Name: determination of the settling rate of clay/cyanobacterial floccules
Uploaded: 2018-06-11T14:00:00
Description: Watch this Scientific Journal Video about Determination of the Settling Rate of Clay/Cyanobacterial Floccules at JoVE.com

The authors gratefully acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (05448, 165831 and 213411).

1. Preparing Cyanobacterial Cultures
<ol>
	<li>Preparing inoculation cultures using solid media
	<ol>
		<li>Obtain axenic cyanobacterial cells from the American Type Culture Collection or Pasteur Culture Collection. For example, the unicellular, marine Synechococcus sp. PCC 7002 was obtained from the Pasteur Culture Collection, it will be referred to hereafter as Synechococcus.</li>
		<li>Maintain Synechococcus cells on plates containing solid media (A+ liquid media<sup class...

<ol>
	<li>Macquaker, H. S., Keller, M. A., Davies, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algal+blooms+and+"marine+snow":+mechanisms+that+enhance+preservation+of+organic+carbon+in+ancient+fine-grained+sediments.">Algal blooms and "marine snow": mechanisms that enhance preservation of organic carbon in ancient fine-grained sediments.</a> J. Sediment. Res. 80, 934-942 (2010).</li><li>Tyson, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sedimentation+rate,+dilution,+preservation+and+total+organic+carbon:+some+results+of+a+modeling+study.">Sedimentation rate, dilution, preservation and total organic carbon: some results of a modeling study.</a> Org. Geochem. 32, 333-339 (2001).</li><li>Piper, D. Z., Calvert, S. 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+marine+biogeochemical+perspective+on+black+shale+deposition.">A marine biogeochemical perspective on black shale deposition.</a> Earth-Sci. Rev. 95, 63-96 (2009).</li><li>Sengco, M. R., Li, A. S., Tugend, K., Kulis, D., Anderson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+red-+and+brown-tide+cells+using+clay+flocculation+I.+Laboratory+culture+experiments+with+Gymnodiniumbreve+and+Aureococcus+anophagefferens.">Removal of red- and brown-tide cells using clay flocculation I. Laboratory culture experiments with Gymnodiniumbreve and Aureococcus anophagefferens.</a> Mar. Ecol. Prog. Ser. 210, 41-53 (2001).</li><li>Guenther, M., Bozelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+algae-clay+aggregation.">Factors influencing algae-clay aggregation.</a> Hydrobiologia. 523, 217-223 (2004).</li><li>Archambault, M. -. C., Grant, J., Bricelj, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+efficiency+of+the+dinoflagellate+Heterocapsa+triquetra+by+phosphatic+clay+and+implications+for+the+mitigation+of+harmful+algal+blooms.">Removal efficiency of the dinoflagellate Heterocapsa triquetra by phosphatic clay and implications for the mitigation of harmful algal blooms.</a> Mar. Ecol. Prog. Ser. 253, 97-109 (2003).</li><li>Beaulieu, S. E., Sengco, M. R., Anderson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+clay+to+control+harmful+algal+blooms:+deposition+and+resuspension+of+clay/algal+flocs.">Using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs.</a> Harmful Algae. 4, 123-138 (2005).</li><li>de Magalh&#227;es, L., Noyma, N., Furtado, L., Mucci, M., van Oosterhout, F., Husza, V., Marinho, M., L&#252;rling, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+coagulants+and+ballast+compounds+in+removal+of+cyanobacteria+(Microcystis)+from+water+of+the+tropical+lagoon+Jacarepagu&#225;+(Rio+de+Janeiro,+Brazil).">Efficacy of coagulants and ballast compounds in removal of cyanobacteria (Microcystis) from water of the tropical lagoon Jacarepagu&#225; (Rio de Janeiro, Brazil).</a> Estuaries and Coasts. 40, 121-133 (2017).</li><li>Li, L., Pan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+method+for+flocculating+harmful+algal+blooms+in+marine+and+fresh+waters+using+modified+sand.">A universal method for flocculating harmful algal blooms in marine and fresh waters using modified sand.</a> Environ. Sci. Tech. 47, 4555-4562 (2013).</li><li>Miranda, M., Noyma, N., Pacheco, F. S., de Magalh&#227;es, L., Pinto, E., Santos, S., Soares, M., Huszar, V., L&#252;rling, M., Marinho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+combined+coagulant+and+ballast+to+remove+harmful+cyanobacterial+blooms+in+a+tropical+shallow+system.">The efficiency of combined coagulant and ballast to remove harmful cyanobacterial blooms in a tropical shallow system.</a> Harmful Algae. 65, 27-39 (2017).</li><li>Pan, G., Chen, J., Anderson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+local+sands+for+the+mitigation+of+harmful+algal+blooms.">Modified local sands for the mitigation of harmful algal blooms.</a> Harmful Algae. 