Max Planck Institute for Dynamics of Complex Technical Systems assisted in meeting the publication costs of this article.

1. Material preparation
<ol>
	<li>Cut and perforate two rectangular pieces of Teflon of the same size as the end plates by using a cutting press; take care and ensure that the holes are in the exact position where the bolts should be placed.</li>
	<li>Using the same procedure cut Teflon gaskets considering the outer and inner dimensions of the flow field, and the position of the holes where the screws should be placed.</li>
	<li>Cut the gas diffusion layers using a metal frame fitting the size of the gaskets.</li>
	<li>Cut the excess Nafion from the catalyst coated membrane (CCM) in order to adjust it to the size of the bipolar plates. Make holes in the membrane at the positions where the screws should go through with the help of the metal frame used previously. Take care to center the frame before making the holes.</li>
</ol>
2. Fuel cell assembly
<ol>
	<li>Place the cathode bipolar plate on a smooth and sturdy surface with the flow field side up.</li>
	<li>Place the gasket on top. Make sure it aligns with the screw holes.</li>
	<li>Place the cathode GDL in the middle of the gasket and put the CCM on top. Make sure the CCM is aligned with the screw holes.</li>
	<li>Place the anode GDL and gasket on top. Make sure the gasket aligns with the screw holes and the GDL is placed in the middle.</li>
	<li>Place the anode bipolar plate on top (flow field side down) and use screws to clamp the parts together. 
	NOTE: The bipolar plates must not be strongly tightened. The purpose of the screws is just to keep aligned the different parts.</li>
	<li>Place the cathode stainless steel end plate on a smooth and sturdy surface.</li>
	<li>Place the rectangular Teflon piece and the copper current collector on top. Make sure they align with the bolt holes.</li>
	<li>Slot the cathode side of the cell unit assembled in step 2.1 on the cathode current collector taking into account the notches in the flow fields.</li>
	<li>Slot the anode side of the unit on the anode current collector, position the Teflon gaskets and finish with the anode stainless steel end plate on top.</li>
	<li>Place the insulating sleeves, the O-ring and the bolts in the holes of the anode end plates; insert the bolts in the holes.</li>
	<li>Position the insulation sleeves and the O-ring; finish by placing the nuts on the bolts on the cathode side.</li>
	<li>Tighten the bolts crosswise using a torque-wrench until you reach the recommended torque value of 5 N&#183;m. 5 crosswise cycles are suggested; start by low torque value (1 N&#183;m) and increase by 1 N&#183;m in each subsequent cycle.</li>
</ol>
3. Integration of a fuel cell with the periphery
<ol>
	<li>Place the fuel cell in the heating box and connect the inlets and outlets to the periphery. Use snoop liquid to check for leakages.</li>
	<li>Insert the thermocouple in the cathode end plate.</li>
	<li>Interface the fuel cell with the potentiostat; choose 2 electrode-configuration. Connect the cables marked as RE and CE to the anode side and the ones marked as WE and SE to the cathode side.</li>
	<li>Start the software used to control the cell periphery; a scheme of the experimental setup is visualized (see schematic in Figure 1). Choose the values of the anode and cathode inlet gas flow rates and open the valves. In the experiments shown in this protocol, flow rates of 850, 300 and 300 mL/min were used for hydrogen (anode side), nitrogen and oxygen (cathode side) respectively.</li>
	<li>Choose the temperature of the inlet gases and turn on the heating tapes. Wait until the set point temperature is reached. In all experiments in this protocol, the set point temperature of the inlet gases at the anode and cathode side was 68 &#176;C.</li>
	<li>Set the temperatures of the thermostats to define the desired dew point temperature of the inlet gases; turn the thermostats on.</li>
	<li>Set the chosen temperature of the fuel cell on the control panel of the heating box. Then, turn the heating on. In the experiments described in this protocol a fuel cell temperature of 80 &#176;C was set.</li>
	<li>Wait until the set point temperature of the fuel cell is reached; check the humidification state of the inlet gases; check the fuel cell open circuit cell potential. The open circuit cell potential value on the display of the potentiostat should be between 1 and 1.2 V.</li>
</ol>
4. Fuel cell start up procedure
