Name: experimental column setup for studying anaerobic biogeochemical interactions between iron (oxy)hydroxides, trace elements, and bacteria
Uploaded: 2017-12-19T12:30:00
Description: Watch this Scientific Journal Video about Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria at JoVE.com

This work was co-funded by BRGM, a postdoctoral grant from the Conseil G&#233;n&#233;ral du Loiret and the Carnot Institute. We also gratefully acknowledge the financial support provided to the PIVOTS project by the R&#233;gion Centre - Val de Loire.

1. Experimental Preparation
<ol>
	<li>Acid-wash all materials (glass, polytetrafluoroethylene (PFTE)) in contact with samples (5 days in 20% nitric acid (HNO3) v/v) followed by 5 days in hydrochloric acid (HCl) 10% v/v). Rinse several times with ultra-pure water and dry under a laminar flow hood prior to use.</li>
	<li>Use polyethylene gloves (or similar) and a fume hood for all steps involving chemicals.</li>
</ol>
2. Prepare Hg and As Spiked Amorphous Iron Oxides
<ol>
	<li>Prepare...

<ol>
	<li>Oremland, R. S., Stolz, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ecology+of+Arsenic.">The Ecology of Arsenic.</a> Science. 300 (5621), 939 (2003).</li><li>Silver, S., Phung, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+enzymes+involved+in+bacterial+oxidation+and+reduction+of+inorganic+arsenic.">Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic.</a> Appl Environ Microbiol. 71 (2), 599-608 (2005).</li><li>Compeau, G. C., Bartha, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfate-Reducing+Bacteria:+Principal+Methylators+of+Mercury+in+Anoxic+Estuarine+Sediment.">Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sediment.</a> Appl. Environ. Microbiol. 50, (1985).</li><li>Fleming, E. J., Mack, E. E., Green, P. G., Nelson, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+Methylation+from+Unexpected+Sources:+Molybdate-Inhibited+Freshwater+Sediments+and+an+Iron-Reducing+Bacterium.">Mercury Methylation from Unexpected Sources: Molybdate-Inhibited Freshwater Sediments and an Iron-Reducing Bacterium.</a> Appl. Environ. Microbiol. 72 (1), 457-464 (2006).</li><li>Barkay, T., Miller, S., Summers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+mercury+resistance+from+atoms+to+ecosystems.">Bacterial mercury resistance from atoms to ecosystems.</a> FEMS Microbiol Rev. 27 (2-3), 355-384 (2003).</li><li>Dixit, S., Hering, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arsenic(V)+and+arsenic(III)+sorption+onto+iron+oxide+minerals:+Implications+for+arsenic+mobility.">Comparison of arsenic(V) and arsenic(III) sorption onto iron oxide minerals: Implications for arsenic mobility.</a> Environ. Sci. Technol. 37, (2003).</li><li>Andersson, H. A., Nriagu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biochemistry of Mercury in the Environnment. , 79-112 (1979).</li><li>Khwaja, A., Bloom, P. R., Brezonik, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+Constants+of+Divalent+Mercury+in+Soil+Humic+Acids+and+Soil+Organic.">Binding Constants of Divalent Mercury in Soil Humic Acids and Soil Organic.</a> Environ. Sci. Technol. 40, (2006).</li><li>Neculita, C. M., Zagury, G. J., Deschenes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+Speciation+in+Highly+Contaminated+Soils+from+Chlor-Alkali+Plants+Using+Chemical+Extractions.">Mercury Speciation in Highly Contaminated Soils from Chlor-Alkali Plants Using Chemical Extractions.</a> J Environ Qual. 34 (1), (2005).