<meta charset="utf8"></head><body>מידול נישות מתאנוטרופ – המערכת האקולוגית של מודל מזרקי השיפוע

<ol>
	<li>Brune, A., Frenzel, P., Cypionka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+at+the+oxic-anoxic+interface:+microbial+activities+and+adaptations.">Life at the oxic-anoxic interface: microbial activities and adaptations.</a> FEMS Microbiol Rev. 24 (5), 691-710 (2000).</li><li>Auman, A. J., Stolyar, S., Costello, A. M., Lidstrom, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+methanotrophic+isolates+from+freshwater+lake+sediment.">Molecular characterization of methanotrophic isolates from freshwater lake sediment.</a> Appl Environ Microbiol. 66 (12), 5259-5266 (2000).</li><li>Whittenbury, R., Phillips, K. C., Wilkinson, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment,+isolation+and+some+properties+of+methane-utilizing+bacteria.">Enrichment, isolation and some properties of methane-utilizing bacteria.</a> J Gen Microbiol. 61 (2), 205-218 (1970).</li><li>Koo, C., Rosenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+aerobic+biological+methane+oxidation.">Biochemistry of aerobic biological methane oxidation.</a> Chem Soc Rev. 50 (5), 3424-3436 (2021).</li><li>Strong, P. J., Xie, S., Clarke, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methane+as+a+resource:+Can+the+methanotrophs+add+value.">Methane as a resource: Can the methanotrophs add value.</a> Environ Sci Technol. 49 (7), 4001-4018 (2015).</li><li>Beals, D. G., Puri, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+methanotroph+phenotypes+to+genotypes+using+a+simple+spatially+resolved+model+ecosystem.">Linking methanotroph phenotypes to genotypes using a simple spatially resolved model ecosystem.</a> ISME J. 18 (1), wrae060 (2024).</li><li>Bussmann, I., Rahalkar, M., Schink, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+methanotrophic+bacteria+in+opposing+gradients+of+methane+and+oxygen.">Cultivation of methanotrophic bacteria in opposing gradients of methane and oxygen.</a> FEMS Microbiol. Ecol. 56 (3), 331-344 (2006).</li><li>Amaral, J. A., Knowles, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+methanotrophs+in+methane+and+oxygen+counter+gradients.">Growth of methanotrophs in methane and oxygen counter gradients.</a> FEMS Microbiol Lett. 126 (3), 215-220 (1995).</li><li>Danilova, O. V., 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=A+new+cell+morphotype+among+methane+oxidizers:+a+spiral-shaped+obligately+microaerophilic+methanotroph+from+northern+low-oxygen+environments.">A new cell morphotype among methane oxidizers: a spiral-shaped obligately microaerophilic methanotroph from northern low-oxygen environments.</a> ISME J. 10 (11), 2734-2743 (2016).</li><li>Ou, F., McGoverin, C., Swift, S., Vanholsbeeck, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+bacterial+cell+enumeration+using+flow+cytometry.">Absolute bacterial cell enumeration using flow cytometry.</a> J Appl Microbiol. 123 (2), 464-477 (2017).</li><li>Krause, S. M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lanthanide-dependent+cross-feeding+of+methane-derived+carbon+is+linked+by+microbial+community+interactions.">Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions.</a> Proc Natl Acad Sci USA. 114 (2), 358-363 (2017).</li><li>Masuko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+analysis+by+a+phenol-sulfuric+acid+method+in+microplate+format.">Carbohydrate analysis by a phenol-sulfuric acid method in microplate format.</a> Anal Biochem. 339 (1), 69-72 (2005).</li><li>Felz, S., Al-Zuhairy, S., Aarstad, O. A., van Loosdrecht, M. C. M., Lin, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+structural+extracellular+polymeric+substances+from+aerobic+granular+sludge.">Extraction of structural extracellular polymeric substances from aerobic granular sludge.</a> J Vis Exp. (115), e54534 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Costa, O. Y. A., Raaijmakers, J. M., Kuramae, 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=Microbial+extracellular+polymeric+substances:+Ecological+function+and+impact+on+soil+aggregation.">Microbial extracellular polymeric substances: Ecological function and impact on soil aggregation.</a> Front Microbiol. 9, 1636 (2018).</li><li>Sinke, A. J. C., Cottaar, F. H. M., Buis, K., Keizer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methane+oxidation+by+methanotrophs+and+its+effects+on+the+phosphate+flux+over+the+sediment-water+interface+in+a+eutrophic+lake.">Methane oxidation by methanotrophs and its effects on the phosphate flux over the sediment-water interface in a eutrophic lake.</a> Microb Ecol. 24 (3), 259-269 (1992).</li><li>Murase, J., Frenzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+methane-driven+microbial+food+web+in+a+wetland+rice+soil.">A methane-driven microbial food web in a wetland rice soil.</a> Environ Microbiol. 9 (12), 3025-3034 (2007).