NOTE: The procedure described in the following section uses a specific software program and potentiostat (Autolab N104, NOVA 2.0 software). However, it can be also performed using other software and potentiostats without changing the main outcomes. The start-up procedure must be performed if a new CCM is used.
<ol>
	<li>Start the Autolab NOVA 2.0 software.
	<ol>
		<li>Select New Procedure in the Action section of the software; the procedure editing page opens.</li>
		<li>In Command, click on the Autolab Control icon ; drag the Autolab Control icon to the workspace section. Then, in Properties, select Mode On Potentiostatic. 
		NOTE: The Autolab NOVA 2.0 software does not differentiate between the terms potentiostatic and voltastatic.</li>
		<li>In Command, select the Cell icon and place it next to the Autolab Control icon. Then, in Properties choose Cell On. Add the Apply icon and in Properties set 0.9 V as Cell Potential with respect to the reference electrode.</li>
		<li>Add the Wait command and set Duration to 1800 s.</li>
		<li>Add the LSV Staircase command from Measurement Cyclic and Linear Sweep Voltammetry. Set the Start Potential to 0.9 V, the Stop Potential to 0.6 V, the Scan Rate to 0.4 mV/s and Step to 0.244 mV.</li>
		<li>Add the Wait command and set Duration to 1800 s.</li>
		<li>Add the LSV Staircase command from Measurement Cyclic and Linear Sweep Voltammetry. Set the Start Potential to 0.6 V, the Stop Potential to 0.9 V, the Scan Rate to 0.4 mV/s and the Step to 0.244 mV.</li>
		<li>Add the Repeat command. In the workspace select the commands from step 4.1.4 (the first Wait command) to step 4.1.7 (the last LSV Staircase command); drag and drop the icons into the Repeat box. In Properties asset the number of repetitions to 20.</li>
	</ol>
	</li>
	<li>Start the cell start up procedure by clicking the Play button.</li>
	<li>After 2 h, if the current is stable at 0.6 V stop the program by pressing on the Stop button. If the current is still changing, let the program run until it terminates.</li>
</ol>
5. Galvanostatic electrochemical impedance spectroscopy experiment
<ol>
	<li>Start the Autolab NOVA 2.0 software.
	<ol>
		<li>Select New Procedure in the Action section of the software; the procedure editing page opens.</li>
		<li>In Command click on the Autolab Control icon ; drag and drop the Autolab Control icon to the workspace section. Then, in Properties select Mode On Galvanostatic.</li>
		<li>Add the Cell On command.</li>
		<li>Add the LSV Staircase command. In Properties set the Start Current to 0 A, the chosen steady state current to Stop Current, the Scan Rate to 0.005 A/s and Step to 0.01 A.</li>
		<li>Insert the Record Signal command; in Properties set the Duration to 7200 s and the Interval Sampling Time to 0.1 s.</li>
		<li>Insert the FRA measurement command window. In Properties set the First applied frequency to 1000 Hz, the Last applied frequency to 0.01 Hz and the Number of frequencies per decade to 5. Set the Amplitude to 5% of the steady state current.</li>
		<li>Add the Cell Off command.</li>
	</ol>
	</li>
	<li>Start the cell galvanostatic EIS program by pressing the Play button.</li>
	<li>Wait until the cell potential value stabilizes by observing the change in the recording window. Then click on the Forward button to start the EIS experiment.</li>
	<li>Check the stability of the system during the experiment and wait until the program is terminated.</li>
</ol>
6. Concentration-alternating frequency response experiment
NOTE: The following instructions describe the procedure for performing cFRA experiments under galvanostatic conditions. However, the procedure would not differ if performing cFRA experiments under voltastatic conditions, apart from setting the galvanostatic to potentiostatic control in the software and fixing a certain cell potential as a steady state instead of current.
<ol>
	<li>Set up the Pyro fiber oxygen sensor for fast dynamic measurements.
	<ol>
		<li>Push gently down on the plunger in the upper part of the Pyro fiber oxygen sensor in order to remove the sensitive part of the fiber from the protective needle and place it in the center of the tubing at the cell inlet.</li>
		<li>Open the Pyro software.</li>
		<li>Click on Options | Advance and choose Enable Fast Sampling.</li>
		<li>Set the Sampling Interval to 0.15 s.</li>
	</ol>
	</li>
	<li>Edit the cFRA procedure by using Autolab NOVA 2.0 software.
	<ol>
		<li>Open the NOVA software and select New Procedure in the Action section ; the software editing page opens.</li>