10, 381-387 (2011).</li><li>Shi, W., Tan, W., Wang, L., Pan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+Microcystis+aeruginosa+using+cationic+starch+modified+soils.">Removal of Microcystis aeruginosa using cationic starch modified soils.</a> Water Research. 97, 19-25 (2016).</li><li>Du, J., Pushkarova, R. A., Smart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-SEM+study+of+aggregate+and+floc+structure+changes+during+clay+settling+and+raking+processes.">A cryo-SEM study of aggregate and floc structure changes during clay settling and raking processes.</a> Int. J. Miner. Process. 93, 66-72 (2009).</li><li>Stevens, S. E., Porter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+in+Agmenellum+quadruplicatum.">Transformation in Agmenellum quadruplicatum.</a> Proc. Natl. Acad. Sci. U. S. A. 77, 6052-6056 (1980).</li><li>Owttrim, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicases+in+cyanobacteria:+biochemical+and+molecular+approaches.">RNA helicases in cyanobacteria: biochemical and molecular approaches.</a> Methods Enzymol. 511, 385-403 (2012).</li><li>Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+Assignments,+Strain+Histories+and+Properties+of+Pure+Cultures+of+Cyanobacteria.">Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria.</a> Microbiology. 111, 1-61 (1979).</li><li>Chamot, D., Owttrim, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cold+shock-induced+RNA+helicase+gene+expression+in+the+cyanobacterium+Anabaena+sp.+strain+PCC+7120.">Regulation of cold shock-induced RNA helicase gene expression in the cyanobacterium Anabaena sp. strain PCC 7120.</a> J. Bacteriol. 182, 1251-1256 (2000).</li><li>Sutherland, B. R., Barrett, K. J., Gingras, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clay+settling+in+fresh+and+salt+water.">Clay settling in fresh and salt water.</a> Environ. Fluid Mech. 15, 147-160 (2014).</li><li>Porra, R. J., Thompson, W. A., Kriedemann, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+accurate+extinction+coefficients+and+simultaneous+equations+for+assaying+chlorophylls+a+and+b+extracted+with+four+different+solvents:+verification+of+the+concentration+of+chlorophyll+standards+by+atomic+absorption+spectroscopy.">Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy.</a> Biochim. Biophys. Acta. 975, 384-394 (1989).</li><li>Liu, Y. X., Alessi, D. S., Owttrim, G. W., Petrash, D. E., Mloszewska, A. M., Lalonde, S. V., Martinez, R. E., Zhou, Q. X., Konhauser, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+reactivity+of+Synechococcus+sp.+PCC+7002:+implications+for+metal+sorption+from+seawater.">Cell surface reactivity of Synechococcus sp. PCC 7002: implications for metal sorption from seawater.</a> Geochim. Cosmochim. Acta. 169, 30-44 (2015).</li><li>Playter, T., Konhauser, K., Owttrim, G., Hodgson, C., Warchola, T., Mloszewska, A. M., Sutherland, B., Bekker, A., Zonneveld, J. -. P., Pemberton, S. G., Gingras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe-clay+interactions+as+a+mechanism+for+the+preservation+of+organic+matter+and+trace+metal+biosignatures+in+black+shales.">Microbe-clay interactions as a mechanism for the preservation of organic matter and trace metal biosignatures in black shales.</a> Chem Geol. 459, 75-90 (2017).</li><li>Verspagen, J. M. H., Visser, P. M., Huisman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+with+clay+causes+sedimentation+of+the+buoyant+cyanobacteria+Microcystis+spp.">Aggregation with clay causes sedimentation of the buoyant cyanobacteria Microcystis spp.</a> Aquat. Microb. Ecol. 44, 165-174 (2006).</li><li>Avnimelech, Y., Troeger, B. W., Reed, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutual+flocculation+of+algae+and+clay:+evidence+and+implications.">Mutual flocculation of algae and clay: evidence and implications.</a> Science. 216, 63-65 (1982).</li><li>Chen, L., Men, X., Ma, M., Li, P., Jiao, Q., Lu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+release+by+Aphanothece+halophytica+inhibits+cyanobacteria/clay+flocculation.">Polysaccharide release by Aphanothece halophytica inhibits cyanobacteria/clay flocculation.</a> J. Phycol. 46, 417-423 (2010).</li><li>Pan, G., Zhang, M. -. M., Chen, H., Zou, H., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+cyanobacterial+blooms+in+Taihu+Lake+using+local+soils.+I.+Equilibrium+and+kinetic+screening+on+the+flocculation+of+Microcystis+aeruginosa+using+commercially+available+clays+and+minerals.">Removal of cyanobacterial blooms in Taihu Lake using local soils. I. Equilibrium and kinetic screening on the flocculation of Microcystis aeruginosa using commercially available clays and minerals.</a> Environ. Poll. 141, 195-200 (2006).</li></ol>