</li><li>Reim, A., L&#252;ke, C., Krause, S., Pratscher, J., Frenzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+millimetre+makes+the+difference:+High-resolution+analysis+of+methane-oxidizing+bacteria+and+their+specific+activity+at+the+oxic-anoxic+interface+in+a+flooded+paddy+soil.">One millimetre makes the difference: High-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic-anoxic interface in a flooded paddy soil.</a> ISME J. 6 (11), 2128-2139 (2012).</li><li>Rahalkar, M., Bussmann, I., Schink, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylosoma+difficile+gen.+nov.,+sp.+nov.,+a+novel+methanotroph+enriched+by+gradient+cultivation+from+littoral+sediment+of+Lake+Constance.">Methylosoma difficile gen. nov., sp. nov., a novel methanotroph enriched by gradient cultivation from littoral sediment of Lake Constance.</a> Int J Syst Evol Microbiol. 57 (5), 1073-1080 (2007).</li><li>Wang, L., Stegemann, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+high-quality+RNA+from+polysaccharide+matrices+using+cetlytrimethylammonium+bromide.">Extraction of high-quality RNA from polysaccharide matrices using cetlytrimethylammonium bromide.</a> Biomaterials. 31 (7), 1612 (2010).</li><li>Liyanage, N. M. N., Chandrasekara, B. C. H. W. M., Bandaranayake, P. C. 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+CTAB+protocol+for+obtaining+high-quality+total+RNA+from+cinnamon+(Cinnamomum+zeylanicum+Blume).">A CTAB protocol for obtaining high-quality total RNA from cinnamon (Cinnamomum zeylanicum Blume).</a> 3 Biotech. 11 (4), 201 (2021).</li><li>Semrau, J. D., DiSpirito, A. A., Gu, W., Yoon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metals+and+methanotrophy.">Metals and methanotrophy.</a> Appl Environ Microbiol. 84 (6), e02289-e02317 (2018).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salinity+significantly+affects+methane+oxidation+and+methanotrophic+community+in+Inner+Mongolia+lake+sediments.">Salinity significantly affects methane oxidation and methanotrophic community in Inner Mongolia lake sediments.</a> Front Microbiol. 13, 1067017 (2023).</li><li>Fox, A. L., Trefry, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+fluxes+from+recent+deposits+of+fine-grained,+organic-rich+sediments+in+a+Florida+estuary.">Nutrient fluxes from recent deposits of fine-grained, organic-rich sediments in a Florida estuary.</a> Front Mar Sci. , (2023).</li><li>He, L., 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=A+methanotrophic+bacterium+to+enable+methane+removal+for+climate+mitigation.">A methanotrophic bacterium to enable methane removal for climate mitigation.</a> Proc Natl Acad Sci. 120 (35), e2310046120 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% Gas mix analytical standard</td>
			<td>Supelco</td>
			<td>22561</td>
			<td>1% each component in nitrogen: carbon monoxide, carbon dioxide, hydrogen, methane and oxygen</td>
		</tr>
		<tr>
			<td>100% Methane</td>
			<td>Airgas</td>
			<td>ME CP300</td>
			<td>chemically pure grade</td>
		</tr>
		<tr>
			<td>15 ppm Gas mix analytical standard</td>
			<td>Supelco</td>
			<td>23470-U</td>
			<td>15 ppm each component in nitrogen: methane, ethane, ethylene, acetylene, propane, propylene, propyne, and n-butane</td>
		</tr>
		<tr>
			<td>1x Nitrate mineral salts&#160;</td>
			<td></td>
			<td>see CAS numbers below</td>
			<td>Dissolve the following in Mili-Q water and autoclave:&#160; 0.2 g/L MgSO4&#183;7H2O, 0.2 g/L CaCl2&#183;6H2O, 1 g/L KNO3, and 30 &#956;M LaCl3. Before use, add trace elements to a 1X final concentration and phosphate buffer (pH 6.8) to a final concentration of 5.8 mM.</td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>BD Biosciences</td>
			<td>305194</td>
			<td>sterile, Luer-Lok</td>
		</tr>
		<tr>
			<td>500x Trace elements</td>
			<td></td>
			<td>see CAS numbers below</td>
			<td>Dissolve the following in Milli-Q water: 1.0 g/L Na2-EDTA, 2.0 g/L FeSO4&#183;7H2O, 0.8 g/L ZnSO4&#183;7H2O, 0.03 g/L MnCl2&#183;4H2O, 0.03 g/L H3BO3, 0.2 g/L CoCl2&#183;6H2O, 0.6 g/L CuCl2&#183;2H2O, 0.02 g/L NiCl2&#183;6H2O, and 0.05 g/L Na2MoO&#183;2H2O.</td>
		</tr>
		<tr>
			<td>96 Well plate</td>
			<td>CELLTREAT</td>
			<td>229596</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>Acid phenol:chloroform:IAA (125:24:1)</td>
			<td>Invitrogen</td>
			<td>AM9720</td>
			<td>&#160;pH 4.5</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160</td>
			<td>molecular biology grade, CAS 9012-36-6</td>
		</tr>
		<tr>
			<td>Aluminum crimp seals</td>
			<td>VWR</td>
			<td>30618-460</td>
			<td>20 mm</td>
		</tr>
		<tr>
			<td>Bead beater</td>
			<td>Qiagen</td>
			<td>9003240</td>
			<td>TissueLyser III</td>
		</tr>
		<tr>
			<td>Butyl rubber stopper</td>
			<td>Chemglass Life Science</td>
			<td>50-143-854</td>
			<td>20 mm, blue</td>
		</tr>
		<tr>
			<td>Chloroform:isoamyl alcohol (24:1)</td>
			<td>Millipore Sigma</td>
			<td>25666</td>
			<td>BioUltra, for molecular biology</td>
		</tr>
		<tr>
			<td>Clark-type O2 microelectrode</td>
			<td>Unisense</td>
			<td>OX-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>Thermo Scientific</td>
