Name: investigating long-distance transport of perfluoroalkyl acids in wheat via a split-root exposure technique
Uploaded: 2022-09-28T14:05:00
Description: Watch this Scientific Journal Video about Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique at JoVE.com

We gratefully acknowledge financial support from the Natural Science Foundation of China (NSFC 21737003), Chinese Universities Scientific Fund (No. 2452021103), and Chinese Postdoctoral Science Foundation (No. 2021M692651, 2021M702680).

Wheat seeds, Triticum aestivum L., were procured (see Table of Materials) and used for the present study.
1. Wheat seedling germination and hydroponic culture
<ol>
	<li>Select similar-sized wheat seeds and disinfect them for 15 min with 8% (w/w) hydrogen peroxide solution.</li>
	<li>Rinse the disinfected seeds with deionized water thoroughly, and then place them on humid filter paper in the dark at room temperature to germinate for 5 days.</li>
	<li...

<ol>
	<li>Lindstrom, A. B., Strynar, M. J., Libelo, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyfluorinated+compounds:+Past,+present,+and+future.">Polyfluorinated compounds: Past, present, and future.</a> Environmental Science &amp; Technology. 45 (19), 7954-7961 (2011).</li><li>Kannan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluoroalkyl+and+polyfluoroalkyl+substances:+Current+and+future+perspectives.">Perfluoroalkyl and polyfluoroalkyl substances: Current and future perspectives.</a> Environmental Chemistry. 8 (4), 333-338 (2011).</li><li>Cui, Q., 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=Occurrence+and+tissue+distribution+of+novel+perfluoroether+carboxylic+and+sulfonic+acids+and+legacy+per/polyfluoroalkyl+substances+in+black-spotted+frog+(Pelophylax+nigromaculatus).">Occurrence and tissue distribution of novel perfluoroether carboxylic and sulfonic acids and legacy per/polyfluoroalkyl substances in black-spotted frog (Pelophylax nigromaculatus).</a> Environmental Science &amp; Technology. 52 (3), 982-990 (2018).</li><li>Negri, 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=Exposure+to+PFOA+and+PFOS+and+fetal+growth:+a+critical+merging+of+toxicological+and+epidemiological+data.">Exposure to PFOA and PFOS and fetal growth: a critical merging of toxicological and epidemiological data.</a> Critical Reviews in Toxicology. 47 (6), 489-515 (2017).</li><li>Chi, Q., Li, Z., Huang, J., Ma, J., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+perfluorooctanoic+acid+and+perfluorooctanesulfonic+acid+with+serum+albumins+by+native+mass+spectrometry,+fluorescence+and+molecular+docking.">Interactions of perfluorooctanoic acid and perfluorooctanesulfonic acid with serum albumins by native mass spectrometry, fluorescence and molecular docking.</a> Chemosphere. 198, 442-449 (2018).</li><li>Zhang, X., Chen, L., Fei, X. C., Ma, Y. S., Gao, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+PFOS+to+serum+albumin+and+DNA:+insight+into+the+molecular+toxicity+of+perfluorochemicals.">Binding of PFOS to serum albumin and DNA: insight into the molecular toxicity of perfluorochemicals.</a> Bmc Molecular Biology. 10, 16 (2009).</li><li>Pan, Y. T., 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=Worldwide+distribution+of+novel+perfluoroether+carboxylic+and+sulfonic+acids+in+surface+water.">Worldwide distribution of novel perfluoroether carboxylic and sulfonic acids in surface water.</a> Environmental Science &amp; Technology. 52 (14), 7621-7629 (2018).</li><li>Knight, E. R., 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=An+investigation+into+the+long-term+binding+and+uptake+of+PFOS,+PFOA+and+PFHxS+in+soil+-+plant+systems.">An investigation into the long-term binding and uptake of PFOS, PFOA and PFHxS in soil - plant systems.