Name: laboratory simulation of an iron(ii)-rich precambrian marine upwelling system to explore the growth of photosynthetic bacteria
Uploaded: 2016-07-24T15:00:00
Description: Watch this Scientific Journal Video about Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria at JoVE.com

Mark Nordhoff assisted in the design and implementation of tubing connections. Ellen Struve helped to select and acquire equipment used.

1. Preparation of Culturing Medium
Note: Information on the required equipment, chemicals and supplies for the preparation of the culture medium is listed in Table 1. Italic alphanumerical codes in brackets refer to the equipment itemized in Table 2 and shown in Figure 1.
<ol>
	<li>Prepare 5 L of Marine Phototroph (MP) medium (referred to hereafter as &#34;medium&#34;) following the protocol of Wu et al.16. Adjust the pH ...

<ol>
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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=Photoferrotrophs+thrive+in+an+Archean+Ocean+analogue.">Photoferrotrophs thrive in an Archean Ocean analogue.</a> PNAS. 105 (41), 15938-15943 (2008).</li><li>Jones, 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=Biogeochemistry+of+manganese+in+ferruginous+Lake+Matano,+Indonesia.">Biogeochemistry of manganese in ferruginous Lake Matano, Indonesia.</a> Biogeosciences. 8 (10), 2977-2991 (2011).</li><li>Lliros, 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=Pelagic+photoferrotrophy+and+iron+cycling+in+a+modern+ferruginous+basin.">Pelagic photoferrotrophy and iron cycling in a modern ferruginous basin.</a> Sci. Rep. 5 (13803), (2015).</li><li>Koeksoy, E., Halama, M., Konhauser, K. O., Kappler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+modern+ferruginous+habitats+to+interpret+Precambrian+banded+iron+formation+deposition.">Using modern ferruginous habitats to interpret Precambrian banded iron formation deposition.</a> Int. J. Astrobiol. , 1-13 (2015).</li><li>Canfield, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+for+Proterozoic+ocean+chemistry.">A new model for Proterozoic ocean chemistry.</a> Nature. 396 (6710), 450-453 (1998).</li><li>Wu, W. F., 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=Characterization+of+the+physiology+and+cell-mineral+interactions+of+the+marine+anoxygenic+phototrophic+Fe(II)+oxidizer+Rhodovulum+iodosum+-+implications+for+Precambrian+Fe(II)+oxidation.">Characterization of the physiology and cell-mineral interactions of the marine anoxygenic phototrophic Fe(II) oxidizer Rhodovulum iodosum - implications for Precambrian Fe(II) oxidation.</a> FEMS Microbiol. Ecol. 88 (3), 503-515 (2014).</li><li>Hungate, R. E., Macy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Roll-Tube+Method+for+Cultivation+of+Strict+Anaerobes.">The Roll-Tube Method for Cultivation of Strict Anaerobes.</a> Bull. Ecol. Res. Comm. 17 (1), 123-126 (1973).</li><li>Van Baalen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+marine+blue-green+algae.">Studies on marine blue-green algae.</a> Bot. mar. 4 (1-2), 129-139 (1962).</li><li>Sakamoto, T., Bryant, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+at+low+temperature+causes+nitrogen+limitation+in+the+cyanobacterium+Synechococcus+sp.+PCC+7002.">Growth at low temperature causes nitrogen limitation in the cyanobacterium Synechococcus sp. PCC 7002.</a> Arch. Microbiol. 169 (1), 10-19 (1998).</li><li>Swanner, E. D., Mloszewska, A. M., Cirpka, O. A., Schoenberg, R., Konhauser, K. O., Kappler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+oxygen+production+in+Archaean+oceans+by+episodes+of+Fe(II)+toxicity.">Modulation of oxygen production in Archaean oceans by episodes of Fe(II) toxicity.</a> Nature Geosci. 8 (2), 126-130 (2015).</li><li>Stookey, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferrozine+-+a+New+Spectrophotometric+Reagent+for+Iron.">Ferrozine - a New Spectrophotometric Reagent for Iron.</a> Anal. Chem. 42 (7), 779-784 (1970).