This work was supported by the Natural Science Foundation of Shanghai, China (No. 18ZR1401000), the Shanghai Pujiang Program (No. 18PJ1400400), and the National Key Research and Development Program of China (No. 2018YFF0215703).

CAUTION: Please carefully read relevant safety data sheets (SDS) of all chemicals and wear proper personal protection equipment (PPE) before use. Some of the chemicals are toxic and irritant. Be careful when handling carbon nanotubes, which may have additional hazards if inhaled or contacted by skin.
1. Preparation of the electroactive titanate-CNT filter
<ol>
	<li>Preparation of titanate nanowires16
	<ol>
		<li>Dissolve 56 g of potassium hydroxide (KOH) in 100 mL of deionized water under vigorous stirring.</li>
		<li>Add 3 g of titanium dioxide (TiO2) powder into the as-dissolved KOH solution.</li>
		<li>Transfer the above solution into a Teflon-lined reactor and keep it at 200 &#176;C for 24 h.</li>
		<li>Wash the obtained white precipitate with 0.1 mol/L hydrochloric acid (HCl) and deionized water until a neutral effluent pH is obtained. Dry the product under vacuum at 60 &#176;C overnight.</li>
		<li>Transfer the products to a tube furnace and heat it to 600 &#176;C for 2 h with a ramp rate of 1 &#176;C/min.</li>
	</ol>
	</li>
	<li>Preparation of titanate-CNT filter17
	<ol>
		<li>Add 20 mg of carbon nanotubes (CNTs) into 40 mL of n-methyl pyrrolidone (NMP). Probe-sonication for 40 min to obtain homogeneous solution.</li>
		<li>Separately, add 20 mg of the as made titanate nanowires into 20 mL of NMP. Perform probe-sonication for 20 min.</li>
		<li>Mix the titanate dispersion solution with the CNT dispersion solution. Filter the mixture solution onto a PTFE membrane, which serves as a support for the titanate-CNT filter.</li>
		<li>Rinse sequentially with 100 mL of ethanol and 200 mL of deionized water. 
		NOTE: A CNT-alone filter can be prepared by a similar route without the addition of titanate nanowires.</li>
	</ol>
	</li>
</ol>
2. Electrochemical filtration of Sb(III)
<ol>
	<li>Description on the experimental apparatus18
	<ol>
		<li>Conduct the sorption experiments in an electrochemistry modified polycarbonate filtration casing (see Figure 1).</li>
		<li>Use a DC power supply to drive the electrochemistry.</li>
		<li>Adopt perforated titanium ring as connector for anodic or cathodic filters.</li>
		<li>Use an insulating silicone rubber as a separator and seal.</li>
	</ol>
	</li>
	<li>Filtration experiments
	<ol>
		<li>Add 2.2 mg of C8H4K2O12Sb2.3H2O into 1000 mL of deionized water to prepare 800 &#956;g/L Sb(III) solution.</li>
		<li>Transfer 100 mL of Sb(III) solution to a 150 mL beaker. Adjust solution pH to 7.</li>
		<li>Place the as-prepared titanate-CNT filter anode into the polycarbonate filtration casing and place another CNT-alone filter as cathode. Seal the casing.</li>
		<li>Pass through the filtration system with Sb(III) solution at a given flow. Apply a DC voltage during filtration.</li>
		<li>Determine the Sbtotal and Sb(III) concentration with atomic fluorescence spectrometer technique17. 
		NOTE: In this process, flow rate and applied voltage can be tuned by a peristaltic pump and a DC power supply, respectively.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Sun, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+microbial+community+in+a+watershed+heavily+contaminated+by+an+active+antimony+(Sb)+mine+in+Southwest+China.">Profiling microbial community in a watershed heavily contaminated by an active antimony (Sb) mine in Southwest China.</a> Science of the Total Environment. 550, 297-308 (2016).</li><li>Herath, I., Vithanage, M., Bundschuh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimony+as+a+global+dilemma:+geochemistry,+mobility,+fate+and+transport.">Antimony as a global dilemma: geochemistry, mobility, fate and transport.</a> Environmental Pollution. 223, 545-559 (2017).</li><li>Pan, L. B., 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=Assessments+of+levels,+potential+ecological+risk,+and+human+health+risk+of+heavy+metals+in+the+soils+from+a+typical+county+in+Shanxi+Province,+China.">Assessments of levels, potential ecological risk, and human health risk of heavy metals in the soils from a typical county in Shanxi Province, China.</a> Environmental Science and Pollution Research. 23, 19330-19340 (2016).</li><li>Huang, S. 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=Sulfide-modified+zerovalent+iron+for+enhanced+antimonite+sequestration:+characterization,+performance,+and+reaction+mechanisms.">Sulfide-modified zerovalent iron for enhanced antimonite sequestration: characterization, performance, and reaction mechanisms.</a> Chemical Engineering Journal. 338, 539-547 (2018).</li><li>Ungureanu, G., Santos, S., Boaventura, R., Botelho, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic+and+antimony+in+water+and+wastewater:+Overview+of+removal+techniques+with+special+reference+to+latest+advances+in+adsorption.">Arsenic and antimony in water and wastewater: Overview of removal techniques with special reference to latest advances in adsorption.