</a> Journal of Hazardous Materials. 404, 124065 (2021).</li><li>Liu, Z. 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=Crop+bioaccumulation+and+human+exposure+of+perfluoroalkyl+acids+through+multi-media+transport+from+a+mega+fluorochemical+industrial+park,+China.">Crop bioaccumulation and human exposure of perfluoroalkyl acids through multi-media transport from a mega fluorochemical industrial park, China.</a> Environment International. 106, 37-47 (2017).</li><li>Mei, W. P., 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=Per-+and+polyfluoroalkyl+substances+(PFASs)+in+the+soil-plant+system:+Sorption,+root+uptake,+and+translocation.">Per- and polyfluoroalkyl substances (PFASs) in the soil-plant system: Sorption, root uptake, and translocation.</a> Environment International. 156, 106642 (2021).</li><li>Wang, W., Rhodes, G., Ge, J., Yu, X., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+accumulation+of+per-+and+polyfluoroalkyl+substances+in+plants.">Uptake and accumulation of per- and polyfluoroalkyl substances in plants.</a> Chemosphere. 261, 127584 (2020).</li><li>Lu, J., Wu, J., Stoffella, P. J., Wilson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+bisphenol+A,+nonylphenol,+and+natural+estrogens+in+vegetables+and+fruits+using+gas+chromatography-tandem+mass+spectrometry.">Analysis of bisphenol A, nonylphenol, and natural estrogens in vegetables and fruits using gas chromatography-tandem mass spectrometry.</a> Journal of Agricultural and Food Chemistry. 61 (1), 84-89 (2013).</li><li>Herschbach, C., Gessler, A., Rennenberg, H., Luttge, U., Beyschlag, W., Budel, B., Francis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Distance+Transport+and+Plant+Internal+Cycling+of+N-+and+S-Compounds.">Long Distance Transport and Plant Internal Cycling of N- and S-Compounds.</a> Progress in Botany 73. , 161-188 (2012).</li><li>Liu, Q., 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=Uptake+kinetics,+accumulation,+and+long-distance+transport+of+organophosphate+esters+in+plants:+Impacts+of+chemical+and+plant+properties.">Uptake kinetics, accumulation, and long-distance transport of organophosphate esters in plants: Impacts of chemical and plant properties.</a> Environmental Science &amp; Technology. 53 (9), 4940-4947 (2019).</li><li>Wang, Z. 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=Xylem-+and+phloem-based+transport+of+CuO+nanoparticles+in+maize+(Zea+mays+L.).">Xylem- and phloem-based transport of CuO nanoparticles in maize (Zea mays L.).</a> Environmental Science &amp; Technology. 46 (8), 4434-4441 (2012).</li><li>Chen, 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=Uptake,+accumulation,+and+translocation+mechanisms+of+steroid+estrogens+in+plants.">Uptake, accumulation, and translocation mechanisms of steroid estrogens in plants.</a> Science of the Total Environment. 753, 141979 (2021).</li><li>Felizeter, S., McLachlan, M. S., de Voogt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+perfluorinated+alkyl+acids+by+hydroponically+grown+lettuce+(Lactuca+sativa).">Uptake of perfluorinated alkyl acids by hydroponically grown lettuce (Lactuca sativa).</a> Environmental Science &amp; Technology. 46 (21), 11735-11743 (2012).</li><li>Zhou, J., 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=Insights+into+uptake,+translocation,+and+transformation+mechanisms+of+perfluorophosphinates+and+perfluorophosphonates+in+wheat+(Triticum+aestivum+L.).">Insights into uptake, translocation, and transformation mechanisms of perfluorophosphinates and perfluorophosphonates in wheat (Triticum aestivum L.).</a> Environmental Science &amp; Technology. 54 (1), 276-285 (2020).</li></ol>

