This research was supported by funding from General Directorate of the State Forests, Warsaw, Poland (project LAS, No OR-2717/27/11). We would like to express our gratitude to the entire research group from the Department of Meteorology, Poznan University of Life Sciences, Poland, involved in this protocol implementation and their help during creating its visual version.

<ol>
	<li>Baldocchi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+fluxes+of+trace+gases+and+energy+between+ecosystems+and+the+atmosphere+-+the+state+and+future+of+the+eddy+covariance+method.">Measuring fluxes of trace gases and energy between ecosystems and the atmosphere - the state and future of the eddy covariance method.</a> Global Change Biology. 20, 3600-3609 (2014).</li><li>Aubinet, M., 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=Estimates+of+the+annual+net+carbon+and+water+exchange+of+European+forests:+the+EUROFLUX+methodology.">Estimates of the annual net carbon and water exchange of European forests: the EUROFLUX methodology.</a> Advances in Ecological Research. 30, 113-174 (2000).</li><li>Aubinet, M., Vesala, T., Papale, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A practical guide to measurements and Data Analysis. , (2012).</li><li>Burba, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Eddy Covariance Method for: Scientific, Industrial, Agricultural, and Regulatory Applications. A Field Book on Measuring Ecosystem Gas Exchange and Areal Emission Rates. , (2013).</li><li>Pan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Large+and+Persistent+Carbon+Sink+in+the+World&#8217;s+Forests.">A Large and Persistent Carbon Sink in the World&#8217;s Forests.</a> Science. 333, 988-993 (2011).</li><li>Wofsy, S. C., 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=Net+exchange+of+CO2+in+a+midlatitude+forest.">Net exchange of CO2 in a midlatitude forest.</a> Science. 260 (5112), 1314-1317 (1993).</li><li>Luyssaert, S., 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=CO2+balance+of+boreal,+temperate,+and+tropical+forests+derived+from+a+global+database.">CO2 balance of boreal, temperate, and tropical forests derived from a global database.</a> Global Change Biology. 13, 2509-2537 (2007).</li><li>Knohl, A., 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=Carbon+dioxide+exchange+of+a+Russian+boreal+forest+after+disturbance+by+wind+throw.">Carbon dioxide exchange of a Russian boreal forest after disturbance by wind throw.</a> Global Change Biology. 8, 231-246 (2002).</li><li>Lindauer, M., 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=Net+ecosystem+exchange+over+a+non-cleared+wind-throw-disturbed+upland+spruce+forest-Measurements+and+simulations.">Net ecosystem exchange over a non-cleared wind-throw-disturbed upland spruce forest-Measurements and simulations.</a> Agricultural and Forest Meteorology. 197, 219-234 (2014).</li><li>Schulze, E. 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Principles and workflow examples for scientific and industrial applications. , (2010).</li><li>Businger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+accuracy+with+which+dry+deposition+could+be+measured+with+current+micrometeorological+techniques.">Evaluation of the accuracy with which dry deposition could be measured with current micrometeorological techniques.</a> Journal of Applied Meteorology and Climatology. 25, 1100-1124 (1986).</li><li>. Eddy Pro Software Instruction Manual Available from: <a target="_blank" href="https://www.licor.com/documents/1ium2zmwm6hl36yz9bu4">https://www.licor.com/documents/1ium2zmwm6hl36yz9bu4</a> (2017)</li><li>Wilczak, J. M., Oncley, S. P., Stage, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+anemometer+tilt+correction+algorithms.">Sonic anemometer tilt correction algorithms.</a> Boundary-Layer Meteorology. 99, 127-150 (2001).</li><li>Foken, T., Lee, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-field+quality+control.">Post-field quality control.</a> Handbook of Micrometeorology: A Guide for Surface Flux Measurements. , (2004).</li><li>Kljun, N., Rotach, M. W., Schmid, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+backward+Lagrangian+footprint+model+for+a+wide+range+of+boundary-layer+stratifications.">A three-dimensional backward Lagrangian footprint model for a wide range of boundary-layer stratifications.</a> Boundary Layer Meteorology. 103, 205-226 (2002).