			<td>R0601</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (Ambion)</td>
			<td>Invitrogen</td>
			<td>AM2222</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>CytoFLEX</td>
		</tr>
		<tr>
			<td>Gas chromatograph (flame ionization detection)</td>
			<td>Agilent</td>
			<td></td>
			<td>6890N</td>
		</tr>
		<tr>
			<td>Gastight analytical syringe</td>
			<td>Hamilton</td>
			<td>81220</td>
			<td>1750 TLL</td>
		</tr>
		<tr>
			<td>Gastight analytical syringe needle</td>
			<td>Hamilton</td>
			<td>7729-07</td>
			<td>22 G, metal hub needle, 2 in, point style 5</td>
		</tr>
		<tr>
			<td>Gas-tight vials</td>
			<td>Labco</td>
			<td>938W</td>
			<td>Exetainer vial: 12 mL, round bottom</td>
		</tr>
		<tr>
			<td>Glass culture tubes</td>
			<td>Bellco Glass</td>
			<td>2048-00150</td>
			<td>18 x 150 mm</td>
		</tr>
		<tr>
			<td>LiCl precipitation solution (7.5 M)</td>
			<td>Invitrogen</td>
			<td>AM9480</td>
			<td></td>
		</tr>
		<tr>
			<td>One-way stopcock</td>
			<td>VWR</td>
			<td>MFLX30600-00</td>
			<td>inlet port: female luer, outlet port: male luer lock</td>
		</tr>
		<tr>
			<td>Petri dish, square</td>
			<td>Fisher Scientific</td>
			<td>FB0875711A</td>
			<td>100 x 100 mm</td>
		</tr>
		<tr>
			<td>Phosphate buffer, 0.2 M (pH 6.8)</td>
			<td></td>
			<td>see CAS numbers below</td>
			<td>Dissolve the following in Milli-Q water and autoclave: 12.24 g/L KH2PO4, 26.29 g/L Na2HPO4 &#183; 7H2O</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE syringe filter tip</td>
			<td>Thermo Scientific</td>
			<td>03-050-469</td>
			<td>hydrophobic, pore size: 0.2 &#181;m, diameter: 4 mm</td>
		</tr>
		<tr>
			<td>Qubit 1x dsDNA High Sensitivity Assay Kit&#160;</td>
			<td>Invitrogen</td>
			<td>Q33230</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer</td>
			<td>Invitrogen</td>
			<td>Q33238</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator-5</td>
			<td>Zymo Research</td>
			<td>R1013</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum stopper</td>
			<td>Fisher Scientific</td>
			<td>03-340-302</td>
			<td>20 mm</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Biosciences</td>
			<td>302995</td>
			<td>Luer-Lock, 10 mL, single use, sterile</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump Systems Inc.</td>
			<td>1000-US</td>
			<td>NE-1000 one channel programmable</td>
		</tr>
		<tr>
			<td>SYTO9, propidium iodide, microspheres</td>
			<td>Invitrogen</td>
			<td>L34856</td>
			<td>LIVE/DEAD BacLight Bacterial Viability Kit</td>
		</tr>
		<tr>
			<td>Zirconia/silica beads</td>
			<td>BioSpec Products</td>
			<td>11079101z</td>
			<td>0.1 mm diameter</td>
		</tr>
		<tr>
			<td>Chemical reagents</td>
			<td>CAS number</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;6H2O</td>
			<td>7774-34-7</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>7791-13-1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated sulfuric acid</td>
			<td>7664-93-9</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CTAB, cetrimonium bromide</td>
			<td>57-09-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl2&#183;2H2O</td>
			<td>10125-13-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>64-17-5</td>
			<td></td>
			<td>&#160;&#160;</td>
		</tr>
		<tr>
			<td>FeSO4&#183;7H2O</td>
			<td>7782-63-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>10043-35-3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>69-63-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>7778-77-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>7757-79-1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LaCl3</td>
			<td>10099-58-8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>10034-99-8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2&#183;4H2O</td>
			<td>13446-34-9</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2CO3, sodium carbonate</td>
			<td>497-19-8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2-EDTA</td>
			<td>139-33-3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 &#183; 7H2O</td>
			<td>7782-85-6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2MoO&#183;2H2O</td>
			<td>10102-40-6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl, sodium chloride</td>
			<td>7647-14-5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NiCl2&#183;6H2O</td>
			<td>7791-20-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol (90% solution in water)</td>
			<td>108-95-2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVP40, polyvinylpyrrolidone</td>
			<td>9003-39-8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>1185-53-1</td>
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			<td>ZnSO4&#183;7H2O</td>
			<td>7446-20-0</td>
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			<td></td>
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			<td>&#946;-Mercaptoethanol</td>
			<td>60-24-2</td>
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agarose-based model ecosystem for cultivating methanotrophs in a methane-oxygen counter gradient