		<li>In Commands select the Control icon and insert it in the workspace. In Properties select Mode On Galvanostatic. Then select the Cell On command and place it next to the Control icon.</li>
		<li>Add the LSV Staircase command from the Measurement Cyclic and Linear Sweep Voltammetry. In Properties set the Start Current to 0.0 A; set as Stop current the steady state current value at which the cFRA experiment should be performed. Then use 0.005 A/s as the Scan Rate and 0.01 A as the Step.</li>
		<li>Insert two Record Signal commands; in Properties set Duration to 7200 s and Interval Sampling Time to 0.05 s. Repeat the same step 20 times by adding a Repetition command. The number of repetitions must be equivalent to the number of signal frequencies that need to be measured. 
		NOTE: Two recording signal windows are convenient for the following reasons: one recording window is used to monitor the transient part of the periodic output signal, while the second one is used to register the steady state part of the periodic output signal. The steady state part of the signal is used for transfer function determinations.</li>
	</ol>
	</li>
	<li>Press the Play button to start the cFRA program.</li>
	<li>In the first set of repetitions, check if the cell potential reaches the steady state value by observing the recording window.</li>
	<li>Open the additional oxygen valve and set the mass flow controller to 5% of the value of the total flow rate of the main feed in order to ensure a linear response (example: set 30 mL/min with 600 mL/min of total flow rate). Then set the switching time of the valve to an initial value of 0.5 s. Press the switching control Start button.</li>
	<li>Monitor the recording window and wait until the cell potential achieves a periodic steady state; then click on the Next button.</li>
	<li>Register the periodic steady state signal in the new recording window for 60 s. Then, click again on the Next button.</li>
	<li>Simultaneously with the previous step 6.7, register the periodic oxygen input. Select the Start button in the sensor software, insert a name which recalls the frequency input (example: 1 Hz), and click on OK. Register the signal for 60 s as in the current output case and press the Stop button.</li>
	<li>Repeat the previous steps 6.6-6.8 at increasing switching time values in order to measure periodic input/output correlations for a frequency range from 8-1000 mHz by taking 8 frequency points per decade. For experiments at a frequency higher than 100 mHz, register input and output for 60 s. At lower frequencies, sample the signals for a range of time equivalent to 5 periods.</li>
</ol>
7. Analysis of cFRA data
<ol>
	<li>Export measured cell potential responses from the Autolab NOVA 2.0 software.
	<ol>
		<li>In the recording window click on the diagram with the measured periodic steady state cell potential output.</li>
		<li>Click on the Show Data | Key | Export buttons. Insert a file name which recalls the frequency of the input (Example: 1 Hz) and click on Save.</li>
		<li>Repeat steps 7.1.1-7.1.2 for each measured cell potential output at each frequency.</li>
	</ol>
	</li>
	<li>Open the Matlab scripts FFT_input.mat and FFT_output.mat. In the Address Folder section insert the specifications of the location of the folder where the measured oxygen pressure and current data files are stored. 
	NOTE: The script was written with the aim of performing the windowing of the collected inputs in order to have an integer number of periodic cycles to analyze, and calculate their Fourier transforms accurately and quickly. Any other procedure which performs the same task does not change the results.</li>
	<li>Run the FFT_PO2.mat and FFT_Pot.mat scripts; check in the plotted diagrams if the computed algorithm works properly (in the time domain, an integer number of input and output cycles should be extracted from the original input and output samples). 
	CAUTION: A Fourier transform based on a non-integer number of periodic cycles could result in misleading analysis of the inputs and outputs resulting in inaccurate cFRA spectra.</li>
	<li>Open the Matlab script cFRA_spectra.mat and run it. Magnitude, phase angle and Nyquist spectra of the cFRA transfer function under galvanostatic conditions are plotted. 
	NOTE: The script calculates the cFRA transfer function by using the Fourier transform values at the fundamental frequency of the oxygen pressure (inputs) and cell potential (outputs) signal using the following equation. 
<img src="/files/ftp_upload/60129/60129eq1.jpg" /></li>
</ol>

