief periods of this project. Bedding use ranged from 1.95 &#8211; 3.37 kg per animal per day and pen density ranged from 3.22 &#8211; 6.13 m2 per animal.&#160;</td>
		</tr>
		<tr>
			<td colspan="7">3Spiehs et al., 2012. Data collected from simulated bedded packs. Bedding materials included corn stover, bean stover, wheat straw, pelleted corn cobs, paper, wood chips, and sawdust.</td>
		</tr>
		<tr>
			<td colspan="7">4Spiehs et al., 2014b. Data collected from simulated bedded packs. Bedding material included corn stover, pine wood chips, wet cedar chips, and dry cedar chips.</td>
		</tr>
		<tr>
			<td colspan="7">5Ayadi et al., 2015b. Data collected from simulated bedded packs using corn stover and bean stover bedding material. Two temperatures were used (40&#730;C and 10&#730;C)</td>
		</tr>
		<tr>
			<td colspan="7">6Spiehs et al., 2017. Data collected from simulated bedded packs using mixtures of bedding material containing 0, 10, 20, 30, 40, 60, 80, 100% pine with the remaining being corn stover.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1. Range of reported dry matter and nutrient composition (dry-matter basis) of bedding/manure material from commercial deep bedded mono-slope facilities (Euken, 2009 and Spiehs et al., 2011) and from studies conducting using the simulated bedded packs (Spiehs et al., 2012, 2014, 2017 and Ayadi et al., 2015).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Static flux chamber method1</td>
			<td colspan="2">Dynamic flux chamber method2</td>
		</tr>
		<tr>
			<td>Ammonia, ppm</td>
			<td>95.8 &#8211; 641.1</td>
			<td>350.8 &#8211; 516.7</td>
			<td>381 - 1584</td>
			<td>386.3 &#8211; 502.3</td>
			<td colspan="2">89.4 &#8211; 166.7</td>
		</tr>
		<tr>
			<td>TRS, ppb</td>
			<td>---</td>
			<td>8.2 &#8211; 165.9</td>
			<td>---</td>
			<td>5.3 &#8211; 11.4</td>
			<td colspan="2">---</td>
		</tr>
		<tr>
			<td>Hydrogen Sulfide, ppb</td>
			<td>---</td>
			<td>---</td>
			<td>---</td>
			<td>---</td>
			<td colspan="2">0.1 &#8211; 18.1</td>
		</tr>
		<tr>
			<td>Carbon dioxide, ppm</td>
			<td>---</td>
			<td>1232 - 2000</td>
			<td>2322 - 6917</td>
			<td>918 - 1158</td>
			<td colspan="2">957 &#8211; 2149</td>
		</tr>
		<tr>
			<td>Methane, ppm</td>
			<td>---</td>
			<td>2.3 &#8211; 3.6</td>
			<td>7.2 &#8211; 87.0</td>
			<td>4.4 &#8211; 6.7</td>
			<td colspan="2">3.2 &#8211; 16.7</td>
		</tr>
		<tr>
			<td>Nitrous oxide, ppm</td>
			<td>---</td>
			<td>0.67 &#8211; 0.72</td>
			<td>0.31 &#8211; 0.77</td>
			<td>0.21 &#8211; 0.23</td>
			<td colspan="2">0.44 &#8211; 0.58</td>
		</tr>
		<tr>
			<td colspan="7">1Spiehs et al., 2011, 2014a, 2015a, 2016a. Data from these studies were collected using acid traps for ammonia, a hand-held sampler of total reduced sulfides, and one sample from the headspace of each simulated bedded pack analyzed on a greenhouse gas GC for carbon dioxide, methane, and nitrous oxide.</td>
		</tr>
		<tr>
			<td colspan="7">2This data represents three studies conducting using varying bedding materials and surface amendments to control odor and gas emission. These studies were conducting using the gas sampling system and are not yet published.</td>
		</tr>
	</tbody>
</table>
Table 2. Range of reported concentrations of ammonia, total reduced sulfides (TRS), hydrogen sulfide, carbon dioxide, methane, and nitrous oxide from commercial deep bedded mono-slope facilities (Spiehs et al., 2011) and from studies conducting using the simulated bedded packs (Spiehs et al., 2014a, 2016 and Ayadi et al., 2015).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57332/57332fig1.jpg" /> 
Figure 1. Simulated bedded packs in plastic containers with stainless steel flux chambers and rubber skits attached and ready for air sampling. The simulated bedded packs are located inside the environmental chambers. <a href="/files/ftp_upload/57332/57332fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure at JoVE.com

Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure

A protocol has been developed to measure gases, odors, and nutrient composition in lab-scaled bedded manure packs, which can be used to study ways to improve air quality in commercial cattle facilities using deep-bedded manure packs.

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope ...

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Nanyang Technological University

Research

JoVE Journal

Environment

6.3K Views.  USDA ARS U.S. Meat Animal Research Center. Een protocol is ontwikkeld voor het meten van de gassen, geuren en nutriënten compositie in lab-scaled bedden mest packs, die kan worden gebruikt voor het bestuderen van manieren ter verbetering van de kwaliteit van de lucht in commerciële vee-faciliteiten met behulp van deep-bedden mest packs.

