This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (# 2015R1A2A2A01006446).

1. Preparation of Surface for Biofilm Formation
<ol>
	<li>Cut 1-mm-thick 304 stainless steel plates into chips (10 &#215; 10 mm2) with a mechanical cutter.</li>
	<li>Perform ultrasonic cleaning of the chips in acetone, methanol, and deionized (DI) water sequentially for 10 min each. Use a solvent-resistant container, made of substances such as glass, to remove organic contamination.</li>
	<li>Rinse the chips with flowing DI water for 3-5 sec.</li>
	<li>Dry the chips using N2 gas flow for 3-5 sec.</li>
</ol>
2. Preparation of Bacterial Culture
<ol>
	<li>Take a P. putida KT2440 (kindly provided by Prof. Sung Kuk Lee, UNIST, South Korea) stock stored in a -80 &#176;C deep-freezer.</li>
	<li>Thaw at room temperature after 1 min, and the top layer of the frozen stock solution turns to slush. Immerse a loop into the melted layer of the stock solution.</li>
	<li>Use this loop to streak the bacteria onto a Luria-Bertani (LB) plate containing 1.5% agar.</li>
	<li>Incubate the plate overnight at 30 &#176;C to grow the bacterial colonies.</li>
	<li>Pick a single colony from the plate using a fresh loop.</li>
	<li>Inoculate 10 ml of LB broth in a 50 ml conical tube with the loop containing the single bacterial colony.</li>
	<li>Incubate the broth in a shaking incubator for 16 hr at 30 &#176;C and 160 rpm.</li>
</ol>
3. Biofilm Formation on the Surface
<ol>
	<li>Pick up each of the prepared chips with tweezers and dip them in 70% ethanol 5 times for 1-2 sec each, to sterilize the surface of each chip. Ensure that the chips are held with tweezers during the dipping.</li>
	<li>Dip each chip in autoclaved DI water and in LB broth sequentially, 5 times for 1-2 sec each, to remove remaining ethanol.</li>
	<li>Place these chips in 6-well culture plates with 2 chips and 5 ml LB broth per well.</li>
	<li>Dilute the bacterial culture until the concentration in the LB broth reaches 8 &#215; 108 cells/ml (optical density at 600 nm wavelength: ~0.8).</li>
	<li>Inoculate each well with 50 &#181;l of the diluted bacterial culture.</li>
	<li>Incubate the plates at 30 &#176;C without shaking for 24 hr for formation of biofilms.</li>
</ol>
4. CO2 Aerosol Cleaning
<ol>
	<li>Dip the biofilm-formed chips in 10 mM ammonium acetate buffer (volatile) 5 times to remove loosely attached and planktonic bacteria.</li>
	<li>Dry these chips in a biosafety cabinet, where air flows mildly.</li>
	<li>Immediately after drying, place a chip on the loading place, which is 20 mm from the CO2 nozzle along the axis of the jet. Tilt the jet axis to a 40&#176; angle.</li>
	<li>Set the stagnation pressures of CO2 and N2 gas to 5.3 MPa and 0.7 MPa, respectively, using gas-pressure regulators.</li>
	<li>Apply the aerosol jet onto the central part of the chip. White aerosols including the solid CO2 should be visible. Turn &#34;on&#34; the solenoid valve for CO2 for 5 sec, and then turn it &#34;off&#34; for 3 sec (cycle: 8 sec) periodically using a manually controlled switch. If a nitrogen purge needs to be used, turn on the solenoid valve for a continuous supply of N2.</li>
	<li>Treat the chips with CO2 aerosols for 16, 40, and 88 sec with and without nitrogen purges. Retain chips without treatment as negative controls.</li>
</ol>
5. Analysis for Removal Efficiency
<ol>
	<li>Prepare 1 &#181;M green fluorescence nucleic acid stain (excitation/emission wavelength: 480/500 nm) in DI water to stain bacterial cells on the control and aerosol-treated chips.</li>
	<li>Place the chips in the staining solution.</li>
	<li>Incubate the chips in an incubator without light at 37 &#176;C for 30 min.</li>
	<li>Following the incubation, gently rinse the chips with flowing DI water to remove excessive fluorescent dye.</li>
	<li>Dry the chips with N2 gas flow.</li>
	<li>Take fluorescence microscopic images of 5 random fields of view (321 &#215; 240 &#181;m2) for each chip using an epifluorescence microscope, a 40X objective lens, and a CCD camera.</li>
	<li>Obtain the fluorescence intensity for each image using an image processing software such as ImageJ. In ImageJ, use the &#34;Subtract Background&#34; function in the &#34;Process&#34; menu, and select &#34;Integrated density&#34; in the &#34;Set Measurements&#34; window in the &#34;Analyze&#34; menu. Do the &#34;Measurement&#34; in the &#34;Analyze&#34; menu to obtain the fluorescence intensity.</li>
	<li>Calculate the biofilm removal efficiency according to the following formula: 100% &#215; (I of control chips - I of treated chips)/(I of control chips), where I is the calculated fluorescence intensity.</li>
	<li>Obtain the average removal efficiencies and standard deviations. Use at least 4 chips for each case.</li>
</ol>