</li><li>Schuster, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+of+mercury+in+the+soil+with+special+emphasis+on+complexation+and+adsorption+processes+-+a+review+of+the+literature.">The behaviour of mercury in the soil with special emphasis on complexation and adsorption processes - a review of the literature.</a> Water Air Soil pollut. 56 (56), 667-680 (1991).</li><li>Wallschl&#228;ger, D., Desai, M. V. M., Spengler, M., Windm&#246;ller, C. C., Wilken, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+humic+substances+dominate+mercury+geochemistry+in+contaminated+floodplain+soils+and+sediments.">How humic substances dominate mercury geochemistry in contaminated floodplain soils and sediments.</a> J. Environ. Qual. 27 (5), (1998).</li><li>Lovley, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissimilatory+Fe(III)+and+Mn(IV)+reduction.">Dissimilatory Fe(III) and Mn(IV) reduction.</a> Microbiol. Mol. Biol. Rev. 55 (2), 259-287 (1991).</li><li>Lovley, D. R., Kashefi, K., Vargas, M., Tor, J. M., Blunt-Harris, 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=Reduction+of+humic+substances+and+Fe(III)+by+hyperthermophilic+microorganisms.">Reduction of humic substances and Fe(III) by hyperthermophilic microorganisms.</a> Chem. Geol. 169 (3-4), 289-298 (2000).</li><li>Hansel, C. 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=Structural+constraints+of+ferric+(hydr)oxides+on+dissimilatory+iron+reduction+and+the+fate+of+Fe(II).">Structural constraints of ferric (hydr)oxides on dissimilatory iron reduction and the fate of Fe(II).</a> Geochimica Cosmochimica Acta. 68, 3217-3229 (2004).</li><li>Thamdrup, B., Fossing, H., J&#248;rgensen, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese,+iron+and+sulfur+cycling+in+a+coastal+marine+sediment+Aarhus+bay,+Denmark.">Manganese, iron and sulfur cycling in a coastal marine sediment Aarhus bay, Denmark.</a> Geochim.Cosmochim. Acta. 58 (23), 5115-5129 (1994).</li><li>Planer-Friedrich, B., London, J., McCleskey, R. B., Nordstrom, D. K., Wallschl&#228;ger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioarsenates+in+Geothermal+Waters+of+Yellowstone+National+Park:+Determination,+Preservation,+and+Geochemical.">Thioarsenates in Geothermal Waters of Yellowstone National Park: Determination, Preservation, and Geochemical.</a> Environ. Sci. Technol. 41 (15), 5245-5251 (2007).</li><li>Burnol, A., 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=Decoupling+of+arsenic+and+iron+release+from+ferrihydrite+suspension+under+reducing+conditions:+a+biogeochemical+model.">Decoupling of arsenic and iron release from ferrihydrite suspension under reducing conditions: a biogeochemical model.</a> Geochem. Trans. 8 (1), 12 (2007).</li><li>Kocar, B. D., 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=Integrated+biogeochemical+and+hydrologic+processes+driving+arsenic+release+from+shallow+sediments+to+groundwaters+of+the+Mekong+delta.">Integrated biogeochemical and hydrologic processes driving arsenic release from shallow sediments to groundwaters of the Mekong delta.</a> Appl. Geochem. 23 (11), (2008).</li><li>Harris-Hellal, J., Grimaldi, M., Garnier-Zarli, E., Bousserrhine, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+mobilization+by+chemical+and+microbial+iron+oxide+reduction+in+soils+of+French+Guyana.">Mercury mobilization by chemical and microbial iron oxide reduction in soils of French Guyana.</a> Biogeochem. 103 (1), (2011).</li><li>Islam, F. 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=Role+of+metal-reducing+bacteria+in+arsenic+release+from+Bengal+delta+sediments.">Role of metal-reducing bacteria in arsenic release from Bengal delta sediments.