Video: Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of animals housed within a confined area enables rapid dissemination of most infectious agents throughout a herd. Rapid containment is key to controlling any infectious disease outbreak, thus depopulation is often undertaken to prevent spread of a pathogen to the larger livestock population. In that circumstance, a large number of livestock carcasses and contaminated manure are generated that require rapid disposal.
Composting lends itself as a rapid-response disposal method for infected carcasses as well as manure and soil that may harbor infectious agents. We designed a bio-contained mortality composting procedure and tested its efficacy for bovine tissue degradation and microbial deactivation. We used materials available on-farm or purchasable from local farm supply stores in order that the system can be implemented at the site of a disease outbreak. In this study, temperatures exceeded 55&deg;C for more than one month and infectious agents implanted in beef cattle carcasses and manure were inactivated within 14 days of composting. After 147 days, carcasses were almost completely degraded. The few long bones remaining were further degraded with an additional composting cycle in open windrows and the final mature compost was suitable for land application. 
Duplicate compost structures (final dimensions 25 m x 5 m x 2.4 m; L x W x H) were constructed using barley straw bales and lined with heavy black silage plastic sheeting. Each was loaded with loose straw, carcasses and manure totaling ~95,000 kg. A 40-cm base layer of loose barley straw was placed in each bunker, onto which were placed 16 feedlot cattle mortalities (average weight 343 kg) aligned transversely at a spacing of approximately 0.5 m. For passive aeration, lengths of flexible, perforated plastic drainage tubing (15 cm diameter) were placed between adjacent carcasses, extending vertically along both inside walls, and with the ends passed though the plastic to the exterior. The carcasses were overlaid with moist aerated feedlot manure (~1.6 m deep) to the top of the bunker. Plastic was folded over the top and sealed with tape to establish a containment barrier and eight aeration vents (50 x 50 x 15 cm) were placed on the top of each structure to promote passive aeration. After 147 days, losses of volume and mass of composted materials averaged 39.8% and 23.7%, respectively, in each structure.

Using readily available materials, this biocontained composting system enables effective on-site disposal of large animal carcasses arising in the event of infectious disease outbreak. This procedure kills most infectious agents in carcasses and contaminated manure. Once infectious agent is confirmed non-viable, mature compost can be spread as fertilizer.

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of ...

Immunology and Infection

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

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 ...

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% fine dust formation within the EU15. But NH3 emissions also mean a considerable loss of nutrients. Many studies on NH3 emission from organic and mineral fertilizer application have been performed in recent decades. Nevertheless, research related to NH3 emissions after application fertilizers is still limited in particular with respect to relationships to emissions, fertilizer type, site conditions and crop growth. Due to the variable response of crops to treatments, effects can only be validated in experimental designs including field replication for statistical testing. The dominating ammonia loss methods yielding quantitative emissions require large field areas, expensive equipment or current supply, which restricts their application in replicated field trials. This protocol describes a new methodology for the measurement of NH3 emissions on many plots linking a simple semi-quantitative measuring method used in all plots, with a quantitative method by simultaneous measurements using both methods on selected plots. As a semi-quantitative measurement method passive samplers are used. The second method is a dynamic chamber method (Dynamic Tube Method) to obtain a transfer quotient, which converts the semi-quantitative losses of the passive sampler to quantitative losses (kg nitrogen ha-1). The principle underlying this approach is that passive samplers placed in a homogeneous experimental field have the same NH3 absorption behavior under identical environmental conditions. Therefore, a transfer co-efficient obtained from single passive samplers can be used to scale the values of all passive samplers used in the same field trial. The method proved valid under a wide range of experimental conditions and is recommended to be used under conditions with bare soil or small canopies (&#60;0.3 m). Results obtained from experiments with taller plants should be treated more carefully.

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Ammonia emissions are a major threat to the environment by eutrophication, soil acidification and fine particle formation and stem mainly from agricultural sources. This method allows ammonia loss measurements in replicated field trials enabling statistical analysis of emissions and of relationships between crop development and emissions.

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% ...

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Biochemistry

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

Respirometry Protocol for Anaerobic Digestion of Food Waste and Sludge

Measuring Biomethane Potential of Food Scrap Waste Anaerobically Co&#45;Digested with Waste&#45;Activated Sludge Using Respirometry

The use of respirometry to study the biokinetics of microbiota treating wastewater or digesting wastewater sludges has become more prevalent over the last few decades. The use of respirometry to examine the biokinetics of anaerobic microbiota co-digesting organic waste streams such as wastewater sludge and food scrap is an area of active research. To date, no visualized protocol has been published on the topic. Accordingly, in this protocol, we configured a respirometer to measure methane production and flow rate over time using three different food-to-microorganism (F:M) ratios and food scrap waste and waste-activated sludge as substrates. The resulting data, coupled with substrate utilization measurements, provides the basis for understanding how different substrate concentrations influence the rate at which anaerobic microbiota produce methane. Additionally, this protocol presents a method to develop biokinetic parameters (e.g., methane production rate constant and yield). Others can use this respirometry protocol to examine organic degradation under anaerobic conditions and develop microbial parameters.

Author Spotlight: Advancing Anaerobic Microbiota Research Using a Novel Respirometry Protocol

This protocol describes a best practice for determining methane production and microbial kinetic parameters using respirometry for anaerobic microbiota co-digesting food scrap waste and waste-activated sludge.

The use of respirometry to study the biokinetics of microbiota treating wastewater or digesting wastewater sludges has become more prevalent over the last ...

Modeling Methanotroph Niches – The Gradient Syringe Model Ecosystem

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists' understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs' natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.

Author Spotlight: Designing Simple and Inexpensive Techniques to Grow Methane-Oxidizing Bacteria in the Laboratory

A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure

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