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.

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