Microorganisms living at an anoxic-oxic interface often serve important ecological roles1. One example is aerobic methane-oxidizing bacteria (methanotrophs), which exist in counter gradients of methane and oxygen in soils and sediments2. These microorganisms possess unique metabolic and physiological characteristics that enable them to exploit the gas gradients present in their environments and have been the subject of ongoing research for decades3,4,5. Currently, most published research about methanotrophs and methane-oxidizing communities is based on work with homogenous planktonic cultures that often fail to capture the spatial and chemical gradients that are inherent to their natural microbial habitats. This limitation hinders our understanding of microbial physiology and our ability to link genomic information to phenotypic traits.
This protocol reports a simple, laboratory-based model ecosystem that creates reproducible conditions for studying both specific methanotrophs, such as Methylomonas sp. strain LW13, and methane-oxidizing communities directly from environmental soil samples. Importantly, cultivation in the gradient syringe results in counter gradient-specific phenotypes that are not present in homogenous planktonic cultures6, highlighting the system&#39;s ability to unveil new aspects of methanotroph physiology. Inspired by previously published model ecosystems7,8,9, the gradient syringe is a simplified method that can be used to collect chemical and molecular information from microorganisms cultured using this approach.
The reported procedures for genetic, chemical, and molecular analyses have been modified to work reliably on microbial cultures grown within a semi-solid agarose matrix. These procedures may also be useful for analyzing bacteria grown in other semi-solid agarose-based systems, such as those used for bacterial soft agar swimming assays. Adapting these analyses to spatially resolved contexts may open new avenues for studying microbial life in more ecologically relevant environments.

