Name: in situ chemotaxis assay to examine microbial behavior in aquatic ecosystems
Uploaded: 2020-05-05T14:45:00
Description: Watch this Scientific Journal Video about In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems at JoVE.com

This research was funded in part by the Gordon and Betty Moore Foundation Marine Microbiology Initiative, through grant GBMF3801 to J.R.S. and R.S., and an Investigator Award (GBMF3783) to R.S., as well as an Australian Research Council Fellowship (DE160100636) to J.B.R., an award from the Simons Foundation to B.S.L. (594111), and a grant from the Simons Foundation (542395) to R.S. as part of the Principles of Microbial Ecosystems (PriME) Collaborative.

We recommend executing section 1 prior to field experiments to optimize results.
1. Laboratory optimization
NOTE: The volumes described in the optimization procedure are sufficient for a single ISCA (composed of 20 wells).
<ol>
	<li>Preparation of the chemical of interest 
	NOTE: The optimal concentration for each chemoattractant often needs to be determined under laboratory conditions prior to field deployments. The chemical concentration field will decrease...

<ol>
	<li>Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+microbes+see+a+sea+of+gradients.">Marine microbes see a sea of gradients.</a> Science. 338, 628-633 (2012).</li><li>Raina, J. B., Fernandez, V., Lambert, B., Stocker, R., Seymour, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microbial+motility+and+chemotaxis+in+symbiosis.">The role of microbial motility and chemotaxis in symbiosis.</a> Nature Reviews Microbiology. 17, 284-294 (2019).</li><li>Chet, I., Asketh, P., Mitchell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repulsion+of+bacteria+from+marine+surfaces.">Repulsion of bacteria from marine surfaces.</a> Applied Microbiology. 30, 1043-1045 (1975).</li><li>Smriga, S., Fernandez, V. I., Mitchell, J. G., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+toward+phytoplankton+drives+organic+matter+partitioning+among+marine+bacteria.">Chemotaxis toward phytoplankton drives organic matter partitioning among marine bacteria.</a> PNAS. 113, 1576-1581 (2016).</li><li>Matilla, M., Krell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bacterial+chemotaxis+on+host+infection+and+pathogenicity.">The effect of bacterial chemotaxis on host infection and pathogenicity.</a> FEMS Microbiology Reviews. 42, (2018).</li><li>Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+in+bacteria.">Chemotaxis in bacteria.</a> Science. 153, 708-716 (1966).</li><li>Adler, J., Dahl, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+measuring+the+motility+of+bacteria+and+for+comparing+random+and+non-random+motility.">A method for measuring the motility of bacteria and for comparing random and non-random motility.</a> Journal of General Microbiology. 46, 161-173 (1967).</li><li>Ahmed, T., Shimizu, T. S., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+bacterial+chemotaxis.">Microfluidics for bacterial chemotaxis.</a> Integrative Biology. 2, 604-629 (2010).</li><li>Hol, F. J. H., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooming+in+to+see+the+bigger+picture:+microfluidic+and+nanofabrication+tools+to+study+bacteria.">Zooming in to see the bigger picture: microfluidic and nanofabrication tools to study bacteria.</a> Science. 346, 1251821 (2014).</li><li>Rusconi, R., Garren, M., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+expanding+the+frontiers+of+microbial+ecology.">Microfluidics expanding the frontiers of microbial ecology.</a> Annual Review of Biophysics. 43, 65-91 (2014).</li><li>Lambert, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidics-based+in+situ+chemotaxis+assay+to+study+the+behaviour+of+aquatic+microbial+communities.">A microfluidics-based in situ chemotaxis assay to study the behaviour of aquatic microbial communities.</a> Nature Microbiology. 2, 1344-1349 (2017).</li><li>Marie, D., Partensky, F., Jacquet, S., Vaulot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+and+cell+cycle+analysis+of+natural+populations+of+marine+picoplankton+by+flow+cytometry+using+the+nucleic+acid+stain+SYBR+Green+I.">Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I.</a> Applied Environmental Microbiology. 63, 186-193 (1997).</li><li>Rinke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+genomes+from+uncultivated+environmental+microorganisms+using+FACS-based+single-cell+genomics.">Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics.</a> Nature Protocols. 9, 1038-1048 (2014).</li><li>Gaworzewska, E. T., Carlile, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+chemotaxis+of+Rhizobium+leguminosarum+and+other+bacteria+towards+root+exudates+from+legumes+and+other+plants.">Positive chemotaxis of Rhizobium leguminosarum and other bacteria towards root exudates from legumes and other plants.</a> Microbiology. , (1982).</li><li>Walker, T. S., Bais, H. P., Grotewold, E., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudation+and+rhizosphere+biology.">Root exudation and rhizosphere biology.</a> Plant Physiology. 132, 44-51 (2003).</li></ol>

At the scale of aquatic microorganisms, the environment is far from homogenous and is often characterized by physical/chemical gradients that structure microbial communities1,15. The capacity of motile microorganisms to use behavior (i.e., chemotaxis) facilitates foraging within these heterogeneous microenvironments1. Studying chemotaxis directly in the environment has the potential to identify important interspecific interactions and chem...