</li><li>Foken, T., Wichura, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+quality+assessment+of+surface-based+flux+measurements.">Tools for quality assessment of surface-based flux measurements.</a> Agricultural and Forest Meteorology. 78, 83-105 (1996).</li><li>Mauder, M., Foken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+post-field+data+processing+on+eddy+covariance+flux+estimates+and+energy+balance+closure.">Impact of post-field data processing on eddy covariance flux estimates and energy balance closure.</a> Meteorologische Zeitschrift. 15, 597-609 (2006).</li><li>Gu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+threshold+determination+for+nighttime+eddy+flux+filtering.">Objective threshold determination for nighttime eddy flux filtering.</a> Agricultural and Forest Meteorology. 128 (3-4), 179-197 (2005).</li><li>Papale, D., 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=Towards+a+standardized+processing+of+Net+Ecosystem+Exchange+measured+with+eddy+covariance+technique:+algorithms+and+uncertainty+estimation.">Towards a standardized processing of Net Ecosystem Exchange measured with eddy covariance technique: algorithms and uncertainty estimation.</a> Biogeosciences. 3 (4), 571-583 (2006).</li><li>Barr, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interannual+variability+in+the+leaf+area+index+of+a+boreal+aspen-hazelnut+forest+in+relation+to+net+ecosystem+production.">Interannual variability in the leaf area index of a boreal aspen-hazelnut forest in relation to net ecosystem production.</a> Agricultural and Forest Meteorology. 126, 237-255 (2004).</li><li>Krishnan, P., Black, T. A., Jassal, R. S., Chen, B., Nesic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interannual+variability+of+the+carbon+balance+of+three+different-aged+Douglas-fir+stands+in+the+Pacific+Northwest.">Interannual variability of the carbon balance of three different-aged Douglas-fir stands in the Pacific Northwest.</a> Journal of Geophysical Research. 114, 1-18 (2009).</li><li>Reichstein, M., 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=On+the+separation+of+net+ecosystem+exchange+into+assimilation+and+ecosystem+respiration:+Review+and+improved+algorithm.">On the separation of net ecosystem exchange into assimilation and ecosystem respiration: Review and improved algorithm.</a> Global Change Biology. 11, 1424-1439 (2005).</li><li>Falge, E., 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=Gap+filling+strategies+for+defensible+annual+sums+of+net+ecosystem+exchange.">Gap filling strategies for defensible annual sums of net ecosystem exchange.</a> Agricultural and Forest Meteorology. 107, 43-69 (2001).</li><li>Ooba, M., Hirano, T., Mogami, J. 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A., Hall, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+variation+of+carbon+dioxide+exchange+rates+above+and+below+a+boreal+jack+pine+forest.">Seasonal variation of carbon dioxide exchange rates above and below a boreal jack pine forest.</a> Agricultural and Forest Meteorology. 83, 147-170 (1997).</li><li>Lloyd, J., Taylor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Temperature+Dependence+of+Soil+Respiration.">On the Temperature Dependence of Soil Respiration.</a> Functional Ecology. 8, 315-323 (1994).</li><li>Lasslop, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+net+ecosystem+exchange+into+assimilation+and+respiration+using+a+light+response+curve+approach:+critical+issues+and+global+evaluation.">Separation of net ecosystem exchange into assimilation and respiration using a light response curve approach: critical issues and global evaluation.</a> Global Change Biology. 16, 187-208 (2010).</li><li>Kljun, N., Calanca, P., Rotach, M. 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The authors would like to mention, that presented protocol is mostly a simplification of a well-known and widely described issues regarding EC measurements. All sufficient references were given when required. Our main aim was to promote the use of this method, as well as our new and unique adjustable, electrically operated mast for EC measurements, among non-specialists with a step-by-step approach. We hope, that it makes it easier to realize and imagine that however strict requirements need to be met, EC technique can be satisfactorily applied also in non-typical, spatially limited ecosystems. With already broad literature concerning EC theory and methodology, presented protocol can potentially also be an encouragement to further knowledge acquisition on this subject.