</a> Nature. 430, (2004).</li><li>Schultz-Zunkel, C., Rinklebe, J., Bork, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+element+release+patterns+from+three+floodplain+soils+under+simulated+oxidized-reduced+cycles.">Trace element release patterns from three floodplain soils under simulated oxidized-reduced cycles.</a> Ecol. Eng. 83, 485-495 (2015).</li><li>Nickson, R. 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=Mechanisms+of+arsenic+release+to+groundwater,+bangladesh+and+West+Bengal.">Mechanisms of arsenic release to groundwater, bangladesh and West Bengal.</a> App. Geochem. 15, 403-413 (2000).</li><li>Varsanyi, I., 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=Arsenic,+iron+and+organic+matter+in+sediments+and+groundwater+in+the+Pannonian+basin,+Hungary.">Arsenic, iron and organic matter in sediments and groundwater in the Pannonian basin, Hungary.</a> App. Geochem. 21, 949-963 (2006).</li><li>Hellal, 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=Mercury+mobilization+and+speciation+linked+to+bacterial+iron+oxide+and+sulfate+reduction:+A+column+study+to+mimic+reactive+transfer+in+an+anoxic+aquifer.">Mercury mobilization and speciation linked to bacterial iron oxide and sulfate reduction: A column study to mimic reactive transfer in an anoxic aquifer.</a> J. Contam. Hydrol. 180, 56-68 (2015).</li><li>Battaglia-Brunet, F., Dictor, M. C., Garrido, F., Crouzet, C., Morin, D., Dekeyser, K., Clarens, M., Baranger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+arsenic(III)-oxidizing+bacterial+population:+selection,+characterization,+and+performance+in+reactors.">An arsenic(III)-oxidizing bacterial population: selection, characterization, and performance in reactors.</a> J Appl. Microbiol. 93 (2002), 656-667 (2002).</li><li>Salvato, N., Pirola, 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+mercury+traces+by+means+of+solid+sample+atomic+absorption+spectrometry.">Analysis of mercury traces by means of solid sample atomic absorption spectrometry.</a> Microchim Acta. 123 (1), 63-71 (1996).</li><li>Huguet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Caract&#233;risation biog&#233;ochimique et potentiel de m&#233;thylation du mercure de biofilms en milieu tropical (retenue de Petit Saut et estuaire du Sinnamary, Guyane Fran&#231;aise). . , (2009).</li><li>Mamindy-Pajany, 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=Arsenic+in+Marina+Sediments+from+the+Mediterranean+Coast:+Speciation+in+the+Solid+Phase+and+Occurrence+of+Thioarsenates.">Arsenic in Marina Sediments from the Mediterranean Coast: Speciation in the Solid Phase and Occurrence of Thioarsenates.</a> Soil Sed. Contam. 22, 984-1002 (2013).</li><li>dos Santos Afonso, 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=Reductive+dissolution+of+iron(III)+(hydro)oxides+by+hydrogen+sulfide.">Reductive dissolution of iron(III) (hydro)oxides by hydrogen sulfide.</a> Langmuir. 8, 1671-1675 (1992).</li><li>Postma, D., 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=Redox+zonation:+equilibrium+constraints+on+the+Fe(III)/SO4-reduction+interface.">Redox zonation: equilibrium constraints on the Fe(III)/SO4-reduction interface.</a> Geochem Cosmochim. Acta. 60, 3169-3175 (1996).</li><li>Kumar, N., 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=Sulfur+and+oxygen+isotope+tracing+in+zero+valent+iron+based+In+situ+remediation+system+for+metal+contaminants.">Sulfur and oxygen isotope tracing in zero valent iron based In situ remediation system for metal contaminants.</a> Chemosphere. 90, 1366-1371 (2013).</li></ol>

