We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

1. Preparation of Glassware and Filter Housings
<ol style="margin-left: 40px;">
	<li>Unless otherwise noted, sterilize all glassware, filter housings and solutions at 121 &#176;C for 15 min. To ensure sterility, cover all openings or exposed surfaces with either aluminum foil or tape-secured paper prior to sterilization.</li>
	<li>Assemble filtration apparatus by attaching filter housing (47 mm diameter) to 1 L side arm Erlenmeyer flask. Collect filters required: 47 mm diameter electropositive/electronegative disc filters.</li>
	<li>Prepare 1 L of 1.5% beef extract (flocculating; produces floc particles when pH of solution is lowered to 3.5, or non-flocculating; which does not aggregate at pH 3.5), with 0.05 M glycine, by dissolving 15 g of beef extract in 1 L of deionized water and adding 3.75 g glycine. 
	NOTE: Non-flocculating beef extracts will require the addition of celite prior to tertiary concentration.</li>
	<li>Add elution additive sodium polyphosphate to beef extract at a 0.1% concentration.</li>
	<li>Add 1 M hydrochloric acid solution, drop wise, to mixing beef extract, pH solution to desired pH. Autoclave beef extract for 15-30 min at 121 &#176;C.</li>
	<li>Measure 0.1 g of fine, medium, or large particle celite (for use with non-flocculating beef extract only).</li>
	<li>Prepare vacuum/suction by attaching compatible tubing to Erlenmeyer side-arm flask and to the vacuum outlet.</li>
</ol>
2. Preparation of Solutions and Virus Stock
<ol style="margin-left: 40px;">
	<li>Prepare 1 L of 1 M HCl.</li>
	<li>Prepare 1 L of 1 M NaOH.</li>
	<li>Prepare 1x PBS solution (137 mM NaCl, 2.7 mM KCl, 1.47 mM K2PO4, and 4.3 mM NaH2PO4).
	<ol>
		<li>Adjust pH of 1x PBS to 9.0 using 1 M NaOH.</li>
	</ol>
	</li>
	<li>Prepare and dilute stock virus to 104-105 most probable number (MPN) ml-1 by mixing 1 ml of stock virus with 9 ml of PBS (pH 7), as previously described 8,13. Store at -80 &#176;C in 1 ml aliquots.
	<ol>
		<li>Sterilize virus stock by syringe filtering (0.22 &#956;m pore size) to remove potential contaminants.</li>
	</ol>
	</li>
	<li>Dechlorinated Tap Water
	<ol>
		<li>Measure 1 L of sterile tap water using a sterile graduated cylinder and pour into a 2 L beaker containing magnetic stir bar.</li>
		<li>Add 0.7 g of sodium thiosulfate and mix until granules dissolve. Adjust pH of tap water to 7-7.5 if needed using 1 M HCl or 1 M NaOH solution.</li>
		<li>Thaw virus spike (1 ml) and add entire volume of spike to 1 L of dechlorinated sterile tap water. Mix for 10 min.</li>
		<li>Prepare a method blank (dechlorinated tap water without adenovirus) with each experiment and process in the same manner as the spiked sample.</li>
	</ol>
	</li>
</ol>
3. Preparation of Millipore Filter Apparatus
<ol style="margin-left: 40px;">
	<li>Remove sterile covering over filter housing. Ensure proper fit between filter housing upper bowl and lower filter screen to avoid sample leakage during filtration.</li>