</a> Journal of Environmental Management. 151, 326-342 (2015).</li><li>Zou, J. 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=Three-dimensional+reduced+graphene+oxide+coupled+with+Mn3O4+for+highly+efficient+removal+of+Sb(III)+and+Sb(V)+from+water.">Three-dimensional reduced graphene oxide coupled with Mn3O4 for highly efficient removal of Sb(III) and Sb(V) from water.</a> Acs Applied Materials &amp; Interfaces. 8, 18140-18149 (2016).</li><li>Saleh, T. A., Sari, A., Tuzen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+adsorption+of+antimony(III)+from+aqueous+solutions+by+polyamide-graphene+composite+as+a+novel+adsorbent.">Effective adsorption of antimony(III) from aqueous solutions by polyamide-graphene composite as a novel adsorbent.</a> Chemical Engineering Journal. 307, 230-238 (2017).</li><li>Yan, Y. Z., An, Q. D., Xiao, Z. Y., Zheng, W., Zhai, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+core-shell/bead-like+alginate@PEI+with+exceptional+adsorption+capacity,+recycling+performance+toward+batch+and+column+sorption+of+Cr(VI).">Flexible core-shell/bead-like alginate@PEI with exceptional adsorption capacity, recycling performance toward batch and column sorption of Cr(VI).</a> Chemical Engineering Journal. 313, 475-486 (2017).</li><li>Fu, L., Shozugawa, K., Matsuo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+antimony+(III)+in+soil+by+manganese+(IV)+oxide+using+X-ray+absorption+fine+structure.">Oxidation of antimony (III) in soil by manganese (IV) oxide using X-ray absorption fine structure.</a> Journal of Environmental Sciences. 73, 31-37 (2018).</li><li>Liu, W., 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=Adsorption+of+Pb2+,+Cd2+,+Cu2++and+Cr3++onto+titanate+nanotubes:+competition+and+effect+of+inorganic+ions.">Adsorption of Pb2+, Cd2+, Cu2+ and Cr3+ onto titanate nanotubes: competition and effect of inorganic ions.</a> Science of the Total Environment. 456, 171-180 (2013).</li><li>Wu, B., 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=Dynamic+study+of+Cr(VI)+removal+performance+and+mechanism+from+water+using+multilayer+material+coated+nanoscale+zerovalent+iron.">Dynamic study of Cr(VI) removal performance and mechanism from water using multilayer material coated nanoscale zerovalent iron.</a> Environmental Pollution. 240, 717-724 (2018).</li><li>Shan, C., Ma, Z. Y., Tong, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+removal+of+trace+antimony(III)+through+adsorption+by+hematite+modified+magnetic+nanoparticles.">Efficient removal of trace antimony(III) through adsorption by hematite modified magnetic nanoparticles.</a> Journal of Hazardous Materials. 268, 229-236 (2014).</li><li>Luo, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+antimonite+(Sb(III))+and+antimonate+(Sb(V))+from+aqueous+solution+using+carbon+nanofibers+that+are+decorated+with+zirconium+oxide+(ZrO2).">Removal of antimonite (Sb(III)) and antimonate (Sb(V)) from aqueous solution using carbon nanofibers that are decorated with zirconium oxide (ZrO2).</a> Environmental Science &amp; Technology. 49, 11115-11124 (2015).</li><li>Liu, Y. B., 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=Golden+carbon+nanotube+membrane+for+continuous+flow+catalysis.">Golden carbon nanotube membrane for continuous flow catalysis.</a> Industrial &amp; Engineering Chemistry Research. 56, 2999-3007 (2017).</li><li>Ma, B. W., 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=Enhanced+antimony(V)+removal+using+synergistic+effects+of+Fe+hydrolytic+flocs+and+ultrafiltration+membrane+with+sludge+discharge+evaluation.">Enhanced antimony(V) removal using synergistic effects of Fe hydrolytic flocs and ultrafiltration membrane with sludge discharge evaluation.</a> Water Research. 121, 171-177 (2017).</li><li>Yuan, Z. Y., Zhang, X. B., Su, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate+hydrothermal+synthesis+of+potassium+titanate+nanowires.">Moderate hydrothermal synthesis of potassium titanate nanowires.</a> Applied Physics a-Materials Science &amp; Processing. 78, 1063-1066 (2004).</li><li>Liu, Y. B., 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=Electroactive+modified+carbon+nanotube+filter+for+simultaneous+detoxification+and+sequestration+of+Sb(III).">Electroactive modified carbon nanotube filter for simultaneous detoxification and sequestration of Sb(III).</a> Environmental Science &amp; Technology. 53, 1527-1535 (2019).</li><li>Gao, G., Vecitis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+carbon+nanotube+filter+oxidative+performance+as+a+function+of+surface+chemistry.">Electrochemical carbon nanotube filter oxidative performance as a function of surface chemistry.</a> Environmental Science &amp; Technology. 45, 9726-9734 (2011).</li><li>Liu, Y. B., 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=Simultaneous+oxidation+and+sorption+of+highly+toxic+Sb(III)+using+a+dual-functional+electroactive+filter.">Simultaneous oxidation and sorption of highly toxic Sb(III) using a dual-functional electroactive filter.</a> Environmental Pollution. 251, 72-80 (2019).</li></ol>