To ensure the accuracy of this method, careful operation must be taken to ensure that the spiked solution in tube B does not contaminate the unspiked solution in tube A. The given concentration of target PFAAs in the present study was relatively higher than their concentration in the real environment, ensuring to monitor target PFAAs in wheat and unspiked solution using LC-MS/MS. 
There are limitations to this method. Since only one wheat seedling was used in each treatment group and the root ...

Perfluoroalkyl acids (PFAAs) are widely utilized in various commercial and industrial products due to their excellent physicochemical properties, including surface activity and thermal and chemical stability1,2,3. Perfluorooctane sulfonic acid (PFOS) and perfluorooctane acid (PFOA) are the two most important PFAAs used worldwide4,5,6, although these compounds were listed in the international Stockholm Convention in 2009 and 20197,8, respectively. Due to their persistence and widespread use, PFOS and PFOA have been widely detected in various environmental matrices. The concentrations of PFOA and PFOS in surface water from different worldwide rivers and lakes are 0.15-52.8 ng/L and 0.09-29.7 ng/L, respectively9. Due to the use of groundwater or reclaimed water for irrigation and also using biosolids as fertilizer, PFOA and PFOS are widely present in the soil, ranging between 0.01-123 &#181;g/kg and 0.003-162 &#181;g/kg, respectively10, which could introduce a large amount of PFAAs into plants and pose potential risks to human health. The PFAA (C4-C8) concentrations in agricultural soil and grain (wheat and maize) show a positive linear correlation11. Therefore, it is imperative to investigate the accumulation and translocation of PFAAs within plants.
The translocation of PFAAs in plants firstly occurs from the roots to the aboveground tissues, and the translocation of PFAAs from the roots to the edible tissues is regarded as long-distance transport12,13. Previous studies have detected bisphenol A, nonylphenol, and natural estrogens in vegetables and fruits14, which implies that these chemicals might migrate via the phloem. Hence, uncovering the translocation of PFAAs in plants is important to assess their potential risk. However, the accumulation and translocation of PFAAs are impacted by their bioavailability in the soil, so it is not easy to evaluate the translocation ability of target PFAAs in plants. Additionally, hydroponic experiments are generally limited by several factors, making it more difficult to acquire the edible tissues of plants. Typically, the phloem was collected directly from plants to observe the translocation of organic compounds through long distances in plants, whereas it is difficult to acquire phloems from plant seedlings15. Hence, a simple and effective method, the split-root technique, was introduced to study the translocation of PFAAs in plants during relatively short-term exposure. As for the split-root investigation, the roots in one plant seedling are separated into two parts; one part is put into the nutrient solution containing target PFAAs (tube A), and the other is placed in the nutrient solution in the absence of PFAAs (tube B). After exposure for several days, the PFAAs in tube B are measured by LC-MS/MS. The concentration of PFAAs in tube B discloses the translocation potential of PFAAs through the phloem within plants16,17,18.
The split-root experiment has been reported for studying the long-distance translocation of many compounds in plants, such as CuO nanoparticles17, steroid estrogens18, and organophosphate esters16. These studies provided evidence that these compounds could transfer via the phloem to the edible parts of plants. However, whether PFAAs could aid in translocation in plants and the impact of compound properties need to be further explored. Based on these reports, the split-root experiment was conducted in the present study to disclose the long-distance transport of PFAAs in wheat.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACQUITY UPLC BEH C18 column</td>
			<td>Waters, Milford, MA</td>
			<td></td>
			<td>Liquid chromatographic column</td>
		</tr>
		<tr>
			<td>Cleanert PEP cartridge</td>
			<td>Bonna- Angel Technologies, China</td>
			<td></td>
			<td>Solid phase extraction column</td>
		</tr>
		<tr>
			<td>Clearnert Pesticarb cartridge</td>
			<td>Bonna- Angel Technologies, China</td>
			<td></td>
			<td>Solid phase extraction column</td>
		</tr>
		<tr>
			<td>LC-MS/MS(Waters Acquity UPLC i-Class Coupled to Xevo TQ-S)</td>
			<td>Waters, Milford, MA</td>
			<td></td>
			<td>Liquid chromatography and mass spectrometry</td>
		</tr>
		<tr>
			<td>Lyophilizer&#160;</td>
			<td>Boyikang Instrument Ltd., Beijing, China</td>
			<td>FD-1A50</td>
			<td>Freeze-dried sample</td>
		</tr>
		<tr>
			<td>Masslynx</td>
			<td>Waters, Milford, MA</td>
			<td></td>
			<td>data analysis software</td>
		</tr>
		<tr>
			<td>Methyl tert-butyl ether</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td></td>
			<td>use for extracting target compounds from plant tissues</td>
		</tr>
		<tr>
			<td>MPFAC-MXA</td>
			<td>Wellington Laboratories (Ontario, Canada)</td>
			<td>PFACMXA0518</td>
			<td>the internal standards</td>
		</tr>
		<tr>
			<td>PFAC-MXB</td>
			<td>Wellington Laboratories (Ontario, Canada)</td>
			<td>PFACMXB0219</td>
			<td>mixture of PFAA calibration standards</td>
		</tr>
		<tr>
			<td>PFOA</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td>335-67-1</td>
			<td>a represent PFAAs</td>
		</tr>
		<tr>
			<td>PFOS</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td>2795-39-3</td>
			<td>a represent PFAAs</td>
		</tr>
		<tr>
			<td>Sodium carbonate buffer</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td></td>
			<td>use for extracting target compounds from plant tissues</td>
		</tr>
		<tr>
			<td>Tetrabutylammonium hydrogen sulfate</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td></td>
			<td>use for extracting target compounds from plant tissues</td>
		</tr>
		<tr>
			<td>Wheat seeds</td>
			<td>Chinese Academy of Agricultural Sciences (Beijing,China)&#160;</td>
			<td></td>
			<td>Triticum aestivum L.</td>
		</tr>
	</tbody>
</table>