	<li>Remove sterile covering from 1 L Erlenmeyer flask. Ensure that the cork of the filter unit fits snugly in top opening of Erlenmeyer flask, creating a good seal.</li>
	<li>Remove top bowl of filter unit from lower filter screen, and place 47 mm electropositive filter disk squarely over screen with sterile forceps. Attach filter housing bowl to bottom screen and lock into place by twisting counterclockwise.</li>
</ol>
4. Tap Water Filtration (Primary Concentration)
<ol style="margin-left: 40px;">
	<li>Measure 100 ml of virus spiked tap water using sterile graduated cylinder.</li>
	<li>Pour 100 ml of virus spiked tap water into filter housing containing either a 47 mm pleated glass/cellulose filter or a nano-alumina/glass fiber disk filter.</li>
	<li>Allow water to slowly pass through filter. Apply gentle vacuum to remove remaining residuals of tap water from filter surface.</li>
	<li>If using trypsin treatment, add 5 ml of 0.25% trypsin to filter surface and incubate for 5 min.</li>
	<li>Wash trypsin from filter surface using 50 ml of sterile deionized water.</li>
</ol>
5. Virus Elution from Filter
<ol style="margin-left: 40px;">
	<li>Remove filter housing from 2 L Erlenmeyer flask, and place onto 250 ml side-arm flask.</li>
	<li>Measure 100 ml of beef extract using graduated cylinder. Slowly pour 100 ml of beef extract into filter housing.</li>
	<li>Allow beef extract to pour through filter, capturing beef extract in a small (250 ml) Erlenmeyer flask. Apply vacuum to pull through residual beef extract from filter.</li>
</ol>
6. Celite Secondary Concentration
<ol style="margin-left: 40px;">
	<li>Transfer flasks containing the eluted viruses to a stir plate. Add sterile magnetic stir bar and place on stir plate, mixing to create slight vortex.</li>
	<li>Add 0.1 g of celite powder to 100 ml of beef extract and allow celite to disperse. Place sterile pH probe into beef extract.</li>
	<li>Add drop wise 1 M HCl to beef extract to achieve pH 4.0. Allow to slowly mix for 10 min.</li>
	<li>Place 47 mm pre-filter onto filter housing (as previously described) using a 2 L Erlenmeyer flask.</li>
	<li>Pour 100 ml beef extract/celite mixture over pre-filter. Allow beef extract to pour through. Apply slight vacuum to pull through residual beef extract from filter.</li>
	<li>Place metal tube clip on end of filter housing spout. Attach sterile 15 ml polypropylene collection tube to metal tube clip. Place filter housing back into Erlenmeyer flask.</li>
	<li>Add 5 ml of 1x PBS buffer, pH 9.0 over celite collected on pre-filter.</li>
	<li>Allow PBS to drip through pre-filter. Apply slight vacuum to pull through residual PBS into 15 ml collection tube.</li>
</ol>
7. Organic Flocculation Secondary Concentration
<ol style="margin-left: 40px;">
	<li>Transfer the beakers containing the eluted virus to a stir plate. Add sterile magnetic stir bar to 100 ml of beef extract and mix to create slight vortex.</li>
	<li>Place sterile pH probe into beef extract.</li>
	<li>Add 1 M HCl drop wise to beef extract and adjust pH to 3.5. Mix for 30 min.</li>
	<li>Pour the eluate into 250 ml centrifuge conical tubes and centrifuge at 2,500 x g for 15 min at 4 &#186;C.</li>