Diverse microorganisms use motility and chemotaxis to exploit patchy nutrient environments, find hosts, or avoid deleterious conditions1,2,3. These microbial behaviours can in turn influence rates of chemical transformation4 and promote symbiotic partnerships across terrestrial, freshwater, and marine ecosystems2,5.
Chemotaxis has been extensively studied under laboratory conditions for the past 60 years6. The first quantitative method to study chemotaxis, the capillary assay, involves a capillary tube filled with a putative chemoattractant immersed in a suspension of bacteria6. Diffusion of the chemical out of the tube creates a chemical gradient, and chemotactic bacteria respond to this gradient by migrating into the tube7. Since the development of the capillary assay, still widely used today, many other techniques have been developed to study chemotaxis under increasingly controlled physical/chemical conditions, with the most recent involving the use of microfluidics8,9,10.
Microfluidics, together with high-speed video microscopy, enables tracking of the behavior of single cells in response to carefully controlled gradients. Although these techniques have vastly improved our understanding of chemotaxis, they have been restricted to laboratory use and do not translate easily to field deployment in environmental systems. As a consequence, the capacity of natural communities of bacteria to use chemotaxis within natural ecosystems has not been examined; thus, current understanding of the potential ecological importance of chemotaxis is biased toward artificial laboratory conditions and a limited number of laboratory-cultured bacterial isolates. The recently developed ISCA overcomes these limitations11.
The ISCA builds on the general principle of the capillary assay; however, it makes use of modern microfabrication techniques to deliver a highly replicated, easily deployable experimental platform for the quantification of chemotaxis toward compounds of interest in the natural environment. It also allows identification and characterization of chemotactic microorganisms by direct isolation or molecular techniques. While the first working device was self-fabricated and constructed of glass and PDMS11, the latest injection-molded version is composed of polycarbonate, using a highly standardized fabrication procedure (for interest in the latest version of the device, the corresponding authors can be contacted).
The ISCA is credit card-sized and consists of 20 wells distributed in a 5 x 4 well array, each linked to the external aquatic environment by a small port (800 &#181;m in diameter; Figure 1). Putative chemoattractants loaded into the wells diffuse into the environment via the port, and chemotactic microbes respond by swimming through the port into the well. As many factors can influence the outcome of an ISCA experiment in the natural environment, this step-by-step protocol will help new users overcome potential hurdles and facilitate effective deployments.