<ol>
	<li>Yuan, X., Wang, H., Sun, J. C., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AC+impedance+technique+in+PEM+fuel+cell+diagnosis+-+a+review.">AC impedance technique in PEM fuel cell diagnosis - a review.</a> International Journal of Hydrogen Energy. 32 (7), 4365-4380 (2007).</li><li>Niya, S. M. R., Hoorfar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+proton+exchange+membrane+fuel+cells+using+electrochemical+impedance+spectroscopy+technique+-+a+review.">Study of proton exchange membrane fuel cells using electrochemical impedance spectroscopy technique - a review.</a> Journal of Power Sources. 240 (8), 281-293 (2013).</li><li>Niroumand, A. M., Merida, W., Eikerling, M., Safi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+voltage+oscillations+as+diagnostic+tool+for+PEFC+cathode.">Pressure voltage oscillations as diagnostic tool for PEFC cathode.</a> Electrochemistry Communications. 12 (1), 122-124 (2010).</li><li>Engebretsen, 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=Electro-thermal+impedance+spectroscopy+applied+to+an+open-cathode+polymer+electrolyte+fuel+cell.">Electro-thermal impedance spectroscopy applied to an open-cathode polymer electrolyte fuel cell.</a> Journal of Power Sources. 302, 210-214 (2014).</li><li>Engebretsen, E., Mason, T. J., Shearing, P. R., Hinds, G., Brett, D. J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+pressure+impedance+spectroscopy+applied+to+the+study+of+polymer+electrolyte+fuel+cells.">Electrochemical pressure impedance spectroscopy applied to the study of polymer electrolyte fuel cells.</a> Electrochemistry Communications. 75, 60-63 (2016).</li><li>Sorrentino, A., Vidakovi&#263;-Koch, T., Hanke-Rauschenbach, R., Sundmacher, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+frequency+response+analysis:+A+new+method+for+studying+polymer+electrolyte+membrane+fuel+cell+dynamics.">Concentration frequency response analysis: A new method for studying polymer electrolyte membrane fuel cell dynamics.</a> Electrochimica Acta. 243, 53-64 (2017).</li><li>Sorrentino, A., Vidakovi&#263;-Koch, T., Sundmacher, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+mass+transport+dynamics+in+polymer+electrolyte+membrane+fuel+cells+using+concentration-alternating+frequency+response+analysis.">Studying mass transport dynamics in polymer electrolyte membrane fuel cells using concentration-alternating frequency response analysis.</a> Journal of Power Sources. 412, 331-335 (2019).</li><li>Pivac, I., Barbir, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+phenomena+at+low+frequencies+in+impedance+spectra+of+proton+exchange+membrane+fuel+cells-A+review.">Inductive phenomena at low frequencies in impedance spectra of proton exchange membrane fuel cells-A review.</a> Journal of Power Sources. 326, 112-119 (2016).</li><li>Benziger, J., Chia, J. E., Kimbal, E., Kevrekidis, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+Dynamics+in+a+Parallel+Flow+Channel+PEM+Fuel+Cell.">Reaction Dynamics in a Parallel Flow Channel PEM Fuel Cell.</a> Journal of Electrochemical Society. 154, B835-B844 (2007).</li><li>Rannow, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+Efficient+Control+of+Hydraulic+Systems+Using+On/Off+Valves.">Achieving Efficient Control of Hydraulic Systems Using On/Off Valves.</a> Doctoral Dissertation. , (2016).</li></ol>