<ol>
	<li>Jain, A., Gupta, Y., Agrawal, R., Khare, P., Jain, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+-+A+microbial+life+perspective:+A+critical+review.">Biofilms - A microbial life perspective: A critical review.</a> Crit. Rev. Ther. Drug. 24 (5), 393-443 (2007).</li><li>Bott, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofouling+control+with+ultrasound.">Biofouling control with ultrasound.</a> Heat Transfer Eng. 21 (3), 43-49 (2000).</li><li>Meyer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+prevention,+removal+and+killing+of+biofilms.">Approaches to prevention, removal and killing of biofilms.</a> Int. Biodeterior. Biodegradation. 51 (4), 249-253 (2003).</li><li>Hong, S. H., 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+electric+currents+on+bacterial+detachment+and+inactivation.">Effect of electric currents on bacterial detachment and inactivation.</a> Biotechnol. Bioeng. 100 (2), 379-386 (2008).</li><li>Nandakumar, K., Obika, H., Utsumi, A., Ooie, T., Yano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+laser+ablation+of+laboratory+developed+biofilms+using+an+Nd:YAG+laser+of+532+nm+wavelength.">In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength.</a> Biotechnol. Bioeng. 86 (7), 729-736 (2004).</li><li>Gibson, H., Taylor, J. H., Hall, K. E., Holah, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+cleaning+techniques+used+in+the+food+industry+in+terms+of+the+removal+of+bacterial+biofilms.">Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms.</a> J. Appl. Microbiol. 87 (1), 41-48 (1999).</li><li>Kang, M. Y., Jeong, H. W., Kim, J., Lee, J. W., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+biofilms+using+carbon+dioxide+aerosols.">Removal of biofilms using carbon dioxide aerosols.</a> J. Aerosol Sci. 41 (11), 1044-1051 (2010).</li><li>Cha, M., Hong, S., Kang, M. Y., Lee, J. W., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-phase+removal+of+biofilms+from+various+surfaces+using+carbon+dioxide+aerosols.">Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols.</a> Biofouling. 28 (7), 681-686 (2012).</li><li>Dwidar, M., Hong, S., Cha, M., Jang, J., Mitchell, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+application+of+bacterial+predation+and+carbon+dioxide+aerosols+to+effectively+remove+biofilms.">Combined application of bacterial predation and carbon dioxide aerosols to effectively remove biofilms.</a> Biofouling. 28 (7), 671-680 (2012).</li><li>Cha, M., Hong, S., Lee, S. Y., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+different-age+biofilms+using+carbon+dioxide+aerosols.">Removal of different-age biofilms using carbon dioxide aerosols.</a> Biotechnol. Bioprocess Eng. 19 (3), 503-509 (2014).</li><li>Singh, R., Monnappa, A. K., Hong, S., Mitchell, R. J., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Carbon+Dioxide+Aerosols+on+the+Viability+of+Escherichia+coli+during+Biofilm+Dispersal.">Effects of Carbon Dioxide Aerosols on the Viability of Escherichia coli during Biofilm Dispersal.</a> Sci. Rep. 5, 13766 (2015).</li><li>Sherman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Dioxide+Snow+Cleaning.">Carbon Dioxide Snow Cleaning.</a> Particul. Sci.Technol. 25 (1), 37-57 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Previously, we conducted optimization studies on CO2 gas pressure, jet angle, and distance to the solid surface in CO2 aerosol cleaning7. Unlike our previous studies, in the present study, a nitrogen purge was not included in the aerosol (Figure 1). Moreover, 304 stainless steel was used in this protocol, since it is one of the most common stainless steels and is widely used in the food industry. The polished surface is beneficial for fluorescence analysis because of a un...

