<meta charset="utf8"></head><body>Metanotrof Nişlerinin Modellenmesi – Gradyan Şırınga Modeli Ekosistemi

<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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4&#183;7H2O</td>
			<td>7446-20-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>60-24-2</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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>Yazar Spotlight: Laboratuvarda Metan Oksitleyici Bakteri Yetiştirmek için Basit ve Ucuz Teknikler Tasarlamak

Metan-oksijen karşı gradyanında metanotrof yetiştirmek için agaroz tabanlı model ekosistem

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

408 Görüntüleme.  University of Utah. Laboratuvarda metan oksitleyici bakterileri yetiştirmek için doğal ortama daha çok benzeyen basit ve ucuz bir yol tasarlamak istedik. Bunu, standart laboratuvar kültürü koşullarında eksik olan bakteri fenotiplerini ortaya çıkarmak ve nihayetinde bu fenotipleri genetik belirleyicilerine bağlamak için yapmak istedik. Gradyan şırınga, bir metan-oksijen karşı gradyanında metanotrofları kültürlemek için daha önce açıklanan yöntemlerin ba...

Video: Yazar Spotlight: Laboratuvarda Metan Oksitleyici Bakteri Yetiştirmek için Basit ve Ucuz Teknikler Tasarlamak

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.