<meta charset="utf8"></head><body>מחבר זרקור: תכנון טכניקות פשוטות וזולות לגידול חיידקים מחמצני מתאן במעבדה

מודל אקולוגי מבוסס אגרוז לטיפוח מתאנוטרופים בשיפוע מונה מתאן-חמצן

We wanted to design a simple, inexpensive way to grow methane-oxidizing bacteria in the lab that more closely resembles the natural environment. We wanted to do this to uncover bacterial phenotypes that are missing from standard laboratory culture conditions, and ultimately link these phenotypes to their genetic determinants. The gradient syringe is the simplified version of previously described methods to culture methanotrophs in a methane-oxygen counter gradient.This method doesn't require continual flows of gas substrates, which allows multiple replicates to be run in parallel. We also can perform biochemical assays directly on the bacteria cultured within the agarose. Researchers have virtually unlimited access to bacterial genome sequences, but it is still difficult to put all of this information into context.Our findings show that it is critical to consider the environment in which a bacterium evolved, to better understand the role of individual genes. We plan to use techniques like comparative metabolomics and proteomics to learn more about how methanotrophs respond to their position within the methane-oxygen counter gradient. We also have plans to culture multiple strains in the same gradient syringe to see how they interact in a spatially resolved context.

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

agarose-based-model-ecosystem-for-cultivating-methanotrophs-methane

Research

JoVE Journal

Environment

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

335 Views.  University of Utah. רצינו לתכנן דרך פשוטה וזולה לגדל חיידקים מחמצני מתאן במעבדה שדומים יותר לסביבה הטבעית. רצינו לעשות זאת כדי לחשוף פנוטיפים חיידקיים שחסרים בתנאי תרבית מעבדה סטנדרטיים, ובסופו של דבר לקשר את הפנוטיפים האלה לגורמים הגנטיים שלהם. מזרק השיפוע הוא הגרסה הפשוט...

Video: מחבר זרקור: תכנון טכניקות פשוטות וזולות לגידול חיידקים מחמצני מתאן במעבדה

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists' understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs' natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.

A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.