The experimental column setup proved to be a convenient laboratory device to study anaerobic biogeochemical processes in continuous conditions. Continuous column systems allow working in conditions closer to those of real aquifers than slurry batch systems or microcosms. Continuous systems can simulate the movement of groundwater through aquifer sediments.
The most critical step within the protocol is preparing the TE-iron (oxy)hydroxides and the mixture with silica gel and sand, which needs t...

Understanding and predicting trace element (TE) mobility and biogeochemistry in the environment is essential in order to monitor, develop, and apply appropriate management decisions for polluted sites. This especially applies in the case of toxic TEs such as arsenic (As) and mercury (Hg). The fate and speciation of these TEs in soil or aquifers are closely related to physico-chemical conditions, such as Eh and pH, but also to microbial activities that can play either a direct role on speciation or an indirect role on mobility.
Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). This affects As toxicity, since As(III) is the most toxic form of As, and mobility, since As(III) is more mobile than As(V), which can readily adsorb to iron (oxy)hydroxides or organic matter1,2. Likewise, bacteria are strongly involved in mercury cycling, either through its methylation, mainly by sulfate and iron reducing bacteria3,4, forming the neurotoxin monomethyl mercury (readily bioaccumulated in the food chain), or through its reduction to volatile elementary Hg (Hg&#176;)5.
Both As and Hg fates are also strongly linked to soil or aquifer composition, since compounds such as organic matter or iron (oxy)hydroxides can influence their sequestration and bioavailability. As(V) adsorbs well to iron (oxy)hydroxides6, whereas Hg has a very high affinity for organic matter (OM; mainly for thiol groups) but also for colloidal iron or manganese (oxy)hydroxides in OM depleted environments7,8,9,10,11.
Bacterial activities can then influence the fate of TEs adsorbed to (oxy)hydroxides or organic matter through the reduction of iron (oxy)hydroxides or the mineralization of organic matter. Direct iron reduction by bacteria is the dominant pathway for iron reduction in sulfur depleted zones12,13, Fe(III) being used as a terminal electron acceptor, whereas indirectly, Fe(III) can be reduced to Fe(II) by sulfide formed by a bacterial sulfate reduction14. Moreover, the presence of sulfate can also modify Hg and As speciation through the formation of complexes such as thio-arsenates15 with As or metacinnabar with Hg.
Thus, a better understanding of the impact of iron and sulfate cycling on the fate of TE, such as Hg and As, could help us to better manage contaminated sites and maintain soil and water quality. Data could also contribute to reinforcing existing metal-mobility models. Microbial Fe(III)-reduction16,17,18 can cause the desorption of TE. Theoretically, the indirect reduction of iron (oxy)hydroxides by sulfide produced by the microbial reduction of sulfate could also impact TE mobility. However, the extent and kinetics of these reactions are generally studied in batch homogenous systems or batch microcosms16,18,19,20. The drawback of batch experiments is the lack of dissociation of the occurring phenomena; indeed, activity is based on and limited by the resources present in the batch and only gives a final result of the shifts in speciation and adsorption. Using a column approach enables the renewal of inflowing media and the monitoring of the fate of TE over time and space. These conditions are more realistic when compared to an aquifer, where real phenomena are closely linked to continuous percolation conditions. Moreover, heterogeneous iron (oxy)hydroxide occurrence in aquifer sediments is common21,23, and the spatial changes in the mineralogical and chemical composition of the solid phases certainly drives microbial activities.
To elucidate the influence of these heterogeneities on geo-microbial phenomena and the fate of iron-associated TE, we developed a laboratory, a continuously-fed column representing a simplified model aquifer. The column is filled to create an iron-depleted zone at the column entrance and an iron-rich zone at the top. Regular sampling ports enable us to study each zone individually as well as interface-associated phenomena. An example of the application of this experimental device for the study of Hg fate and speciation is already available24. Here we give a detailed description of the experimental setup and a second example of its application focused on the behavior of As in contaminated aquifers.