</li><li>Fitch, M. W., Koros, W. J., Nolen, R. L., Carnes, 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=Permeation+of+Several+Gases+through+Elastomers,+with+Emphasis+on+the+Deuterium+Hydrogen+Pair.">Permeation of Several Gases through Elastomers, with Emphasis on the Deuterium Hydrogen Pair.</a> J. Appl. Polym. Sci. 47 (6), 1033-1046 (1993).</li><li>Smith, A. J. B., Beukes, N. J., Gutzmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Composition+and+Depositional+Environments+of+Mesoarchean+Iron+Formations+of+the+West+Rand+Group+of+the+Witwatersrand+Supergroup,+South+Africa.">The Composition and Depositional Environments of Mesoarchean Iron Formations of the West Rand Group of the Witwatersrand Supergroup, South Africa.</a> Econ. Geol. 108 (1), 111-134 (2013).</li><li>Johnson, C. M., Beard, B. L., Klein, C., Beukes, N. J., Roden, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+isotopes+constrain+biologic+and+abiologic+processes+in+banded+iron+formation+genesis.">Iron isotopes constrain biologic and abiologic processes in banded iron formation genesis.</a> Geochim. Cosmochim. Acta. 72 (1), 151-169 (2008).</li><li>Krepski, S. T., Emerson, D., Hredzak-Showalter, P. L., Luther, G. W., Chan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+biogenic+iron+oxides+records+microbial+physiology+and+environmental+conditions:+toward+interpreting+iron+microfossils.">Morphology of biogenic iron oxides records microbial physiology and environmental conditions: toward interpreting iron microfossils.</a> Geobiology. 11 (5), 457-471 (2013).</li><li>Posth, N. R., Konhauser, K. O., Kappler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+processes+in+banded+iron+formation+deposition.">Microbiological processes in banded iron formation deposition.</a> Sedimentology. 60 (7), 1733-1754 (2013).</li><li>Maliva, R. G., Knoll, A. H., Simonson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secular+change+in+the+Precambrian+silica+cycle:+Insights+from+chert+petrology.">Secular change in the Precambrian silica cycle: Insights from chert petrology.</a> Geol. Soc. Am. Bull. 117 (7-8), 835-845 (2005).</li><li>Kappler, A., Pasquero, C., Konhauser, K. O., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+of+banded+iron+formations+by+anoxygenic+phototrophic+Fe(II)-oxidizing+bacteria.">Deposition of banded iron formations by anoxygenic phototrophic Fe(II)-oxidizing bacteria.</a> Geology. 33 (11), 865-868 (2005).</li><li>Krepski, S. T., Hanson, T. E., Chan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+novel+biomineral+stalk-forming+iron-oxidizing+bacterium+from+a+circumneutral+groundwater+seep.">Isolation and characterization of a novel biomineral stalk-forming iron-oxidizing bacterium from a circumneutral groundwater seep.</a> Environ. Microbiol. 14 (7), 1671-1680 (2012).</li><li>Czaja, A. D., Johnson, C. M., Beard, B. L., Roden, E. E., Li, W. Q., Moorbath, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Fe+oxidation+controlled+deposition+of+banded+iron+formation+in+the+ca.+3770+Ma+Isua+Supracrustal+Belt+(West+Greenland).">Biological Fe oxidation controlled deposition of banded iron formation in the ca. 3770 Ma Isua Supracrustal Belt (West Greenland).</a> Earth. Planet. Sci. Lett. 363 (1), 192-203 (2013).</li><li>Melton, E. D., Schmidt, C., Kappler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+iron(II)+oxidation+in+littoral+freshwater+lake+sediment:+the+potential+for+competition+between+phototrophic+vs.+nitrate-reducing+iron(II)-oxidizers.">Microbial iron(II) oxidation in littoral freshwater lake sediment: the potential for competition between phototrophic vs. nitrate-reducing iron(II)-oxidizers.</a> Front. Microbiol. 3 (197), 1-12 (2012).</li></ol>

Microbial communities in the Precambrian ocean were regulated by, or modified as a result of, their activity and the prevailing geochemical conditions. In interpreting the origins of BIF, researchers generally infer the presence or activity of microorganisms based on the sedimentology or geochemistry of BIF, e.g., Smith et al.23 and Johnson et al.24. The study of modern organisms in modern environments that have geochemical analogs to ancient environments is also a valuabl...