Flocculation catalyzed by cyanobacterial cell-clay interaction has attracted a lot of interest in the fields of ecology and engineering2,3,4,5,6,7,8,9,10,11,1...

Using present environmental conditions and processes to infer past depositional mechanisms has long been an underpinning of sedimentology. While modern depositional analogues, such as the Black Sea, have been used to understand the deposition of organic-rich, fine-grained deposits, laboratory experiments have the potential to shed additional light on the origin of shale deposits. One area of inquiry in the genesis of black shales is the deposition rate and mechanism of original formation. Traditionally, it has been hypothesized that black shales formed in environments where the sedimentation rate, primary productivity, and organic matter respiration rates promote the preservation of organic matter in the sediment1,2,3. However, the role of cyanobacterial and clay flocculation has largely remained unconsidered. This mechanism of flocculation would allow for rapid deposition of organic-rich, fine-grained sediments to occur, and does not necessitate low-oxygen. Considering this premise, this protocol has two goals: 1) measure the sedimentation rate of cyanobacterial/clay floccules, and 2) visualize the sedimentation process in real time. This methodology, in addition to geochemical analysis, has been used to demonstrate that cyanobacterial/clay flocculation may in fact be an important mechanism for shale formation1. While originally intended for modelling shale deposition, this method is applicable to other disciplines such as biology and environmental remediation where the influence of clay input on bacterial metabolism and population need to be measured.
Numerous studies have been conducted to observe the flocculation of cyanobacteria and clay, for mitigating harmful algal blooms2,3,4,5,6,7,8,9,10,11,12. However, while measuring cell concentration over time, these studies have not applied cyanobacteria/clay flocculation to modelling the deposition of the rock record. As such, these studies lack a visual component, which can be critical when modelling past sedimentological processes. Additionally, the majority of studies utilize cell-counting (e.g., Pan et al.11), which can be laborious. Our method, with recent advances in measuring cyanobacterial flocculation, determines the changes in cyanobacterial cell concentration by measuring chlorophyll a (Chl a) at discrete time intervals. Pairing Chl a measurement with visual data is a new approach, which can be used to infer depositional conditions. The images generated can also be used to calculate sedimentation rate after the work from Du et al.13. The combination of visual and numerical data strengthens the reliability of the results. Furthermore, we outline additional protocols allowing for the sedimentation of dead biomass and clay to also be observed. This is important when considering past sedimentological environments, where live and dead biomass may have co-occurred. Differences in the behavior of dead biomass during the flocculation (for example, decrease in flocculation rate) would potentially have sedimentological implications.