	<li>Pour off supernatant taking care not to not to disrupt the pellet. Resuspend the pellet in 5 ml of 1x PBS, pH 9.</li>
	<li>Centrifuge the suspension at 4,000 x g for 10 min at 4 &#186;C. Pour off the supernatant and discard the pellet.</li>
	<li>Adjust the pH to 7-7.5, filter sterilize through 0.22 &#181;m syringe filter and freeze at -80 &#176;C.</li>
</ol>
8. Viral Nucleic Acid Extraction
<ol style="margin-left: 40px;">
	<li>Extract viral nucleic acids using an appropriate commercial kit according to manufacturer&#8217;s instructions.</li>
</ol>
9. AdV 40/41 qPCR
<ol style="margin-left: 40px;">
	<li>Use primers and probes, as well as reaction mix and thermal cycling profiles as published 8,13. 
	NOTE: Estimated limit of detection for the assay is approximately 5 qPCR units per reaction.</li>
	<li>Add components of PCR amplification mixtures at concentrations as follows: 10 mM Tris (pH 8.3), 50 mM KCl, 4.5 mM MgCl2, 10 mM dNTPs, 10 &#181;M primers and 1 &#181;M probe and 0.5 &#181;l of polymerase so that final volumes of 45 &#181;l can be added to appropriate number of wells within a 96-well PCR reaction plate.</li>
	<li>Load 45 &#181;l of PCR amplification mixture to each appropriate well of a 96-well, fast reaction PCR plate.</li>
	<li>Add 5 &#181;l of DNA extracted sample to appropriate wells of the PCR plate.</li>
	<li>Cover plate with heat resistant cover seal, spin the plate to move any droplets to bottom of PCR plate wells.</li>
	<li>Set PCR amplification cycles as follows: 95 &#176;C for 10 min, followed by 40 cycles of 95 &#176;C for 15 sec and 60 &#176;C for 1 min.</li>
</ol>
10. Data Analyses
<ol style="margin-left: 40px;">
	<li>Analyze triplicate measurements of the serial 5-fold dilutions of the samples spanning 1:5 to 1:625 dilution range in order to determine the most probable number (MPN) of the viral particles, as previously described 8,13.</li>
	<li>Analyze serial 5-fold dilutions of adenovirus experimental spikes spanning 1:5 to 1:15,625 dilution range in order to determine the most probable number (MPN) of the viral particles.</li>
	<li>Using the EPA MPN calculator (http://www.epa.gov/nerlcwww/online.html), adjust the calculator to account for type of dilution (1:5 or 1:10), number of dilutions performed, and number of replicate samples in dilution series.</li>
	<li>Perform log10 transformation of the data, followed by normalizing to a unit of volume (e.g., 1 ml, 100 ml, etc.). Calculate recoveries for given experiment by dividing number of viral particles present in sample concentrates with number of viral particles present in experimental spikes.</li>
	<li>Evaluate the effect of the different experimental variables (e.g., different celite types, pH ranges, different filter types and different secondary concentration techniques) on AdV recoveries by performing parametric or non-parametric statistical tests (e.g., paired t-test, one or two-way ANOVA) depending on the distribution of the data and the experimental design.</li>
</ol>