The Precambrian (4.6 to 0.541 Gy ago) atmosphere experienced a gradual build-up of photosynthetically produced oxygen (O2), perhaps punctuated by step changes at the so-called &#34;Great Oxidation Event&#34; (GOE) at approximately 2.4 Gy ago, and again in the Neoproterozoic (1 to 0.541 Gy ago) as atmospheric O2 approached modern levels1. Cyanobacteria are the evolutionary remnants of the first organisms capable of oxygenic photosynthesis2. Geochemical evidence and modeling studies support the role of shallow coastal environments in harboring active communities of cyanobacteria or organisms capable of oxygenic photosynthesis or oxygenic phototrophs, generating local oxygen oases in the surface ocean below a predominantly anoxic atmosphere3-5.
The deposition of Banded Iron Formations (BIFs) from seawater throughout the Precambrian points to iron(II) (Fe(II)) as a major geochemical constituent of seawater, at least locally, during their deposition. Some of the largest BIFs are deep-water deposits, forming off the continental shelf and slope. The amount of Fe deposited is incompatible from a mass balance standpoint with predominantly continental (i.e., weathering) source. Therefore, much of the Fe must have been supplied from hydrothermal alteration of mafic or ultramafic seafloor crust6. Estimates of the rate of Fe deposited outboard of coastal environments are consistent with Fe(II) supplied to the surface ocean via upwelling7. In order for Fe to be transported in upwelling currents, must have been present in the reduced, mobile form &#8212; as Fe(II). The average oxidation state of Fe preserved in BIF is 2.4 8 and it is generally thought that BIF preserve Fe deposited as Fe(III), formed when upwelling Fe(II) was oxidized, possibly by oxygen. Therefore, exploring potential Fe(II) oxidation mechanisms along slope environments is important to understand how BIF formed. Moreover, refined geochemical characterization of marine sediments has identified that ferruginous conditions, where Fe(II) was present in an anoxic water column, were a persistent feature of the oceans throughout the Precambrian, and may not have been limited to just the time and place where BIF were deposited9. Therefore, for at least two billion years of Earth&#39;s history, redox interfaces between Fe(II) and O2 in the shallow oceans were likely commonplace.
Numerous studies utilize modern sites that are chemical and/or biological analogs of different features of the Precambrian ocean. A good example are ferruginous lakes where Fe(II) is stable and present in sunlit surface waters while photosynthetic activity (including by cyanobacteria) was detected10-13. The results of these studies provide insight into the geochemical and microbial characteristics of an oxic to anoxic/ferruginous chemocline. However these sites are generally physically stratified with little vertical mixing14, rather than the chemical interfaces occurring in an upwelling system, and are thought to support the most oxygen production in Precambrian time4.
A natural analogue to explore the development of a marine oxygen oasis beneath an anoxic atmosphere, and at an Fe(II)-rich upwelling system in sunlit surface water column is not available on the modern Earth. Therefore, a laboratory system that can simulate a ferruginous upwelling zone and also support the growth of cyanobacteria and photoferrotrophs is needed. The understanding and identification of microbial processes and their interaction with an upwelling aqueous medium that represents Precambrian seawater promotes the understanding and can complement the information gained from the rock record in order to fully understand the distinctive biogeochemical processes on ancient Earth.
Toward that end, a laboratory-scale column was designed in which Fe(II)-rich seawater medium (pH neutral) was pumped into the bottom of the column, and pumped out from the top. Illumination was provided at the top to create a 4 cm wide &#34;photic zone&#34; that supported the growth of cyanobacteria in the top 3 cm. Natural environments are generally stratified and stabilized by physicochemical gradients, like salinity or temperature. In order to stabilize the water column on a lab-scale, the column cylinder was packed with a porous glass bead matrix that helped to maintain the establishment of geochemical patterns that developed during the experiment. A continuous N2/CO2 gas flow was applied to flush the headspace of the column in order to maintain an anoxic atmosphere reflective of an ocean prior to the GOE15. After a constant flux of Fe(II) was established, cyanobacteria were inoculated throughout the column, and their growth was monitored by cell counts on