The authors have nothing to disclose.

In contrast to classical EIS, cFRA is a diagnostic tool focused on the characterization of dynamics related to the different mass transport phenomena occurring in the fuel cell. It is not able to detect any transients having a time constant below the oxygen diffusion in the electrode, as for example the charging/discharging of the double layer6. Therefore, unlike EIS where several phenomena are coupled, cFRA can help to identify patterns related to specific dynamics more clearly. This would decrea...

Characterizing the dynamic behavior of a PEM fuel cell is important in order to understand which mechanisms dominate the transient operational states lowering the performance of the cell. Electrochemical impedance spectroscopy (EIS) is the most commonly used methodology for studying PEM fuel cell dynamics, due to its ability to separate different process contributions to the overall dynamic performance1,2. However, transient processes with similar time constants are often coupled in the EIS spectra, making it difficult to interpret them. For this reason, in the past transient diagnostic tools based on the application of non-electrical inputs with the aim of detecting the impact of a few or individual dynamics have been developed and proposed3,4,5,6,7.
A novel frequency response technique based on concentration perturbation input and electrical outputs named concentration-alternating frequency response analysis (cFRA) has been developed in our group. The potential of cFRA as a selective diagnostic tool has been investigated theoretically and experimentally6,7. It was found that cFRA can separate different kinds of mass transport phenomena and discriminate between the different states of operation of the cell. In this protocol, we focus on the step-by-step description of the procedure for performing cFRA experiments. The assembling of the cell, its conditioning and the experimental setup for creating a feed with periodic concentration perturbation, as well as the data analysis will be shown and discussed in detail. Finally, the most critical points of the procedure will be highlighted and several strategies for improving the quality and selectivity of cFRA spectra will be pinpointed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Membrane Electrode Assemby N115 25,8 cm2</td>
			<td>QuinTech</td>
			<td>EC-NM-115</td>
			<td>cathode/anode loding: 1mg Pt/cm2</td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>Metrhohm</td>
			<td>PGSTAT302N</td>
			<td></td>
		</tr>
		<tr>
			<td>Booster</td>
			<td>Metrohm</td>
			<td>BOOSTER20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Retractable fiber oxygen sensor</td>
			<td>Pyro Science</td>
			<td>OXR430-UHS</td>
			<td></td>
		</tr>
		<tr>
			<td>Dew Point and Temperature Meter</td>
			<td>VAISALA</td>
			<td>DMT340</td>
			<td></td>
		</tr>
		<tr>
			<td>Software process control system</td>
			<td>Siemens</td>
			<td>Simatic PCS 7</td>
			<td></td>
		</tr>
		<tr>
			<td>Software MATLAB2012a</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen</td>
			<td>Linde</td>
			<td>Hydrogen 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>Linde</td>
			<td>Nitrogen 5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Linde</td>
			<td>Oxygen 5.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

a guide to concentration alternating frequency response analysis of fuel cells

An experimental setup capable of generating a periodic concentration input perturbation of oxygen was used to perform concentration-alternating frequency response analysis (cFRA) on proton-exchange membrane (PEM) fuel cells. During cFRA experiments, the modulated concentration feed was sent to the cathode of the cell at different frequencies. The electric response, which can be cell potential or current depending on the control applied on the cell, was registered in order to formulate a frequency response transfer function. Unlike traditional electrochemical impedance spectroscopy (EIS), the novel cFRA methodology makes it possible to separate the contribution of different mass transport phenomena from the kinetic charge transfer processes in the frequency response spectra of the cell. Moreover, cFRA is able to differentiate between varying humidification states of the cathode. In this protocol, the focus is on the detailed description of the procedure to perform cFRA experiments. The most critical steps of the measurements and future improvements to the technique are discussed.

The preliminary analysis of the fuel cell dynamics based on EIS spectra is shown in Figure 2. EIS magnitude (Figure 2A) and phase Bode plots (Figure 2B) spectra are measured at three different steady state current densities under galvanostatic control. As expected, all main transient processes are observed: the double layer charging/discharging in the high frequency ...