Biofilms are complex bacterial community structures in which bacterial cells are embedded within self-produced matrices of extracellular polymeric substances, held together, and protected from the external environment. Biofilms can present a public health risk and cause economic losses because they can form on various surfaces, including medical implant materials and devices, food processing equipment, and heat exchangers. In fact, biofilms have been found to be associated with 65% of all bacterial infectious diseases in humans, according to the Centers for Disease Control and Prevention1.
Autoclaves and disinfectants such as chlorine have generally been used for the inactivation of biofilms. However, the use of an autoclave is limited for surfaces that can neither withstand high temperature steam nor be placed into the autoclave. Disinfectants are not suitable for surfaces sensitive to chemical treatment or prone to collecting toxic oxidation products2. In addition, the biofilm should not only be inactivated, but also removed in order to prevent the attachment of new cells onto the surface, thereby forming a new biofilm3. However, it is difficult to remove biofilms using methods based on viscous fluid shear because the flow velocity near the surface is almost zero and the shear force usually cannot overcome the adhesive forces of micron- and submicron-sized substances. Moreover, the biofilm matrix is known to act as a physical and chemical barrier1.
Many physical and mechanical techniques have been developed to remove biofilms from surfaces, including ultrasonic vibration1, electric currents4, laser irradiation5, and high-pressure water sprays6. Each technique has its own pros and cons. Ultrasonic vibration and electric current can be used to control biofilm formation; however, they require a particular configuration and conductive surfaces, respectively, requiring additional shear stress1, 4. Laser irradiation can be applied to a limited area and to hard surfaces; however, some live and dead cells remain on the surface5. High-pressure water sprays effectively remove biofilms; however, their high momentum can cause damage to soft substrates6.
Biofilm removal using CO2 aerosols has been previously proposed. It has shown promising results, with high removal efficiencies within a short time7-11. CO2 aerosols are generated by adiabatic expansion of a high-pressure CO2 gas through a nozzle, and they are applied to the surfaces contaminated with a biofilm. This cleaning technique utilizes the momentum transfer of solid CO2 particles and the solvent action of the melted CO2 liquids, followed by the aerodynamic shear force of the CO2 gas12. Compared with high-pressure N2 gas jets, CO2 aerosol jets at the same stagnation pressure are much more effective in removing E. coli biofilms7. Moreover, although the momentum of the solid CO2 that is delivered to the bacteria is considerably high, the momentum of the total aerosol jet applied to the solid surface is substantially lower than that of water jets. Therefore, damage-free cleaning is possible using this CO2 aerosol technique.
In this protocol, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from a polished stainless steel surface. In fact, nitrogen purges have been used in many CO2 aerosol studies to increase the removal efficiency by reducing the moisture condensation generated during the cleaning, even though heating with a hot plate or infrared lamps and employing dry boxes have also been adopted12. The surface biofilm formation and the optimized cleaning procedures are described below. The removal efficiency was evaluated by measuring the fluorescence intensity of the biofilm on the surface.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">304 stainless steel</td>
			<td style="width:71px;">Steelni 
			(South Korea)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Polished and diced ones</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasonic cleaner</td>
			<td style="width:71px;">Branson 
			(USA)</td>
			<td style="width:119px;">5510E-DTH</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Luria-Bertani (LB)</td>
			<td style="width:71px;">Becton, Dickinson and Company 
			(USA)</td>
			<td style="width:119px;">244620</td>
			<td>500 g</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Agar</td>
			<td style="width:71px;">Becton, Dickinson and Company 
			(USA)</td>
			<td style="width:119px;">214010</td>
			<td>500 g</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">6-well culture plate</td>
			<td style="width:71px;">SPL Life Sciences 
			(South Korea)</td>
			<td style="width:119px;">32006</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ammonium acetate buffer</td>
			<td style="width:71px;">Sigma-Aldrich 
			(USA)</td>
			<td style="width:119px;">667404</td>
			<td>10 mM</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Dual gas unit</td>
			<td style="width:71px;">Applied Surface Technologies 
			(USA)</td>
			<td style="width:119px;">K6-10DG</td>
			<td style="width:167px;">One nozzle for CO2 gas 
			&#38; 8 nozzles for N2 gas</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">SYTO9</td>
			<td style="width:71px;">Thermo Fissher Scientific 
			(USA)</td>
			<td style="width:119px;">Invitrogen</td>
			<td style="width:167px;">Excitaion: 480 nm 
			Emission: 500 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Epifluorescence microscope&#160;</td>
			<td style="width:71px;">Nikon (Japan)</td>
			<td style="width:119px;">Eclipse 80i</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40&#215; objective lens</td>
			<td style="width:71px;">Nikon 
			(Japan)</td>
			<td style="width:119px;">Plan Fluor</td>
			<td>NA: 0.75</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">CCD camera&#160;</td>
			<td style="width:71px;">Photometrics 
			(USA)</td>
			<td style="width:119px;">Cool SNAP HQ2</td>
			<td>Monochrome</td>
		</tr>
	</tbody>
</table>