We have nothing to disclose.

The key to this technology is to fabricate an electroactive conductive and porous hybrid filter with high Sb-specificity. To do this, special care should be paid to the fabrication process. The amount of titanate nanowires need to be precisely controlled due to the "trade-off" effect between the filter's electrical conductivity and surface area.
In addition, it should be also noted that a proper applied voltage is necessary. Once the applied voltage is too high (e.g., &#62;3 V), other competit...

Recently, the environmental pollution caused by emerging antimony (Sb) has attracted much attention1,2. Extensive studies demonstrate that Sb compounds pose high toxicity to human and microorganisms, although present in low concentrations in the environment3,4. Even worse, conventional physicochemical or biological methods are usually ineffective to remove these emerging contaminants due to their low concentrations and high toxicity5. The most abundant species of Sb are Sb(V) and Sb(III), of which the latter form is more toxic.
Among the currently available treatment methods, adsorption is believed to be a promising and feasible alternative due to its high efficiency, low cost, and simplicity6,7. Till now, several nanoscale sorbents with tunable microstructures, large specific surface area and Sb specificity have been developed, such as TiO28, MnO29, titanate10, zerovalent iron11, iron oxides and other binary metal oxides12,13. A common problem when dealing with nanoscale adsorbents is the post-separation issue due to their small particle size. One strategy to address this issue is to load these nano-sorbents onto macro/micro-scale supports14. Another challenging issue restricting the wide application of adsorption technology is the poor mass transport caused by limited concentration of target compounds/molecules15. This issue may be partially addressed by adopting a membrane design and convention could enhance the mass transport significantly. Recent efforts have been devoted to develop advanced treatment systems that combine adsorption and oxidation in a single unit for effective Sb(III) removal. Here, we show how an electroactive titanate-carbon nanotube (titanate-CNT) filter was rationally designed and applied for the simultaneously adsorption and sequestration of toxic Sb(III). By fine-tuning the titanate loading amount, applied voltage, and flow rate, we demonstrate how the Sb(III) oxidation rate and sequestration efficiency can be tailored correspondingly. Although the fabrication and application of the electroactive filter is shown in this protocol, similar designs can also apply to the treatment of other heavy metal ions.
Minor changes in the fabrication process and reagents may cause significant changes in the morphology and performance of the final system. For instance, the hydrothermal time, temperature, and chemical purity have been shown to affect the microstructures of these nanoscale adsorbents. The flow rate of the adsorbate solution also determines the residence time within a flow-through system as well as the removal efficiency of target compounds. With clear identification of these key impacting parameters, a reproducible synthesis protocol can be secured and a stable removal efficiency of Sb(III) can be achieved. This protocol aims to provide detailed experience on the fabrication of dual-functional hybrid filters as well as their applications towards the removal of toxic heavy metal ions in a flow-through manner.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Atomic fluorescence spectrometer</td>
			<td>Ruili Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon nanotubes (CNT)</td>
			<td>TimesNano Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DC power supply</td>
			<td>Dahua Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 96%</td>
			<td>Sinopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 36%</td>
			<td>Sinopharm</td>
			<td></td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>L-antimony potassium tartrate</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>N-methyl-2-pyrrolidinone (NMP), 99.5%</td>
			<td>Sinopharm</td>
			<td></td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>Potassium hydroxide, 85%</td>
			<td>Sinopharm</td>
			<td></td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium dioxide powders</td>
			<td>Sinopharm</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a dual-functional electroactive filter towards simultaneously sb(iii) oxidation and sequestration