investigating long-distance transport of perfluoroalkyl acids in wheat via a split-root exposure technique

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is imperative to investigate the accumulation and translocation of PFAAs within plants. Long-distance transport is an important pathway for PFAAs transferred from the plant leaves to the edible tissues through the phloem. However, it was previously difficult to assess the translocation potential of organic contamination in a short-term exposure period. The split-root experiment provides a solution to effectively uncover the long-distance translocation of PFAAs using a hydroponic experiment, which, in this study, was carried out in two 50 mL centrifuge tubes (A and B), of which centrifuge tube A had 50 mL of one-quarter strength Hoagland sterile nutrient solution, while centrifuge tube B had the same amount of nutrient concentration, and the target PFAAs (perfluorooctane sulfonic acid, PFOS, and perfluorooctane acid, PFOA) added at a given concentration. A whole wheat root was manually separated into two parts and inserted carefully into tubes A and B. The concentration of PFAAs in the roots, shoots of wheat, and solutions in tubes A and B were evaluated using LC-MS/MS, respectively, after being cultured in an incubator for 7 days and harvested. The results suggested that PFOA and PFOS experience a similar long-distance transport process through the phloem from the shoot to the root and could be released into the ambient environment. Thus, the split-root technique can be used to evaluate the long-distance transport of different chemicals.

The split-root experiment investigated the long-distance transport of PFAAs in wheat. As shown in Figure 2A,C, both PFOA and PFOS could be taken up by the wheat root and transferred to the shoot. PFOS and PFOA were not detected in the wheat root and solution in tube A of the blank control. It was found that PFOS and PFOA were detected in the wheat roots cultured in the unspiked solution, with a concentration of 0.26 ng/g &#177; 0.02 ng/g and 0.64 ng/g &#177; 0.05 ng/g dry we...

Watch this Scientific Journal Video about Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique at JoVE.com

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

The present protocol describes a simple and efficient method for the long-distance transport of perfluoroalkyl acids in wheat.

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is ...