biofilm removal using carbon dioxide aerosols without nitrogen purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

CO2 aerosols were used to remove the P. putida biofilms from SUS304 surfaces (Figure 1). Most of the surfaces were covered with the biofilm after 24 hr of growth. Most of the biofilm was removed using the CO2 aerosols (Figure 2). As expected, Figure 3 shows an increase in biofilm removal efficiency as the CO2 aerosol treatment time increased. For the treatment time of 88 sec, the removal efficiency was determined to be as high as...

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Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

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6.3K Views.  Ulsan National Institute of Science and Technology (UNIST). Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Video: Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

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 ...

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

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 ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

Functionalization and Dispersion of Carbon Nanomaterials Using an Environmentally Friendly Ultrasonicated Ozonolysis Process

Functionalization of carbon nanomaterials is often a critical step that facilitates their integration into larger material systems and devices. In the as-received form, carbon nanomaterials, such as carbon nanotubes (CNTs) or graphene nanoplatelets (GNPs), may contain large agglomerates. Both agglomerates and impurities will diminish the benefits of the unique electrical and mechanical properties offered when CNTs or GNPs are incorporated into polymers or composite material systems. Whilst a variety of methods exist to functionalize carbon nanomaterials and to create stable dispersions, many the processes use harsh chemicals, organic solvents, or surfactants, which are environmentally unfriendly and may increase the processing burden when isolating the nanomaterials for subsequent use. The current research details the use of an alternative, environmentally friendly technique for functionalizing CNTs and GNPs. It produces stable, aqueous dispersions free of harmful chemicals. Both CNTs and GNPs can be added to water at concentrations up to 5 g/L and can be recirculated through a high-powered ultrasonic cell. The simultaneous injection of ozone into the cell progressively oxidizes the carbon nanomaterials, and the combined ultrasonication breaks down agglomerates and immediately exposes fresh material for functionalization. The prepared dispersions are ideally suited for the deposition of thin films onto solid substrates using electrophoretic deposition (EPD). CNTs and GNPs from the aqueous dispersions can be readily used to coat carbon- and glass-reinforcing fibers using EPD for the preparation of hierarchical composite materials.

Here, a novel method for the functionalization and stable dispersion of carbon nanomaterials in aqueous environments is described. Ozone is injected directly into an aqueous dispersion of carbon nanomaterial that is continuously recirculated through a high-powered ultrasonic cell.

Functionalization of carbon nanomaterials is often a critical step that facilitates their integration into larger material systems and devices. In the ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The system comprises a high-pressure rheometer which is connected to a circulation loop. The rheometer has a rotational flow-through measurement cell with two alternative geometries: coaxial cylinder and double gap. The circulation loop contains a mixer, to bring the crude oil sample into equilibrium with CO2, and a gear pump that transports the mixture from the mixer to the rheometer and recycles it back to the mixer. The CO2 and crude oil are brought to equilibrium by stirring and circulation and the rheology of the saturated mixture is measured by the rheometer. The system is used to measure the rheological properties of Zuata crude oil (and its toluene dilution) in equilibrium with CO2 at elevated pressures up to 220 bar and a temperature of 50 &#176;C. The results show that CO2 addition changes the oil rheology significantly, initially reducing the viscosity as the CO2 pressure is increased and then increasing the viscosity above a threshold pressure. The non-Newtonian response of the crude is also seen to change with the addition of CO2.