<table>
	<tbody>
		<tr>
			<td>Acrylic glue</td>
			<td>Evonik</td>
			<td>1133</td>
			<td>Acrifix 1S 0116</td>
		</tr>
		<tr>
			<td>Acrylic sheet</td>
			<td>McMaster-Carr</td>
			<td>8505K725</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Adhesive tape</td>
			<td>Scotch</td>
			<td>3M 810</td>
			<td>Scotch Magic tape</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Systec</td>
			<td>D-200</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75002451</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Bungee cord</td>
			<td>Paracord Planet</td>
			<td>667569184000</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Centrifuge tube - 2 mL</td>
			<td>Sigma Aldrich</td>
			<td>BR780546-500EA</td>
			<td>Eppendorf tube</td>
		</tr>
		<tr>
			<td>Conical centrifuge tube - 15 mL</td>
			<td>Fisher Scientific</td>
			<td>11507411</td>
			<td>Falcon tube</td>
		</tr>
		<tr>
			<td>Conical centrifuge tube - 50 mL</td>
			<td>Fisher Scientific</td>
			<td>10788561</td>
			<td>Falcon tube</td>
		</tr>
		<tr>
			<td>Deployment arm</td>
			<td>Irwin</td>
			<td>1964719</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Deployment enclosure plug</td>
			<td>Fisher Scientific</td>
			<td>21-236-4</td>
			<td>See alternatives in manuscript</td>
		</tr>
		<tr>
			<td>Disposable wipers</td>
			<td>Kimtech - Fisher Scientific</td>
			<td>06-666</td>
			<td>Kimwipes</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman</td>
			<td>C09756</td>
			<td>CYTOFlex</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25%</td>
			<td>Sigma Aldrich</td>
			<td>G5882</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Green fluorescent dye</td>
			<td>Sigma Aldrich</td>
			<td>S9430</td>
			<td>SYBR Green I - 1:10,000 final dilution</td>
		</tr>
		<tr>
			<td>Hydrophilic GP filter cartridge - 0.2 &#181;m</td>
			<td>Merck</td>
			<td>C3235</td>
			<td>Sterivex filter</td>
		</tr>
		<tr>
			<td>In Situ Chemotaxis Assay (ISCA)</td>
			<td>-</td>
			<td>-</td>
			<td>Contact corresponding authors</td>
		</tr>
		<tr>
			<td>Laser cutter</td>
			<td>Epilog Laser</td>
			<td>Fusion pro 32</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma Aldrich</td>
			<td>L3022</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Marine Broth 2216</td>
			<td>VWR</td>
			<td>90004-006</td>
			<td>Difco</td>
		</tr>
		<tr>
			<td>Nylon slotted flat head screws</td>
			<td>McMaster-Carr</td>
			<td>92929A243</td>
			<td>M 2 &#215; 4 &#215; 8 mm</td>
		</tr>
		<tr>
			<td>Pipette set</td>
			<td>Fisher Scientific</td>
			<td>05-403-151</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 1 mL</td>
			<td>Fisher Scientific</td>
			<td>21-236-2A</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 20 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>21-236-4</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 200 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>21-236-1</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Sea salt</td>
			<td>Sigma Aldrich</td>
			<td>S9883</td>
			<td>For artificial seawater</td>
		</tr>
		<tr>
			<td>Serological pipette - 50 mL</td>
			<td>Sigma Aldrich</td>
			<td>SIAL1490-100EA</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringe filter - 0.02 &#181;m</td>
			<td>Whatman</td>
			<td>WHA68091002</td>
			<td>Anatop filter</td>
		</tr>
		<tr>
			<td>Syringe filter - 0.2 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>10695211</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringe needle 27G</td>
			<td>Henke Sass Wolf</td>
			<td>4710004020</td>
			<td>0.4 &#215; 12 mm</td>
		</tr>
		<tr>
			<td>Syringes - 1 mL</td>
			<td>Codau</td>
			<td>329650</td>
			<td>Insulin Luer U-100</td>
		</tr>
		<tr>
			<td>Syringes - 10 mL</td>
			<td>BD</td>
			<td>303134</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringes - 50 mL</td>
			<td>BD</td>
			<td>15899152</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Tube rack - 15 mL</td>
			<td>Thomas Scientific</td>
			<td>1159V80</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Tube rack - 50 mL</td>
			<td>Thomas Scientific</td>
			<td>1159V80</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Uncoated High-Speed Steel General Purpose Tap</td>
			<td>McMaster-Carr</td>
			<td>8305A77</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Vacuum filter - 0.2 &#181;m</td>
			<td>Merck</td>
			<td>SCGPS05RE</td>
			<td>Steritop filter</td>
		</tr>
	</tbody>
</table>

in situ chemotaxis assay to examine microbial behavior in aquatic ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

This section presents laboratory results using the ISCA to test the chemotactic response of marine microbes to a concentration range of glutamine, an amino acid known to attract soil bacteria14. The concentration of glutamine that elicited the strongest chemotactic response in the laboratory tests was used to perform a chemotaxis assay in the marine environment.
To perform the laboratory tests, seawater communities sampled from coastal water in Sydney, Australia, were e...

Watch this Scientific Journal Video about In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems at JoVE.com