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="665">
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	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">cyanobacteria (in this study: Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803)</td>
			<td style="width:135px;">Pasteur Culture Collection</td>
			<td style="width:147px;">PCC 7002 or PCC 6803</td>
			<td style="width:167px;">used to inoculate the plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">agar</td>
			<td style="width:135px;">Thermo Scientific</td>
			<td style="width:147px;">CM0003</td>
			<td style="width:167px;">used to fill two petri dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Petri plates (standard bacteriology, 100 x 15 mm)</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">82.1473.001</td>
			<td style="width:167px;">2 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 L heat resistant Erlenmeyer flask</td>
			<td style="width:135px;">Pyrex</td>
			<td style="width:147px;">4980-125</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mL heat resistant Erlenmeyer flask</td>
			<td style="width:135px;">Pyrex</td>
			<td style="width:147px;">4980-250</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Nichrome inoculating loop with handle</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">14-956-103</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">tinfoil</td>
			<td style="width:135px;">Reynolds Wrap Aluminum Foil</td>
			<td style="width:147px;">89079-067</td>
			<td style="width:167px;">50 cm required; used to cover foam stopper and neck of erlenmeyer flasks</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">growth media (e.g. A+)</td>
			<td style="width:135px;"></td>
			<td style="width:147px;"></td>
			<td style="width:167px;">1050 mL required; produced using composition described in tables 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bunsen Burner</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">S95941</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">plastic tubing</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">S504591</td>
			<td style="width:167px;">1 m required; used to create the bubbling apparatus</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">sponge stopper</td>
			<td style="width:135px;">Jaece Industries Inc</td>
			<td style="width:147px;">14-127-40E</td>
			<td style="width:167px;">1 required; hole made in center for pipette; used for constructin the bubbling apparatus</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">acrylic sheet&#160;</td>
			<td style="width:135px;">Home Depot</td>
			<td style="width:147px;">Optix clear acrylic sheet model # MC-102S</td>
			<td style="width:167px;">1 required; used to construct acrylic tank (20 x 30 x 5.1 cm)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">clear waterproof silicone adhesive</td>
			<td style="width:135px;">Home Depot</td>
			<td style="width:147px;">Loctite clear silicone model # 908570</td>
			<td style="width:167px;">1 required; used to construct acrylic tank (20 x 30 x 5.1 cm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">camera or video recorder</td>
			<td style="width:135px;">Panasonic</td>
			<td style="width:147px;">HC-V770 HD camcorder</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tripod</td>
			<td style="width:135px;">Magnus</td>
			<td style="width:147px;">VT-300</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">black cloth</td>
			<td style="width:135px;">primomart</td>
			<td style="width:147px;">&#160;EAN 0726670162199; Part number 680254blacknappedfr</td>
			<td style="width:167px;">1 required; duvetyne light block-out cloth; approximatly 152 x 213 cm to cover tank experiment</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">heat resistant serological pipet</td>
			<td style="width:135px;">corning incorporated C708510</td>
			<td style="width:147px;">13-671-101G</td>
			<td style="width:167px;">1 required; used to create the bubbling apparatus</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">sample vials&#160;</td>
			<td style="width:135px;">Dynalon</td>
			<td style="width:147px;">S30467</td>
			<td style="width:167px;">at least 12 (will vary with time interval chosen)</td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;">heat resistant glass pipette</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">Corning Incorporated C708510, 13-671-101G</td>
			<td style="width:167px;">1 required; used to create the bubbling apparatus; Polystyrene serological pipet would also work, but should be connected to the tubing and stopper after the rest of the apparatus is autoclaved.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">microcentrifuge</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:147px;">22 62 120-3</td>
			<td style="width:167px;">&#160;1 required;Comparable products may be used if capable of centrifuging 1.5 -2 mL microfuge tubes at 13,000 x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">vortex machine (Vortex-Genie 2)</td>
			<td style="width:135px;">Scientific Industries, Inc</td>
			<td style="width:147px;">SI-0236</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">100% methanol</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">A412-500 SDS</td>
			<td style="width:167px;">at least 12 mL (1mL per sample) required; Caution: Flammable, toxic. Wear gloves and safety glasses. Do not use or store near ignition source. Alternate sources may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cuvettes (1.6&#160; mL, polystyrene)</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">67.742</td>
			<td style="width:167px;">at least 12 required</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">spectrophotometer</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">222-271600</td>
			<td style="width:167px;">1 required; Pharmacia Biotech Novaspec ll could also be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">light bulbs</td>
			<td style="width:135px;">Home Depot</td>
			<td style="width:147px;">model # 451807; internet #205477895; store SKU #1001061538</td>
			<td style="width:167px;">6-8 bulbs required to provide light for the tank experiments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pipette (Pipetman Classic P1000</td>
			<td style="width:135px;">Gilson</td>
			<td style="width:147px;">F123602</td>
			<td style="width:167px;">used to collect samples</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">37 % Hydrochloric acid</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:147px;">258148</td>
			<td style="width:167px;">Caution: Corrosive and toxic. Wear lab coat, safety glasses and acid-resistant gloves while using. Prepared to 4 N before use by dilution into deionized water in a chemical fumehood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Foam stopper (small)</td>
			<td style="width:135px;">Canlab</td>
			<td style="width:147px;">T 1385</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Foam stopper (large)</td>
			<td style="width:135px;">Canlab</td>
			<td style="width:147px;">T 1387</td>
			<td style="width:167px;">Requires some intact stoppers and some with a single hole through the centre</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">30 &#176;C incubator/growth room with continuous illumination</td>
			<td style="width:135px;"></td>
			<td style="width:147px;"></td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">70 % Ethanol</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">BP8201500</td>
			<td style="width:167px;">30 mL&#160; required;Caution: Toxic and flammable. Wear lab coat and safety glasses</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">hydrophobic air filter (Midisart 2000, 0.2 &#181;m)</td>
			<td style="width:135px;">Sartorius</td>
			<td style="width:147px;">17805</td>
			<td style="width:167px;">1 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">clay (e.g. kaolin)</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:147px;">MFCD00062311</td>
			<td style="width:167px;">at least 50 g required</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">microfuge tubes (2 mL, polypropylene)</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">72.695.500</td>
			<td style="width:167px;">Comparable products may be used. At least 12 (will vary with time interval chosen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1000 &#181;L pipet tips</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:147px;">70.762</td>
			<td style="width:167px;">1 required</td>
		</tr>
	</tbody>
</table>