samples removed through sampling ports. Oxygen was monitored in situ by placing oxygen-sensitive optode foils onto the inner wall of the column cylinder and measurements were made with an optical fiber from outside the column. Aqueous Fe speciation was quantified by removing samples from depth-resolved horizontal sampling ports and analyzed with the Ferrozine method. The abiotic control experiments and results demonstrate proof-of-concept &#8212; that a laboratory scale analog of the ancient water column, maintained in isolation from the atmosphere, is achievable. Cyanobacteria grew and produced oxygen, and the reactions between Fe(II) and oxygen were resolvable. Herein, the methodology for design, preparation, assembly, execution, and sampling of such a column are presented, along with results from an 84 hr run of the column while inoculated with the marine cyanobacterium Synechococcus sp. PCC 7002.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Widdel flask (5 L)</td>
			<td>Ochs</td>
			<td>110015</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Glass bottles (5 L)</td>
			<td>Rotilabo</td>
			<td>Y682.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Glass pipettes (5 mL)</td>
			<td></td>
			<td>51714</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>0.22 &#181;m Steritop filter unit (0.22&#160;&#181;m Polyethersulfone membrane)</td>
			<td>Millipore</td>
			<td>X337.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Luer Lock glass syringe, filled with cotton</td>
			<td></td>
			<td>C681.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Luer Lock stainless steel needles (150 mm, 1.0 mm ID)</td>
			<td></td>
			<td>201015</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>433209</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>208094</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>KBr</td>
			<td>Sigma</td>
			<td>P3691</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Glass cylinder</td>
			<td></td>
			<td>Y310.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td></td>
			<td>7377.2</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Glass beads (&#248; 0.55 - 0.7&#160;mm)</td>
			<td></td>
			<td>11079105</td>
			<td>biospec.com</td>
		</tr>
		<tr>
			<td>Butyl rubber stopper (&#248; 1.2 cm)</td>
			<td></td>
			<td>271024</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Petri Dish, glass (&#248; 8.0 cm)</td>
			<td></td>
			<td>T939.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Polymers glue</td>
			<td></td>
			<td>OTTOSEAL S68</td>
			<td>adchem.de</td>
		</tr>
		<tr>
			<td>Optical oxygen sensor foil (for oxygen analysis, see below)</td>
			<td></td>
			<td>&#8211; on request &#8211;</td>
			<td>presens.de</td>
		</tr>
		<tr>
			<td>Rubber tubing (35 mm, 7 mm ID)</td>
			<td></td>
			<td>770350</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Luer Lock tube connector (3.0 mm, luer lock male = LLM)</td>
			<td></td>
			<td>P343.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Luer Lock tube connector (3.0 mm, luer lock female = LLF)</td>
			<td></td>
			<td>P335.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Rubber tubing (25 mm, 0.72 mm ID)</td>
			<td></td>
			<td>2600185</td>
			<td>newageindustries.com</td>
		</tr>
		<tr>
			<td>Rubber tubing (50 mm, 7 mm ID)</td>
			<td></td>
			<td>770350</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Luer Lock stainless steel needle (150 mm, 1.0 mm ID)</td>
			<td></td>
			<td>201015</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Luer Lock glass syringe (10 mL)</td>
			<td></td>
			<td>C680.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Loose cotton&#160;</td>
			<td></td>
			<td>&#8211;</td>
			<td></td>
		</tr>
		<tr>
			<td>Butyl rubber stopper (&#248; 1.75 cm)</td>
			<td></td>
			<td>271050</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Stainless steel needle (40 mm, 1.0 mm ID)</td>
			<td>Sterican</td>
			<td>4665120</td>
			<td>bbraun.de</td>
		</tr>
		<tr>
			<td>Luer Lock stainless steel needle (150 mm, 1.5 mm ID)</td>
			<td></td>
			<td>201520</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>position: Luer Lock female connector part at C.7</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polymers glue</td>
			<td></td>
			<td>OTTOSEAL S68</td>
			<td>adchem.de</td>
		</tr>
		<tr>
			<td>Stainless steel needle (120 mm, 0.7 mm ID)</td>
			<td>Sterican</td>
			<td>4665643</td>
			<td>bbraun.de</td>
		</tr>
		<tr>
			<td>Rubber tubing (40 mm, 0.74 mm ID)</td>
			<td></td>
			<td>2600185</td>
			<td>newageindustries.com</td>
		</tr>
		<tr>
			<td>Heat shrink tubing (35 mm, 3 mm ID shrunk)</td>
			<td></td>
			<td>541458 - 62</td>
			<td>conrad.de</td>
		</tr>