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

Our protocol enables us to robustly quantify microbial chemotaxis directly in their home habitat. And thereby we can isolate microorganisms and we can better quantify and understand the metabolic potential. The in situ chemotaxis assay, or ISCA in short, is a flexible, user-friendly, and simple device that allows new insight into microbial behaviors and therefore the role and function of microorganisms in any environment that has a liquid phase.Demonstrating the procedure for deployment in both the laboratory and in the field is Estelle Clerc, who is currently a PhD student in my research group. Before preparing the chemoattractant, filter the medium with a 0.2 micrometer filter, and autoclave the filtered solution. Next, prepare a 10 millimolar solution of chemoattractant in 1 milliliter of the sterile medium, and filter the chemoattractant solution with a 0.2 micrometer syringe filter to remove any particles and potential contaminants.Then, dilute the filtered chemoattractant stock solution in series, according to the experimental protocol. To fill an ISCA, attach a 27 gauge needle to a 1 milliliter syringe, and load one syringe for each concentration of the filtered chemoattractant. Holding the device with the port facing upwards, slowly inject each concentration of the substance into each of the five wells of one row of the device until a small droplet appears on top of each port.When all of the wells have been filled, transfer 1.5 milliliter of the marine or freshwater bacteria culture of interest in 150 milliliters of the appropriate bacterial culture medium. Next, place two small pieces of double-sided adhesive tape on the flat surface of a 200 milliliter capacity tray. And secure one ISCA onto each piece of tape.Using a 50 milliliter serological pipette, slowly fill the deployment tray with the bacterial solution. And allow the bacteria to respond to the chemoattractant of interest for one hour. To end the analysis, use a new 50 milliliter pipette to gently remove the medium from the ISCA tray.To retrieve the samples, first position the ISCA so that the port faces downward. Next, use a sterile 1 milliliter syringe equipped with a 27 gauge needle to draw the solution from each well. Pulling the samples from each row of the same concentration into a single tube.Then, analyze the samples by flow cytometry to determine the number of bacteria attracted to each chemoattractant concentration. To prepare an ISCA for field deployment, first, collect 5 milliliters of water per ISCA from the field side and filter the water through a 0.2 micrometer syringe filter into a 50 milliliter conical tube. Pass the syringe filtered water through a hydrophilic GP filter cartridge two times, collecting the filtrate in a new 50 milliliter conical centrifuge tube, after each rinse.Then, filter the water through a 0.02 micrometer syringe filter into a new 50 milliliter tube. Use aliquots of the filtrate to resuspend all of the chemoattractants of interest to the desired concentrations in individual 15 milliliter conical tubes. Then use 10 milliliter syringes to filter the resuspended chemoattractants through individual 0.2 micrometer syringe filters into sterile 15 milliliter conical centrifuge tubes to remove any unwanted particles or water insoluble compounds.For ISCA field deployment, screw each ISCA onto piece 9 of the device enclosure and close the enclosure. Seal the enclosure with adhesive tape and use bungee cords to secure the enclosure to a deployment arm and human made structure. Alternatively, the enclosure can be secured with a small weight on a shallow substrate or attached to a net in the Pelagic ocean.Submerge the enclosure completely to start the filling, holding the enclosure firmly to prevent excessive water movement inside. Once the level of the water has reached the top of the enclosure, make sure that no air is trapped inside. When the enclosure is completely full, seal the bottom and top holes with two plugs.Then, leave the ISCA in place for 1 to 3 hours. At the end of the sampling period, remove the enclosure from the water. Place the enclosure over a container to drain out the water, and carefully remove the upper part of the adhesive tape from the front holes.Once the water line has passed below the top of the ISCA, remove the bottom plug and drain the rest of the water. While the ISCAs are still attached to the enclosure, use a 1 milliliter pipette, or a 1 milliliter syringe, to carefully remove the water trapped on top of each ISCA and remove the ISCAs without touching the upper surface. Then use a disposable wipe to remove any remaining liquid on the surface, and retrieve the samples as demonstrated for the laboratory sample retrieval.In this representative in vitro analysis, 1 millimolar was determined to be the optimal glutamine deployment concentration as it induced a significant chemotactic response that was 18 fold higher than the filtered seawater control. Higher and lower concentrations of glutamine also induced significant, but weaker, chemotactic response. In this field deployment analysis, five ISCA replicates filled with 1 millimolar glutamine were deployed for 1 hour at a coastal site near Sydney, Australia.And the glutamine filled ISCA attracted nearly three times more bacteria than the control wells filled with filtered seawater. The most important thing to remember when deploying the ISCA, both in the lab and in the field, is to avoid creating strong fluid flows which will prevent chemotaxis. ISCA derived samples can be used to identify specific organisms or genes, to characterize the genomic potential of chemotactic organisms, and for targeted isolation of specific strains on selective media.

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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 ...

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 ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Simulating Impacts of Ice Storms on Forest Ecosystems

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. Current models suggest that the frequency and intensity of ice storms could increase over the coming decades in response to changes in climate, heightening interest in understanding their impacts. Because of the stochastic nature of ice storms and difficulties in predicting when and where they will occur, most past investigations of the ecological effects of ice storms have been based on case studies following major storms. Since intense ice storms are exceedingly rare events it is impractical to study them by waiting for their natural occurrence. Here we present a novel alternative experimental approach, involving the simulation of glaze ice events on forest plots under field conditions. With this method, water is pumped from a stream or lake and sprayed above the forest canopy when air temperatures are below freezing. The water rains down and freezes upon contact with cold surfaces. As the ice accumulates on trees, the boles and branches bend and break; damage that can be quantified through comparisons with untreated reference stands. The experimental approach described is advantageous because it enables control over the timing and amount of ice applied. Creating ice storms of different frequency and intensity makes it possible to identify critical ecological thresholds necessary for predicting and preparing for ice storm impacts.

Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. ...