determination of the settling rate of clay/cyanobacterial floccules

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction of clay particles with reactive, planktonic cyanobacterial cells to the sedimentary record is under studied. This interaction is a potentially major contributor to shale depositional models. Within a lab setting, the flocculation and sedimentation rates of these materials can be examined and measured in a controlled environment. Here, we detail a protocol for measuring the sedimentation rate of cyanobacterial/clay mixtures. This methodology is demonstrated through the description of two sample experiments: the first uses kaolin (a dehydrated form of kaolinite) and Synechococcus sp. PCC 7002 (a marine coccoid cyanobacteria), and the second uses kaolin and Synechocystis sp. PCC 6803 (a freshwater coccoid cyanobacteria). Cyanobacterial cultures are mixed with varying amounts of clay within a specially designed tank apparatus optimized to allow continuous, real-time video and photographic recording. The sampling procedures are detailed as well as a post-collection protocol for precise measurement of chlorophyll a from which the concentration of cyanobacterial cells remaining in suspension can be determined. Through experimental replication, a profile is constructed that displays sedimentation rate.

When exposed to clay, cyanobacterial cells are brought out of suspension22. This is demonstrated in the representative results given here. To determine the effect of clay on cyanobacterial populations and to observe the sedimentation rates, two experiments were conducted during which Synechococcus and Synechocystis were exposed to 50 g/L kaolin clay (Table 5&#8211;6, Figure 2&#8211;3)...