		<tr>
			<td>Tube clamp</td>
			<td></td>
			<td>STHC-C-500-4</td>
			<td>tekproducts.com</td>
		</tr>
		<tr>
			<td>Luer Lock tube connector (1.0 mm, LLF)</td>
			<td></td>
			<td>P334.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Luer Lock plastic cap (LLM)</td>
			<td></td>
			<td>CT69.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Glass bottle (5 L)</td>
			<td>Rotilabo</td>
			<td>Y682.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Butyl rubber stopper (for GL45)</td>
			<td></td>
			<td>444704</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Stainless steel capillary (300 mm, 0.74 mm ID)</td>
			<td></td>
			<td>56736</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Stainless steel capillary (50 mm, 0.74 mm ID)</td>
			<td></td>
			<td>56737</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Shrink tubing (35 mm, 3 mm ID shrunk)</td>
			<td></td>
			<td>541458 - 62</td>
			<td>conrad.de</td>
		</tr>
		<tr>
			<td>Rubber tubing (100 mm, 0.74 mm ID)</td>
			<td></td>
			<td>2600185</td>
			<td>newageindustries.com</td>
		</tr>
		<tr>
			<td>Luer Lock tube connector (1.0 mm, LLF)</td>
			<td></td>
			<td>P334.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Luer Lock glass syringe (10 mL)</td>
			<td></td>
			<td>C680.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Loose cotton&#160;</td>
			<td></td>
			<td>&#8211;</td>
			<td></td>
		</tr>
		<tr>
			<td>Butyl rubber stopper (&#248; 1.75 cm)</td>
			<td></td>
			<td>271050</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Stainless Steel needle (40 mm, 0.8 mm ID)</td>
			<td>Sterican</td>
			<td>4657519</td>
			<td>bbraun.de</td>
		</tr>
		<tr>
			<td>Luer Lock glass syringe (5 mL)</td>
			<td></td>
			<td>C679.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Butyl rubber stopper (&#248; 1.75 mm)</td>
			<td></td>
			<td>271050</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Stainless steel needle (40 mm, 0.8 mm ID)</td>
			<td>Sterican</td>
			<td>4657519</td>
			<td>bbraun.de</td>
		</tr>
		<tr>
			<td>Rubber tubing (40 mm, 0.74 mm ID)</td>
			<td></td>
			<td>2600185</td>
			<td>newageindustries.com</td>
		</tr>
		<tr>
			<td>Glass bottle (2 L)</td>
			<td>Rotilabo</td>
			<td>X716.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Butyl rubber stopper (for GL45)</td>
			<td></td>
			<td>444704</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Stainless steel capillary (50 mm, 0.74 mm ID)</td>
			<td></td>
			<td>56736</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Rubber tubing (30 mm x 0.74 mm ID)</td>
			<td></td>
			<td>2600185</td>
			<td>newageindustries.com</td>
		</tr>
		<tr>
			<td>Rubber tubing (100 mm x 0.74 mm ID)</td>
			<td></td>
			<td>2600185</td>
			<td>newageindustries.com</td>
		</tr>
		<tr>
			<td>Luer Lock tube connector (1.0 mm, LLF)</td>
			<td></td>
			<td>P334.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Luer Lock 3-way connector (LLF, 2x LLM)</td>
			<td></td>
			<td>6134</td>
			<td>cadenceinc.com</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Samsung</td>
			<td>SI-P8V151DB1US</td>
			<td>samsung.com</td>
		</tr>
		<tr>
			<td>Peristalic pump</td>
			<td>Ismatec</td>
			<td>EW-78017-35</td>
			<td>coleparmer.com</td>
		</tr>
		<tr>
			<td>Pumping tubing (0.89 mm ID)</td>
			<td></td>
			<td>EW-97628-26</td>
			<td>coleparmer.com</td>
		</tr>
		<tr>
			<td>Stainless steel capillary (200 mm, 0.74 mm ID)</td>
			<td></td>
			<td>56736</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Stainless steel capillary (400 mm, 0.74 mm ID)</td>
			<td></td>
			<td>56737</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Supel-Inert Foil (Tedlar - PFC) gas pack (10 L)</td>
			<td></td>
			<td>30240-U</td>
			<td>sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Rubber tube (30 mm, 6 mm ID)</td>
			<td></td>
			<td>770300</td>
			<td>labor-ochs.de</td>
		</tr>
		<tr>
			<td>Luer Lock tube connector (3.0 mm, LLM)</td>
			<td></td>
			<td>P343.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Luer Lock tube connector (3.0 mm, LLF)</td>
			<td></td>
			<td>P335.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Gas-tight syringe (20 mL)</td>
			<td></td>
			<td>C681.1</td>
			<td>carlroth.com</td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td>&#8211;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optic oxygen meter for oxygen quantification</td>
			<td>Presens</td>
			<td>TR-FB-10-01</td>
			<td>presens.de</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td></td>
			<td>&#8211;</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone glue for oxygen optodes</td>
			<td>Presens</td>
			<td>PS1</td>
			<td>presens.de</td>
		</tr>
	</tbody>
</table>