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Preparation of Biomass-based Mesoporous Carbon with Higher Nitrogen-/Oxygen-chelating Adsorption for Cu(II) Through Microwave Pre-Pyrolysis

An environment-friendly technique for synthesizing biomass-based mesoporous activated carbon with high nitrogen-/oxygen-chelating adsorption for Cu(II) is proposed. Bagasse impregnated with phosphoric acid is utilized as the precursor. To pyrolyze the precursor, two separate heating modes are used: microwave pyrolysis and conventional electric-heating pyrolysis. The resulting bagasse-derived carbon samples are modified with nitrification and reduction modification. Nitrogen (N)/oxygen (O) functional groups are simultaneously introduced to the surface of activated carbon, enhancing its adsorption of Cu(II) by complexing and ion-exchange. Characterization and copper adsorption experiments are performed to investigate the physicochemical properties of four prepared carbon samples and determine which heating method favors the subsequent modification for doping of N/O functional groups. In this technique, based on analyzing data of nitrogen adsorption, Fourier transform infrared spectroscopy, and batch adsorption experiments, it is proven that microwave-pyrolyzed carbon has more defect sites and, therefore, time-saving effective microwave pyrolysis contributes more N/O species to the carbon, although it leads to a lower specific surface area. This technique offers a promising route to synthesis adsorbents with higher nitrogen and oxygen content and a higher adsorption capacity of heavy-metal ions in wastewater remediation applications.

Preparation of Biomass-based Mesoporous Carbon with Higher Nitrogen-/Oxygen-chelating Adsorption for CuII Through Microwave Pre-Pyrolysis

Here, we present a protocol to synthesize nitrogen/oxygen dual-doped mesoporous carbon from biomass by chemical activation in different pyrolysis modes followed by modification. We demonstrate that the microwave pyrolysis benefits the subsequent modification process to simultaneously introduce more nitrogen and oxygen functional groups on the carbon.

An environment-friendly technique for synthesizing biomass-based mesoporous activated carbon with high nitrogen-/oxygen-chelating adsorption for Cu(II) is ...

Measurement of Aerosols Optical Thickness of the Atmosphere using the GLOBE Handheld Sun Photometer

Here, we describe the measurement of aerosol optical thickness using the GLOBE handheld sun photometer. Aerosol optical thickness (AOT) was measured at Xavier University of Louisiana (XULA, 29.96&#176; N, 90.11&#176; W and 3 m above sea level). The measurements were done at two different wavelengths, 505 nm and 625 nm. AOT measurements were done 6 times a day (7 AM, 9 AM, 11 AM, solar noon, 3 PM and 5 PM). The data shown in this paper are the monthly average AOT values taken at solar noon. During each measurement time; at least five values of the sunlight voltage V and the dark voltage Vdark are taken for each channel. The mean for these five measurements is taken as the average for that measurement time. Other meteorological data such as temperature, surface pressure, rainfall and relative humidity are also measured at the same time. The whole protocol is completed within a time span of 10&#8211;15 min. The measured AOT values at 505 nm and 625 nm are then used to extrapolate the AOT values for wavelengths 667 nm, 551 nm, 532 nm and 490 nm. The measured and extrapolated AOT values were then compared with values from the nearest AERONET station at Wave CIS site 6 (AERONET, 28.87&#176; N, 90.48&#176; W and 33 m above sea level), which is about 96 km south of XULA. In this study we tracked the annual and daily variations of AOT for a 12 month period from September 2017 to August 2018. We also compared AOT data from two independently calibrated GLOBE handheld sun photometers at the XULA site. The data show that the two instruments are in excellent agreement.

The goal of the methods presented here is to measure aerosol optical thickness of the atmosphere. The sun photometer is pointed at the sun and the largest voltage reading obtained on an in-built digital voltmeter is recorded. Atmospheric measurements such as barometric pressure and relative humidity are also performed.

Here, we describe the measurement of aerosol optical thickness using the GLOBE handheld sun photometer. Aerosol optical thickness (AOT) was measured at ...