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No competing financial interests. 
The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Electropositive filters are useful in concentrating viruses from water; however these filters can differ in their structure and composition which could in turn alter their effectiveness. Compounding this problem, capsid structures and charges vary between virus strains requiring concentration techniques be tailored to ensure optimal recovery 15. Through simple modifications of the existing concentration techniques (e.g., electropositive filters, beef extract elution), more effective concentration of t...

Human enteric viruses are important causative agents of waterborne diseases 1-3, but are generally present in low numbers in contaminated environmental waters, making their detection difficult without concentration. Procedures used to concentrate viruses typically include a filtration step, followed by filter elution, and secondary concentration of the filter eluate. A common filtration procedure relies on use of charged membranes such as electropositive filters (recently reviewed in 4,5). These filters rely on capturing viruses suspended in water using electrostatic interactions between the filter surface (positively charged) and targeted virus particles (negatively charged). Two electropositive filters that are commercially available rely on this technology, the glass/cellulose and nano-alumina/glass fiber filters. The glass/cellulose filter costs are up to 10 times that of the nano-alumina/glass fiber, which limit the use of the glass/cellulose filters for routine virus monitoring. Recent studies have concluded differences are nominal between these two filters in recovery of enteroviruses from ambient water 6,7, justifying the use of a cheaper filter alternative. Other filter options such as electronegative and glass-wool filters have been studied, however, they either require the pretreatment of source water (electronegative filters) or are not commercially available (glass-wool filters). The development of virus concentration procedures has mostly focused on optimizing primary concentration techniques (filters) in order to improve virus recoveries from water. However, secondary concentration procedures, which reduce the volume of eluant typically from 1 L to milliliter volumes, can also have a significant impact on virus recoveries 8.
Secondary concentration of enteric viruses typically relies on a flocculating agent such as some types of beef extract (organic flocculation) or skimmed milk flocculation 9-12 to remove virus particles from filter surfaces. Recently, another secondary concentration procedure using beef extract coupled with the addition of celite (fine particle) has shown promise for recovering adenovirus, enterovirus, and norovirus 8,13,14. Celite concentration works under similar principles to that of the organic flocculation method in that virus particles attach to and are released from particles (floc or celite) by altering the pH of the suspension solution. Comparisons between these two secondary concentration techniques have been evaluated in recovery of spiked adenovirus (AdV) types 40 and 41 8. This study concluded that the two secondary concentration techniques were statistically similar in recovery of adenoviruses. However, the organic flocculation method requires a 30 min. incubation at pH 3.5, while the celite technique requires a shorter incubation (10 min) at pH 4.0. The organic flocculation also requires the use of expensive laboratory equipment (centrifuges) to collect floc particles during tertiary concentration, the celite technique in contrast uses only basic laboratory equipment (vacuum filtration) to separate celite particles from suspension.
Certain combinations of filters and secondary elution techniques can also affect virus recoveries. One study concluded that certain combinations of primary (electropositive filters) and secondary concentration techniques (celite or organic flocculation) had a significant impact of recovery of adenovirus 13. These findings suggest that optimization is required in order to optimally recover target virus from a given water matrix when using these techniques. Optimization is a time consuming, arduous process many researchers actively avoid since numerous variables will be evaluated (filter type/brand, pH elution solution, celite/organic flocculation).
For this study, a procedure was developed to identify optimal conditions for virus concentration from water using spiked human adenovirus strains 40 and 41. Presumably, since each virus type displays a unique capsid morphology and specific capsid charge, concentration protocols may need to be optimized for every virus target in order to attain optimal viral recovery. This study provides an approach for AdV 40 and 41 concentration by: 1) evaluating virus recoveries in tap water using electropositive filter discs followed by 2) evaluation of an established organic flocculation method versus the celite technique as a secondary concentration, and 3) evaluation of elution buffers for tertiary concentration.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:295px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 40 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 41 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Thiosulfate</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">72051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Celites #577</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">22142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">NanoCeram 47mm</td>
			<td style="width:143px;">Argonide</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1MDS 47mm</td>
			<td style="width:143px;">3M</td>
			<td style="width:119px;">6408502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AP-20 Prefilter 47mm</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td style="width:119px;">AP2004700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glycine&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">50046-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Polyphosphate</td>
			<td style="width:143px;">Acros Organics</td>
			<td style="width:119px;">390930010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypsin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">PBS</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hydrochloric Acid</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">A481-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">BBL Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">212303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Difco Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">211520</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:231px;">ABI 7900 Real-time PCR system</td>
			<td style="width:143px;">ABI</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stainless Steel Filter Housing</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td>XX2004720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Blood DNA Extraction Kit</td>
			<td style="width:143px;">Qiagen&#160;</td>
			<td style="width:119px;">51104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EPA MPN Calculator</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.epa.gov/nerlcwww/online.html</td>
		</tr>
	</tbody>
</table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

Celite selection
Three different types of celite were tested prior to the selection of the best performing variant. Celites with fine to medium sized particles produced the highest adenovirus recoveries. Use of larger celites resulted in lower recoveries for both AdV40 and 41 (range 32%-100%) (Figure 1). Average recoveries of adenovirus 40 were 144% &#177; 52% (fine), 115% &#177; 28% (medium) and 82% &#177; 53% (large) particle celites and for AdV41, 132% &#177; 39% (fine), 83...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

a-small-volume-procedure-for-viral-concentration-from-water

Research

JoVE Journal

Environment

10.0K Views.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

Video: A Small Volume Procedure for Viral Concentration from Water

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

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

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

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

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

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 ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

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

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

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

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...