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.

Control experiment
Abiotic control experiments (10 days) demonstrated consistently low oxygen concentrations (O2 &#60;0.15 mg/L) with no significant fluctuations in the Fe(II)-profile throughout the upwelling water column. The formation of precipitates (presumably Fe(III)(oxyhydr-)oxides) in the medium reservoir and the slight decrease in the overall Fe(II) concentration from 500 &#181;M to...

Watch this Scientific Journal Video about Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria at JoVE.com

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.

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 ...

The overall goal of this experimental approach is to simulate a ferruginous upwelling system and to detect geochemical profiles of oxygen and aqueous iron in the water column when cyanobacteria were present and produces oxygen by photosynthesis. This newly developed method can help answer key questions in the field of earth's history such as the oxygenation of the atmosphere and the deposition of banded iron formations. The main advantage of this technique is that geochemical conditions still might have existed in a Precambrian ocean can be simulated on lab scale being investigated and seen to with modern methods.Though this method can provide insight into early earth studies, it can also be applied to other modern environments such as for geo-microbial investigations and chemical fluctuating in sediment bodies. Generally, individuals new to this method will struggle because setting up the experiment is not trivial but the video will provide information on the most critical steps performed in the protocol. Bacteria culturing, early assembly steps of the set up, and notes on sterilizing materials are all described in the text protocol.Note that the cell solution for Synechococcus 7002 must be anoxic for later experiments. Therefore, remove the soluble oxygen by bubbling the solution with anoxic nitrogen carbon dioxide via a 0.22 micron-filtered syringe, having a second needle in the stopper to release over pressure. In order to stop photosynthesis and the production of oxygen, cover the culture bottle.After five minutes of bubbling, flush the head space. To assemble the column, place it on a flat surface and stabilize it with a laboratory stand and clamps. Make sure to work under sterile conditions and carefully remove the aluminium foil from the top opening.Then fill the column with sterilized glass beads. Next, prepare a watch glass to tightly close the column. Apply glue to the inner surface where it will contact the column.Then lightly press it to the top of the column. Let it set for at least six hours. Proceed with making attachments.First, attach the head space gas exchange panel to the head space vent. Then insert the appropriate butyl rubber stoppers into the cotton filled glass syringes. Following this, connect the Luer Lock glass syringe to the corresponding stainless steel needle.Then connect the anoxic gas line to the head space gas exchange panel using another Luer Lock stainless steel needle. After making a few more connections, be sure to connect the medium distribution panel to the butyl stopper for the medium bottle. Flush the column instillation and capillary system at low pressure with anoxic gas.Let gas escape via the medium bottle capillary, the three way connector, and the sampling ports. Flush gas through the instillation for at least 20 minutes. To connect the stopper to the medium bottle, first close the anoxic gas flow with a hose clamp on the corresponding tube.Then lightly lift the butyl rubber stopper off the medium bottle. Next, insert a sterile cotton filled syringe with a bent needle into the head space of the medium bottle blowing gas as 50 millibar and flush the head space with anoxic gas. Then quickly remove and replace the rubber stopper with the prepared stopper with the capillary.This must be performed quickly and with strict attention to sterility to avoid adding oxygen and bacteria. Next, remove the long metal needle to close the lid. Then change the long metal needle of the glass syringe to a disposable needle and inject anoxic gas into the stopper to flush the head space of the medium bottle.Do this for at least four minutes. Then close the gas line and pull the injection needle out of the stopper. The remaining over pressure of gas will escape via the glass syringe.To proceed, connect the syringe to a gas pack loaded with anoxic gas while putting