The phloem was collected directly from plants to observe the translocation of organic compounds through long distances, whereas it is difficult to acquire phloem from plant seedlings. A simple and effective method, the split-root technique, was introduced to study the translocation of PFAAs in plants, during relatively short-term exposure. This method can also disclose the long-distance transport of different compounds in plants.The given concentration of target PFAAs must be relatively higher than their concentration in the real environment, ensuring the monitoring of the target PFAAs in wheat in unspiked solution. Begin by selecting the similar-sized Triticum aestivum, or wheat seeds, and disinfect them for 15 minutes with an 8%hydrogen peroxide solution. Thoroughly rinse the disinfected seeds with deionized water.Place the seeds on the humid filter paper in the dark at room temperature for 5 days for germination. Select approximately 9 germinated seedlings, of uniform size, and transfer them to plastic beakers with 250 milliliters of 1/4 strength of Hoagland's solution. Before the treatment, cultivate the seedlings in growth chambers for 7 days with a cycle of 14 hours at 22 degrees Celsius, and 10 hours at 27 degrees Celsius.For seedling cultivation, prepare 2 centrifuge tubes, A and B, each containing 50 milliliters of 1/4 strength of Hoagland's solution. Dissolve the commercial perfluorooctane acid, or PFOA, and perfluorooctanesulfonic acid, or PFOS, in methanol, and dilute it with the sterile nutrient solution as the stock solution, then add the stock solution to tube B at 100 micrograms per liter of PFOA and PFOS. Next, separate the whole roots of a wheat seedling into 2 equal parts using tweezers with the roots connected to the same shoot, then insert them into tubes A and B respectively.Seal the 2 tubes with aluminum foil before culturing them in an incubator for 7 days with a cycle of 14 hours at 22 degrees Celsius, and 10 hours at 27 degrees Celsius. After 7 days, collect the wheat seedlings and separate them into 3 parts using scissors, such as shoots and roots cultured in the spiked solution of perfluoroakyl acids, or PFAAs, in unspiked solution. Freeze dry the plant samples in a lyophilizer at minus 55 degrees Celsius for 48 hours.After 2 days, homogenize. Weigh the root and shoot samples. Collect the spiked and unspiked solution samples.In a 15-milliliter polypropylene tube, add 2 milliliters of sodium carbonate buffer, 1 milliliter of tetrabutylammonium hydrogen sulfate, 5 milliliters of methyl tert-butyl ether, and the homogenized root or shoot. When done, shake the tube at 250 RPM for 20 minutes, before centrifugation at 2000 g for 10 minutes at room temperature to obtain the supernatant organic phase. After the second extraction, pool the similar treatment extracts and vaporize them to dryness in a gentle nitrogen stream, then reconstitute with 5 milliliters of methanol and vortex them, maintaining the same speed for approximately 30 seconds.Condition the PestiCarb cartridge with 5 milliliters of 0.1%ammonium hydroxide in methanol, 5 milliliters of water and 5 milliliters of methanol. Add 5 milliliters of extracting methanol solution through the PestiCarb cartridge to remove the pigment. Elute the cartridge with 5 milliliters of methanol and collect in the sample tubes.Evaporate the collected 10 milliliters of methanol solution to dryness and reconstitute with 200 microliters of methanol before vortexing, then centrifuge the sample at 10, 000 g for 20 minutes at room temperature. Condition the polar-enhanced polymer extraction cartridge with 5 milliliters of methanol and 5 milliliters of water to activate. Add 1 milliliter of the spiked solution or 50 milliliters of the un spiked solution samples through the cartridge.Elude the target PFAAs with 10 milliliters of methanol. Evaporate the extract with a gentle nitrogen stream and then reconstitute the evaporated extract with 200 microliters of methanol for analysis. The split-root experiment investigated the long-distance transport of PFAAs in wheat.It was observed that PFOA and PFOS were taken up by the wheat root with a concentration of 11.94 nanograms per gram and 30.67 nanograms per gram dry weight in spiked solution respectively, and were transferred to the shoots with a concentration of 5.01 nanograms per gram and 4.17 nanograms per gram dry weight. It was found that PFOA and PFOS were detected in wheat roots cultured in the unspiked solution with a concentration of 0.26 nanograms per gram and 0.64 nanograms per gram dry weight respectively. It suggested that PFOA and PFOS could experience long-distance transport through the phloem from the shoot to the root.It was noted that PFOA and PFOS were also found in the unspiked nutrient solution with a concentration of 17.8 nanograms per liter and 28.5 nanograms per liter respectively, suggesting that PFOA and PFOS could pass through the root Casparian strip. Precaution must be taken to ensure that the spiked solution in tube B does not contaminate the unspiked solution in tube A.The visual demonstration of this method can provide a simple operation to disclose the long-distance transport of PFAAs in wheat.

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Environment

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Implementation of Portable Emissions Measurement Systems (PEMS) for the Real-driving Emissions (RDE) Regulation in Europe

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. However, on the road, ambient and driving conditions can vary over a wide range, sometimes causing emissions to be higher than those measured in the laboratory. For this reason, the European Commission has developed a complementary Real-Driving Emissions (RDE) test procedure using the Portable Emissions Measurement Systems (PEMS) to verify gaseous pollutant and particle number emissions during a wide range of normal operating conditions on the road. This paper presents the newly-adopted RDE test procedure, differentiating six steps: 1) vehicle selection, 2) vehicle preparation, 3) trip design, 4) trip execution, 5) trip verification, and 6) calculation of emissions. Of these steps, vehicle preparation and trip execution are described in greater detail. Examples of trip verification and the calculations of emissions are given.

Implementation of Portable Emissions Measurement Systems PEMS for the Real-driving Emissions RDE Regulation in Europe

The European Commission has developed a Real-Driving Emissions (RDE) test procedure to verify pollutant emissions during real-world vehicle operation using the Portable Emissions Measurement Systems (PEMS). This paper presents the experimental procedures required by the newly-adopted RDE test.

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...