Watch this Scientific Journal Video about A Guide to Concentration Alternating Frequency Response Analysis of Fuel Cells at JoVE.com

A Guide to Concentration Alternating Frequency Response Analysis of Fuel Cells

We present a protocol for concentration alternating frequency response analysis of fuel cells, a promising new method of studying fuel cell dynamics.

An experimental setup capable of generating a periodic concentration input perturbation of oxygen was used to perform concentration-alternating frequency ...

a-guide-to-concentration-alternating-frequency-response-analysis-fuel

Research

Education

JoVE Core

JoVE Journal

Electrical Engineering

Environment

Unit 1

Frequency Response

SCIENTISTS IN ACTION

6.6K Views.  Max Planck Institute for Dynamics of Complex Technical Systems. We present a protocol for concentration alternating frequency response analysis of fuel cells, a promising new method of studying fuel cell dynamics.

Video: A Guide to Concentration Alternating Frequency Response Analysis of Fuel Cells

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

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.

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 ...

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

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 ...

Watershed Planning within a Quantitative Scenario Analysis Framework

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall short as a result of an inability to quantify and predict complex cumulative effects of current and future land use scenarios at relevant spatial scales. The goal of this manuscript is to provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework. Sites are first selected for inclusion within the watershed assessment by identifying sites that fall along independent gradients and combinations of known stressors. Field and laboratory techniques are then used to obtain data on the physical, chemical, and biological effects of multiple land use activities. Multiple linear regression analysis is then used to produce landscape-based cumulative effects models for predicting aquatic conditions. Lastly, methods for incorporating cumulative effects models within a scenario analysis framework for guiding management and regulatory decisions (e.g., permitting and mitigation) within actively developing watersheds are discussed and demonstrated for 2 sub-watersheds within the mountaintop mining region of central Appalachia. The watershed assessment and management approach provided herein enables resource managers to facilitate economic and development activity while protecting aquatic resources and producing opportunity for net ecological benefits through targeted remediation.

There is a critical need for tools and methodologies capable of managing aquatic systems in the face of uncertain future conditions. We provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework.

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

On the Preparation and Testing of Fuel Cell Catalysts Using the Thin Film Rotating Disk Electrode Method

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a high surface area carbon, and to test their performance in thin film rotating disk electrode (TF-RDE) measurements. The TF-RDE methodology is widely used for catalyst screening; nevertheless, the measured performance sometimes considerably differs among research groups. These uncertainties impede the advancement of new catalyst materials and, consequently, several authors discussed possible best practice methods and the importance of benchmarking.
The visual tutorial highlights possible pitfalls in the TF-RDE testing of Pt/C catalysts. A synthesis and testing protocol to assess standard Pt/C catalysts is introduced that can be used together with polycrystalline Pt disks as benchmark catalysts. In particular, this study highlights how the properties of the catalyst film on the glassy carbon (GC) electrode influence the measured performance in TF-RDE testing. To obtain thin, homogeneous catalyst films, not only the catalyst preparation, but also the ink deposition and drying procedures are essential. It is demonstrated that an adjustment of the ink&#39;s pH might be necessary, and how simple control measurements can be used to check film quality. Once reproducible TF-RDE measurements are obtained, determining the Pt loading on the catalyst support (expressed as Pt wt%) and the electrochemical surface area is necessary to normalize the determined reaction rates to either surface area or Pt mass. For the surface area determination, so-called CO stripping, or the determination of the hydrogen underpotential deposition (Hupd) charge, are standard. For the determination of the Pt loading, a straightforward and cheap procedure using digestion in aqua regia with subsequent conversion of Pt(IV) to Pt(II) and UV-vis measurements is introduced.

Preparing and testing Pt/C fuel cell catalysts is subject to continuous discussion in the scientific community with respect to reproducibility and best practice. With the presented work, we intend to present a step-by-step tutorial to make and test Pt/C catalysts, which can serve as benchmark for novel catalyst systems.

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a ...