This protocol presents the eddy covariance (EC) method to be used at non-ideal sites (here a reforested windthrow site): site location and measuring infrastructure setup, net CO2 fluxes computation and post-processing, as well as some issues regarding gap filling and fluxes partitioning procedures.
Even though the EC technique is commonly used at many measuring sites around the world, most of them are non-disturbed ecosystems, where the design and the following data processing can b...

Nowadays, the most commonly used technique in the atmosphere-land ecosystem carbon dioxide (CO2) exchange studies is the eddy covariance (EC) technique1. The EC method has been used for decades, and comprehensive descriptions of issues concerning all the methodological, technical and practical aspects have already been published2,3,4. Compared with other techniques used for similar purposes, the EC method allows for obtaining the spatially and temporally averaged net CO2 fluxes from automatic, point measurements that consider the contribution of all elements in complicated ecosystems, instead of laborious, manual measurements (e.g., chamber techniques) or the requirement of taking many samples1.
Among land ecosystems, forests play the most significant role in C cycling and many scientific activities have focused on investigating their CO2 cycle, carbon storage in woody biomass and their mutual relationships with changing climatic conditions by both direct measurement or modeling5. Many EC sites, including one of the longest flux records6, were set up above different types of forests7. Usually, the site location was carefully chosen before the measurements started, with the goal of the most homogenous and largest area possible. Although, in disturbed forest sites, such as windthrows, the number of EC measuring stations are still insufficient8,9,10. One reason is logistical difficulties in measuring site setup and, most of all, a small number of suddenly appearing locations. In order to obtain the most informative results at windthrow areas, it is crucial to start as soon as possible after such an incidental event, which may cause additional problems. In contrast to untouched forest sites, the EC measurements at windthrow sites are more challenging and can deviate from already established procedures3. Since some extreme wind phenomena create spatially limited areas, there is a need for a thoughtful measuring station location and careful data processing in order to derive as much reliable flux values as possible. Similar difficulties in EC method application have occurred (e.g., Finish studies performed above a long but narrow lake) where measured CO2 fluxes required rigorous data filtering11,12 in order to assure their spatial representativeness.
Hence, the presented protocol is an example of the use of the EC method at non-typical locations, designed not only for windthrow areas, but for all other types of short vegetation with the limited area (e.g., croplands situated between taller vegetation types). The biggest advantage of the proposed methodology is a general description of complicated procedures, requiring advanced knowledge, from the site location choice and instrumentation set up to the final outcome: a complete dataset of high-quality CO2 fluxes. The technical novelty of the measuring protocol is the use of a unique base construction for the EC system placement (e.g., tripod with a defined height that is a &#8220;mini- tower&#8221; with an adjustable, electrically operated mast, allowing changing the final height of sensors according to individual needs).

<table>
	<tbody>
		<tr>
			<td>Adjustable mast with metal rails and electric engine (24 V)</td>
			<td>maszty.net</td>
			<td>-</td>
			<td>Alternative basic construction. To be designed and made by professionals</td>
		</tr>
		<tr>
			<td>EddyPro</td>
			<td>LI-COR, Inc.</td>
			<td>ver. 6.2.0.</td>
			<td>Free commercial software for fluxes calculation. Available on a website: https://www.licor.com/env/products/eddy_covariance/software.html, on request</td>
		</tr>
		<tr>
			<td>Enclosed-path infrared gas analyzer</td>
			<td>LI-COR, Inc.</td>
			<td>LI-7200</td>
			<td>One of two instruments of the eddy covariance system (EC) used for CO2 fluxes measurements. Other types of fast analyzers (&#62;10Hz sampling frequency) can be used</td>
		</tr>
		<tr>
			<td>REddyProc</td>
			<td>-</td>
			<td>-</td>
			<td>Free software for EC fluxes gap filling and partitioning. Available on Max Planck Institute for Biogeochmistry: https://www.bgc-jena.mpg.de/bgi/index.php/Services/REddyProcWeb. Both online tool and R package are provided.</td>
		</tr>
		<tr>
			<td>Short aluminum tower base with concrete foundation</td>
			<td>maszty.net</td>
			<td>-</td>
			<td>Alternative basic construction (pioneering solution). To be designed and made by professionals</td>
		</tr>
		<tr>
			<td>Sonic anemometer</td>
			<td>Gill Instruments</td>
			<td>Gill Windmaster</td>
			<td>One of two instruments of the eddy covariance system (EC) used for wind speed measurements. Other types of three-dimensional sonic anemometers can be used</td>
		</tr>
		<tr>
			<td>Stainless-steel tripod</td>
			<td>Campbel Scientific, Inc.</td>
			<td>CM110 10 ft</td>
			<td>The basic construction for eddy covariance (EC) system. Can be constructed by yourself- materials to be found in a hardware store</td>
		</tr>
		<tr>
			<td>Sunshine sensor</td>
			<td>Delta-T Devices Ltd.</td>
			<td>BF5</td>
			<td>One of the exemplary instruments for photosynthetic photon flux density measurements (PPFD). To be bought from several commercial companies. Remember to place it above the canopy, far from reflective surfaces.</td>
		</tr>
		<tr>
			<td>Thermistors</td>
			<td>Campbel Scientific, Inc.</td>
			<td>T107</td>
			<td>One of the exemplary instruments for soil temperature measurements. To be bought from several commercial companies. It is advisable to have a profile of soil temperature</td>
		</tr>
		<tr>
			<td>Thermohygrometer</td>
			<td>Vaisala Oyj</td>
			<td>HMP155</td>
			<td>One of the exemplary instruments for air temperature and humidity measurements. To be bought from several commercial companies. Remember to place it inside radiation shield at similar height as the EC system.</td>
		</tr>
	</tbody>
</table>