Watch this Scientific Journal Video about Determination of the Settling Rate of Clay/Cyanobacterial Floccules at JoVE.com

Determination of the Settling Rate of Clay/Cyanobacterial Floccules

The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules.

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction ...

This method can help answer key questions in the field of sedimentology such as how organic rich, fine grain sediments can be deposited under oxygenated conditions. The main advantage of this technique is that both quantitative results and a visual record of sedimentation are produced. To begin, prepare Cyanobacterial cultures according to the text protocol.Then rinse one 250 milliliter and two one-liter heat resistant Erlenmeyer flasks with four molar hydrochloric acid solution followed by distilled water. Fill the three flasks with liquid media. Seal the flasks with gas permeable foam stoppers and cover the necks with aluminum foil.Then label and autoclave the flasks at 121 degrees Celsius and 100 kilopascals for 25 minutes. After this, allow them to cool to room temperature. To inoculate the media, flame sterilize an inoculation loop.Then use the loop to transfer the Cyanobacterial culture into the 250 milliliter flask that contains 50 milliliters of medium. After inoculation, resterilize the lip of the 250 milliliter flask and replace the foam stopper and aluminum foil. Next, prepare the growth chamber as outlined in the text.Transfer the inoculated 50 milliliter culture to the growth chamber and incubate with a maintained temperature of 30 degrees Celsius and agitation of 150 rpm. After successful culture growth, place the 50 milliliter culture and the one liter flask containing 400 milliliters of liquid medium in a laminar flow hood. Remove the foam stoppers and foil caps.Use a Bunsen burner to sterilize the rim of each flask. Pour the culture from the 250 milliliter flask into the one liter flask containing 400 milliliters of medium. Then resterilize the neck of the one liter flask and attach the bubbling apparatus.Finally, agitate the one liter flask at 30 degrees Celsius and 150 rpm with the bubbler apparatus attached to an air pump which circulates a humidified air mixture through the solution at the rate of 300 milliliters per minute. Incubate the culture for 120 to 168 hours at 30 degrees Celsius in the growth chamber. Using a graduated cylinder, transfer additional growth medium to the 400 milliliter culture until the final volume is one liter, thus diluting the culture.Add the solution to an acrylic tank. Label two milliliter microcentrifuge tubes and place them near the acrylic tank. Then weigh out 50 grams of kaolin clay.Use a camcorder to record the experiment. Pipette one milliliter of the Cyanobacterial sample into a labeled microcentrifuge tube. Use the sample to determine the Cyanobacterial cell count by measuring the optical density at 750 nanometers.Quickly the pour clay into the tank while vigorously stirring with a stirring stick for 10 to 15 seconds. Then use a P1000 pipette to extract one milliliter of the sample for chlorophyll a determination and record this time as T0.Take additional samples at appropriate time intervals as outlined in the text. Next, use the modified procedure outlined in the text to determine the chlorophyll a concentration in each cell sample.Use a microcentrifuge to pellet the cells from each sample at room temperature for three minutes at 13, 000 times gravity. Then use a P1000 pipette to remove the excess medium. Resuspend the cell pellet with one milliliter of 100%methanol.Incubate the samples at negative 20 degrees Celsius for 24 hours. After centrifuging the samples again, use a spectrophotometer to determine the chlorophyll a concentration. This experiment illustrates the effect of clay on Cyanobacterial populations and their settling rates.Cultures of the Cyanobacteria Synechococcus that are mixed with kaolin clay are seen to have higher settling rates than the cultures containing only Cyanobacteria. After being exposed to the clay, Cyanobacterial cells settle out of suspension within 10 minutes. Cultures of the Cyanobacteria Synechocystis mixed with clay are also seen to have higher settling rates than cultures containing only Cyanobacteria.After being exposed to clay, these Cyanobacterial cells are also brought out of suspension in 10 minutes. After watching this video, you should have a good understanding of how to measure sedimentation rates of Cyanobacteria and clay mixtures which can be used to inform depositional models of black shells.

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