<table border="0" cellpadding="0" cellspacing="0" width="991">
	<tbody>
		<tr height="120">
			<td height="120" style="height:120px;width:276px;">Glass columns&#160;</td>
			<td style="width:171px;">Beaucaverre, France</td>
			<td style="width:127px;">Specific request</td>
			<td style="width:417px;">columns were composed of 3 separate pieces, the main column core with the cooling jacket and the 5 sampling ports (size GL14 with olive) and a top and bottom piece that fits to the main column body and is held in place with a silicone joint and screw (RIN F 40x38 &#38; SVL 42). note: this design was discussed directly with the company. We recommend to find a local glazier.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Septa PTFE/silicone diam 20mm&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>508608</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">PTFE tubing ID 3mm</td>
			<td style="width:171px;">VWR</td>
			<td>228-0745</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Peristaltic pump</td>
			<td style="width:171px;">Dominique Dutsher SAS</td>
			<td style="width:127px;">66493</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Peristaltic pump tubing LMT 55</td>
			<td style="width:171px;">VWR</td>
			<td>224-2250</td>
			<td>Tygon&#174; LMT 55&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Fontainbleau sand&#160; D50=209 &#181;m</td>
			<td>SIBELCO,&#160; France</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:276px;">N2 for bubbling&#160;</td>
			<td style="width:171px;">Air Liquide, France</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Gamma iradiation</td>
			<td>Ionisos, Dagneux, France</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Automatic Mercury Analyser (AMA 254, )</td>
			<td style="width:171px;">Courtage Analyses, France</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Varian SpectrAA 300 Zeeman</td>
			<td style="width:171px;">Agilent&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Name</td>
			<td style="width:171px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Chemicals</td>
			<td style="width:171px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:276px;">HNO3 Supra pur</td>
			<td style="width:171px;">VWR</td>
			<td style="width:127px;">1.00441.1000</td>
			<td>Producer: Merck</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">HCL 30% Supra pur</td>
			<td style="width:171px;">VWR</td>
			<td>1.00318.1000</td>
			<td>Producer: Merck</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:276px;">Hg(NO3)2</td>
			<td style="width:171px;">Merck</td>
			<td>516953</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:276px;">As2O3</td>
			<td style="width:171px;">Merck</td>
			<td style="width:127px;">202673</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:276px;">FeCl3-6H2O</td>
			<td>Merck</td>
			<td>207926</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">silica gel</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>336815-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental column setup for studying anaerobic biogeochemical interactions between iron (oxy)hydroxides, trace elements, and bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Example 1. Impact of iron reduction of As mobility and speciation 
The As column was directly inoculated with groundwater from a site presenting an As concentration higher than the drinking standards (Bracieux, Loire et Cher, France). Groundwater was sampled in sterile bottles, and stored at 5 &#176;C until use. The column was fed from the bottom with this water containing the natural endogenous microbial commun...

Watch this Scientific Journal Video about Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria at JoVE.com