measurements of co2 fluxes at non-ideal eddy covariance sites

This protocol is an example of utilizing the eddy covariance (EC) technique to investigate spatially and temporally averaged net CO2 fluxes (net ecosystem production, NEP), in non-typical ecosystems, on a currently reforested windthrow area in Poland. After a tornado event, a relatively narrow &#8220;corridor&#8221; was created within surviving forest stands, which complicates such kind of experiments. The application of other measuring techniques, such as the chamber method, is even more difficult under these circumstances, because especially at the beginning, fallen trees and in general great heterogeneity of the site provide a challenging platform to perform flux measurements and then to properly upscale obtained results. In comparison with standard EC measurements carried out in untouched forests, the case of windthrow areas requires special consideration when it comes to the site location and data analysis in order to ensure their representativeness. Therefore, here we present a protocol of real-time, continuous CO2 flux measurements at a dynamically changing, non-ideal EC site, which includes (1) site location and instrumentation setup, (2) flux computation, (3) rigorous data filtering and quality control, and (4) gap filling and net fluxes partitioning into CO2 respiration and absorption. The main advantage of the described methodology is that it provides a detailed description of the experimental setup and measurement performance from scratch, which can be applied to other spatially limited ecosystems. It can also be viewed as a list of recommendations on how to deal with unconventional site operation, providing a description for non-specialists. Obtained quality-checked, gap filled, half-hour values of net CO2, as well as absorption and respiration fluxes, can be finally aggregated into daily, monthly, seasonal or annual totals.

One of the crucial steps in flux filtering and quality control at non-ideal EC sites is the assessment of the measured fluxes&#8217; spatial representativeness. The simplest way to perform such analysis, given the fact that calculations were done using commercial, widely applied software, is to include measurements from desired area only, on the basis of wind direction and footprint estimations (see section 3.7). Thus, the wind rose plot, with a chosen wind direction and maximal acceptabl...

Watch this Scientific Journal Video about Measurements of CO2 Fluxes at Non-Ideal Eddy Covariance Sites at JoVE.com

Measurements of CO2 Fluxes at Non-Ideal Eddy Covariance Sites

The presented protocol uses the eddy covariance method at non-typical locations, applicable to all types of short-canopy ecosystems with limited area, on a currently reforested windthrow site in Poland. Details of measuring site setup rules, flux calculations and quality control, and final result analysis, are described.

Measurements of CO2 Fluxes at Non-Ideal Eddy Covariance Sites

This protocol is an example of utilizing the eddy covariance (EC) technique to investigate spatially and temporally averaged net CO2 fluxes (net ecosystem ...

measurements-of-co2-fluxes-at-non-ideal-eddy-covariance-sites

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7.8K Views.  Poznan University of Life Sciences. The presented protocol uses the eddy covariance method at non-typical locations, applicable to all types of short-canopy ecosystems with limited area, on a currently reforested windthrow site in Poland. Details of measuring site setup rules, flux calculations and quality control, and final result analysis, are described.

Video: Measurements of CO2 Fluxes at Non-Ideal Eddy Covariance Sites

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

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 ...

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% ...

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 ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

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 ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...