We have designed a facile method to synthesize a dual-functional electrochemical filter consisting of two 1-D materials: titanate nanowires and carbon nanotubes. The hybrid titanate-CNT filter was prepared by a sonication coupled with a post-filtration route. Due to the synergistic effects of the increased number of exposed sorption sites, electrochemical reactivity, small pore size of the titanate-CNT network coupled with a flow-through design, simultaneous Sb(III) oxidation and sequestration can be readily achieved. Atomic fluorescence spectrometer technology demonstrated that the applied electrical field accelerates the Sb(III) conversion rate and the as-obtained Sb(V) were adsorbed effectively by the titanate nanowires due to their Sb specificity. This protocol provides a practical solution for the removal of highly toxic Sb(III) and other similar heavy metal ions.

The electroactive filtration apparatus employed is an electrochemically modified polycarbonate filtration casing (Figure 1). Field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM) techniques are employed to characterize the morphology of the titanate-CNT filter (Figure 2). To demonstrate the efficacy of the electrochemical filtration system, the change of Sbtotal and Sb valence state as a function of time is det...

Watch this Scientific Journal Video about A Dual-Functional Electroactive Filter Towards Simultaneously SbIII Oxidation and Sequestration at JoVE.com

A Dual-Functional Electroactive Filter Towards Simultaneously SbIII Oxidation and Sequestration

A protocol for the rational design of a dual-functional electroactive filter consisting of carbon nanotubes and titanate nanowires is reported and their environmental applications towards Sb(III) oxidation and sequestration is presented.

A Dual-Functional Electroactive Filter Towards Simultaneously Sb(III) Oxidation and Sequestration

We have designed a facile method to synthesize a dual-functional electrochemical filter consisting of two 1-D materials: titanate nanowires and carbon ...

a-dual-functional-electroactive-filter-towards-simultaneously-sb-iii

Research

JoVE Journal

Environment

5.4K Views.  Donghua University. A protocol for the rational design of a dual-functional electroactive filter consisting of carbon nanotubes and titanate nanowires is reported and their environmental applications towards Sb(III) oxidation and sequestration is presented.

Video: A Dual-Functional Electroactive Filter Towards Simultaneously SbIII Oxidation and Sequestration

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

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

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

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

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

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

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

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

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

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

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

A Protocol for Collecting and Constructing Soil Core Lysimeters

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the landscape has complex and varied soil morphology, an accompanying disparity in flow paths for leachate through the soil macropore and matrix structure is present. The rate of flow through these paths is further affected by antecedent soil moisture. Lysimeters are used to quantify flow rate, volume of water and concentration of nutrients leaching downward through soils. While many lysimeter designs exist, accurately determining the volume of water and mass balance of nutrients is best accomplished with bounded lysimeters that leave the natural soil structure intact. 
Here we present a detailed method for the extraction and construction of soil core lysimeters equipped with soil moisture sensors at 5 cm and 25 cm depths. Lysimeters from four different Coastal Plain soils (Bojac, Evesboro, Quindocqua and Sassafras) were collected on the Delmarva Peninsula and moved to an indoor climate controlled facility. Soils were irrigated once weekly with the equivalent of 2 cm of rainfall to draw down soil nitrate-N concentrations. At the end of the draw down period, poultry litter was applied (162 kg TN ha-1) and leaching was resumed for an additional five weeks. Total recovery of applied irrigation water varied from 71% to 85%. Nitrate-N concentration varied over the course of the study from an average of 27.1 mg L-1 before litter application to 40.3 mg L-1 following litter application. While greatest flux of nutrients was measured in soils dominated by coarse sand (Sassafras) the greatest immediate flux occurred from the finest textured soil with pronounced macropore development (Quindocqua).

A detailed method for extraction and assembly of intact soil core lysimeters and their use for study of leachate and associated loss of nutrients from surface applied poultry litter is demonstrated.

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the ...

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

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

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

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

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...