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

The overall goal of this experimental setup is to study the fate of trace metals under sulfate and/or iron-reducing conditions induced by microbial activities. This method can help answer key questions in the biogeochemistry field such as interactions between key biogeochemical cycles like mercury and arsenic. The main advantage of this technique is that due to the continuous water flow, the dynamics of the biogeochemical reactions and lability of trace elements are dissociated and accessible through the sampling ports.And so, this method can provide insight into the fate of mercury and arsenic as demonstrated here ending to iron oxides. It can also be applied to other trace elements or carrier substrates. Demonstrating the procedure will Hafida Tris, a technician from our laboratory.To begin this procedure, add 500 milliliters of ultrapure water to a glass reactor equipped with a magnetic stir bar. Add 50 grams of iron(III)chloride hexahydrate while stirring. Manually add approximately 50 milliliters of a 10-molar solution of sodium hydroxide to begin precipitation of ferrihydrite.Next, adjust the pH to six and continue stirring for one hour to stabilize. For arsenic-spiked hydroxides and oxyhydroxides, prepare 100 milliliters of arsenic trioxide at a concentration of 10 grams per liter. Then, add 70 milliliters of this solution to the iron oxide solution.Continue stirring for three hours. After this, centrifuge at 2, 000 G for 20 minutes. Remove the supernatant and resuspend the pellet with 50 milliliters of ultrapure water.Repeat the centrifugation ane resuspension two additional times. Recover and store the humid hydroxides and oxyhydroxides as outlined in the text protocol. Keep four grams of silica gel in 40 milliliters of 7%potassium hydroxide on a hot plate stirring with a magnetic stir bar until dissolved.Add 60 milliliters of ultrapure water. Cool the solution on ice to about 20 degrees Celsius to avoid the gel setting too quickly. Titrate the silica gel mixture with phosphoric acid 20%to pH 7.5.Then, quickly mix the liquid silica gel with 320 grams of sterile sand previously mixed with 18.3 grams of either mercury-spiked or arsenic-spiked iron oxides before it solidifies. Using a spatula, break up the gelified mixture. To begin setting up the glass columns, connect the water jackets to a water-cooling system.Set the system to maintain an average temperature of 20 degrees Celsius. Cut lengths of PTFE tubing with a three-millimeter inner diameter making sure there is sufficient length for the column inlet and outlet. Then, add a layer of damp rock wool to the column from the top.Add 320 grams of sterile sand. Add the final layer consisting of the previously mixed sterile sand and spiked hydrated amorphous iron oxides fixed in the silica gel matrix. Next, connect the inflow to peristaltic tubing.Connect the other end of the peristaltic tubing to the water medium supply. Connect an ascendent flow of ultrapure sterile water continuously bubbled by nitrogen gas at low velocity of approximately two milliliters per hour. After this, cover the column with aluminum foil to block out the light.After two weeks, a black-colored precipitate is observed at the interface between the two layers of sand. This black zone progressively invades the top iron hydroxide and oxyhydroxide-enriched zones with the entire upper layer becoming black by the end of the experiment. After 35 days of continuous running, the measured levels of sulfate decrease followed by a transient increase in the total dissolved iron.From day 60 onward, the total dissolved arsenic is seen to rise significantly. On day 54, the physical and chemical parameters are profiled. While the pH remains close to pH seven along the column, the redox potential changes significantly with values nearing negative 200 millivolts in the iron-depleted zone where sulfate reduction is occurring and nearing negative millivolts at the top where bacterial and chemical iron reduction is occurring.Dissolved sulfate concentrations reach to approximately 20 milligrams per liter in the bottom layers and decrease to under one milligram per liter in the iron-rich zone. Sulfate also decreases sharply at the interface between iron-deprived and iron-rich zones indicating a peak of sulfate reduction activity. Meanwhile, arsenic is detected in the upper zone while thioarsenate species are seen closer to the interface and thiosulfate is in the bottom iron-deprived layer.These concentration profiles indicate a peak of sulfate reduction activity at the interface between iron-deprived and iron-rich layers. Following this procedure, other methods like scanning electron microscopy, Raman spectroscopy or activity assays can be performed in order to answer additional questions such as which new minerals are formed or which bacterial communities have developed. Don't forget that working with mercury and arsenic can be extremely hazardous and precautions such gloves and fume hoods should be used while performing this procedure.

experimental-column-setup-for-studying-anaerobic-biogeochemical

Research

JoVE Journal

Environment

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 &#176;C) and thermophilic (55 &#176;C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 &#176;C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4/kgVS&#160;(VS: volatile solids) and 121 L CH4/kgVS&#160;at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the deep ocean. Roles of minerals are critical in many hydrothermal organic geochemical processes. Traditional hydrothermal methodology, which includes using reactors made of gold, titanium, platinum, or stainless-steel, is usually associated with the high cost or undesired metal catalytic effects. Recently, there is a growing tendency for using the cost-effective and inert quartz or fused silica glass tubes in hydrothermal experiments. Here, we provide a protocol for carrying out organic-mineral hydrothermal experiments in silica tubes, and we describe the essential steps in the sample preparation, experimental setup, products separation, and quantitative analysis. We also demonstrate an experiment using a model organic compound, nitrobenzene, to show the effect of an iron-containing mineral, magnetite, on its degradation under a specific hydrothermal condition. This technique can be applied to study complex organic-mineral hydrothermal interactions in a relatively simple laboratory system.

Earth-abundant minerals play important roles in the natural hydrothermal systems. Here, we describe a reliable and cost-effective method for the experimental investigation of organic-mineral interactions under hydrothermal conditions.

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...