light pressure on the gas pack. The gas pack will compensate for the volume loss in the bottle when medium is pumped out of it. Next, connect an empty pre-flushed ten liter gas pack to the free port of the three way connector that is attatched to the discharge bottles.Make sure to keep the valve of the gas pack closed. Now reduce the pressure of the anoxic gas to below 0.1 millibar to maintain a steady outflow of gas from the out-gassing needle. Then set up the pump and release the clamp on the hose to access the media.Now open the valve of the empty gas pack and start the pump. The pack will collect air displaced from the discharge bottles as they are filled with medium. Monitor the medium distribution panel filling up with medium and remove the remaining gas from the panel as it fills by holding it in an inverted position.The gas is released from the capillaries connected to the pump. Adjust the pump to 0.45 liters per day and monitor the column it fills with medium. Lastly, install the light source.Position it two centimeters above the upper end of the column. Then cover the upper ten centimeters of the column with dark tape or aluminium foil. Then cover the whole instillation with a dark textile.For the inoculation, have six syringes with needles long enough to reach the center of the column body. Begin with sterilizing the outside of the butyl rubber stoppers on the six main sampling ports. Use 80%ethanol and flaming.Then flush a syringe with anoxic gas. Next, draw up one milliliter of the anoxic cell solution and inject it into the center of the column through the butyl rubber stoppers on the six sampling ports. If necessary, use sterile tweezers to stabilize the needle for injection.Every 24 hours, take samples from the column. Start at the top port and work down. First, flush the syringe with anoxic gas.Then fill it with anoxic gas. Next, quickly remove the plastic cap and put the syringe almost onto the tube connector. Then eject the gas in the syringe and firmly connect the syringe to the connector.Now remove the clamp and take a one milliliter sample from the column. Reattach the clamp before withdrawing the syringe. Then firmly close the connector with it's cap.Now repeat this process at the next port going down the column. A ten day control experiment with no inoculated bacteria demonstrated consistently low oxygen concentrations with no significant fluctuations in soluble iron throughout the upwelling water column. At day five, the lowest oxygen levels were below 15 milligrams per liter which is essentially anoxic.Prior to inoculation on the day zero, no precipitates were visible thus no oxygen was present that could oxidize soluble iron into iron precipitates. The soluble iron concentrations were constant throughout the upwelling water column. 84 hours after inoculation with cyanobacteria, a green color developed within the top two centimeters of the column indicative of growth.An orange band at negative three centimeters was indicative of precipitating iron due to oxygen production by the cyanobacteria. While prior to inoculation the initial oxygen concentration was essentially zero, after 84 hours of growth, the oxygen profile of the upper column rose significantly. Conversely, a drop in soluble iron from the upper depths was also measured.After watching this video, you should have a good understanding of how to set up and perform the experiment. The collection of liquid samples will allow you both to quantify geochemical parameters and perform cell analysis. Once mastered, the experiment can be set up within one day.After starting the run, changes in the water column can be monitored over a period of several weeks if it is performed properly. Following in this procedure, alternative set ups can be used to answer additional questions regarding insidious reductions and microbial distributions within sediment bodies.

laboratory-simulation-an-ironii-rich-precambrian-marine-upwelling

Research

JoVE Journal

Environment

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. 
This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.

Microbial sulfate reduction is a process of great importance in environmental biotechnology. The success of the sulfidogenic reactors depends among other factors on the microbial composition of the sludge. Here, we present a protocol to develop sulfidogenic sludge from hydrothermal vents sediments in a UASB reactor for reductive dechlorination purposes.

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...