Preparation and Extrusion of Agarose Gradient Syringes for Methanotroph Cultivation and Phenotype Analysis

To begin, obtain a culture plate with Methylomonas species LW13 colonies. Inoculate the colonies in six milliliters of nitrate mineral salts medium in a glass tube. Seal the tube with a serum stopper and aluminum crimp seal.Add methane using a syringe to create a final atmosphere of 50%volume by volume methane in the air. Shake this planktonic liquid culture at 200 revolutions per minute at room temperature until turbid, which takes about one day. Passage the liquid cultures at a one to 10 ratio into fresh media.Continue growing the liquid cultures of methanotrophs to log phase growth, reaching an optical density at 600 nanometers of approximately 0.5. To prepare the syringe, remove the accompanying plungers and place it in a sterile container. Attach a sterile polytetrafluoroethylene or PTFE filter tip to each syringe and place the syringe in a standard test tube rack with the tip facing down.Then, mix one milliliter of the cells with five milliliters of nitrate mineral salts medium and four milliliters of molten agarose in a sterile conical tube. Slowly pour the agarose mixture into the syringe up to the eight milliliter mark and let it solidify. After approximately 15 minutes, cap the syringe with a sterile 20 millimeter rubber butyl stopper.Secure the stoppers with lab tape and label the syringe. Next, fill a large 60-milliliter syringe with 100%methane and attach a PTFE filter tip connected to a sterile 23-gauge needle. Pierce the rubber stopper of the syringe with the agarose mixture with a large syringe, and insert a second sterile needle as a gas outlet.Depress the plunger of the large syringe to allow 20 milliliters of 100%methane to flush through the head space. Remove the outlet needle when there are one to two milliliters of methane left in the syringe to prevent oxygen backflow. Incubate the syringe with agarose mixture and methane at 18 degrees Celsius.To extrude the agarose, replace the PTFE filter tip with a sterile 23-gauge needle and the rubber stopper with the supplied syringe plunger. Slowly depress the plunger to dispense one milliliter increments into separate sterile 1.5-milliliter microcentrifuge tubes. The wild type LW13 strain formed a distinct horizontal band at a specific depth in the gradient syringe where both methane and oxygen concentrations were low.LW13 inoculated in gradient syringes showed a methane-oxygen counter gradient with methane depletion and oxygen consumption corresponding to the depth of the horizontal band. The OAT deletion mutant of LW13 lacked the distinct horizontal band formation observed in the wild type strain, indicating the gene's role in this phenotype.

הכנה ושחול של מזרקי אגרוז הדרגתיים לגידול מתאנוטרופ וניתוח פנוטיפ

Counting Cells in the Gradient Syringe for Studying Methanotroph Growth in Spatially Resolved Conditions

To begin, obtain the extruded agarose segments from gradient syringes, inoculated with either wild type or mutant methylomona species, LW13. Add 0.75 milliliters of 0.85%sodium chloride in water to the extruded agarose sample, and homogenize by vortexing. Further, dilute the sample 1 to 10 in a new micro centrifuge tube with 900 microliters of salt solution.Incubate the samples with three microliters of a one to one mixture of SYTO 9 and propidium iodide stains in the dark at room temperature for 15 minutes. To determine the cells per milliliter of agarose, sonicate the microsphere counting bead suspension in a water bath for five minutes. Then add 10 microliters of the suspension to the sample before flow cytometry analysis.Analyze samples using a flow cytometer. Compare side scatter versus forward scattered op plots between cell-free control samples and inoculated agarose samples to draw bacterial event voltage gates that exclude background agarose particles. To count colony forming units within the gradient syringe, add 800 microliters of nitrate mineral salts medium to the extruded agarose segment and vortex for 10 seconds to aid in pipetting.Prepare a sterile 96-well plate with 180 microliters of nitrate mineral salts medium in each well. Add 20 microliters of diluted agarose sample to each well in the first column and mix. Using a multi-channel pipette, serially dilute 20 microliters of samples tenfold, starting from the first row of wells.Label square grid plate containing nitrate mineral salts auger or media of choice. Using a multichannel pipette, spot five microliters from a column of the 96-well plate onto the auger plate. Flow cytometry using the extruded agarose samples confirmed that the OAT deletion mutant of LW13 showed reduced cell growth as compared to the wild type strain.Complementation of the mutant with the OAT gene restored cell numbers and band formation to levels similar to the wild type, indicating the gene's specific role in band formation.