This research was supported in part by Grants-in-Aid for Scientific Research for challenging exploratory research (J.K.), and Scientific Research (A) (J.K. and S.M.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. A RIKEN FPR fellowship (R.C.E.) provided additional support. The authors express their gratitude to Drs. Eisuke Chikayama, Yasuyo Sekiyama and Mami Okamoto for technical assistance with NMR and statistical analyses.

1. Filter Preparation to Remove Extractables
<ol>
 <li>Use 25-mm diameter 0.22 &mu;m pore-size Durapore PVDF hydrophilic filters (Millipore). Place filters in a clean 500 ml Pyrex beaker using tweezers. Pre-rinse three times with distilled water. Swirl well as you rinse to prevent the filters from sticking to each other. Add 300 ml Milli-Q (Millipore) or equivalent high-quality water. Autoclave to facilitate complete removal of extractables from the filters. </li>
 <li>Pour off the Milli-Q and again triple rinse the f...

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The filtration and metabolite extraction method demonstrated here allows for microbial planktonic biomass to be collected in sufficient amount for NMR metabolomics. While only extraction of aqueous-soluble metabolites using KPi and 1D 1H NMR is demonstrated, other extraction solvents and spectroscopic approaches can be used. One useful example is the use of deuterated methanol as a semi-polar solvent, which has been shown to produce superior NMR spectra from heterogeneous samples and is less sensitive to conta...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>0.22 &mu;m hydrophilic Durapore PVDF filters, 25 mm</td>
			<td>Millipore </td>
			<td>GVWP02500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microanalysis Filter Holder, 25 mm, fritted glass support</td>
			<td>Millipore</td>
			<td>XX1002500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3-place manifold, 47 mm, stainless steel</td>
			<td>Millipore</td>
			<td>XX2504735</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Wako</td>
			<td>169-04245</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Wako</td>
			<td>164-04295</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Deuterium oxide, 2H &gt; 90%</td>
			<td>Campridge Isotope Laboratoties</td>
			<td>DLM-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DSS</td>
			<td>Fluka</td>
			<td>92754</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Automill</td>
			<td>Tokken</td>
			<td>TK-AM4</td>
			<td>Stainless steel crushers included</td>
		</tr>
		<tr>
			<td>Thermomixer comfort</td>
			<td>Eppendorf</td>
			<td>5355 000.011</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagenode</td>
			<td>UCD-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vacuum evaporator</td>
			<td>EYELA</td>
			<td>CVE-3100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NMR</td>
			<td>Bruker </td>
			<td>DRX-500 with 5 mm-TXI probe</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectral binning tool</td>
			<td>Originally developed</td>
			<td>FT2DB</td>
			<td><a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a></td>
		</tr>
		<tr>
			<td>Metabolite annotation tool and database</td>
			<td>Originally developed</td>
			<td>SpinAssign</td>
			<td><a href="http://prime.psc.riken.jp/?action=nmr_search">http://prime.psc.riken.jp/?action=nmr_search</a></td>
		</tr>
	</tbody>
</table>

concentration of metabolites from low-density planktonic communities for environmental metabolomics using nuclear magnetic resonance spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

Watch this Scientific Journal Video about Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

concentration-metabolites-from-low-density-planktonic-communities-for

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Biology

12.7K ビュー.  RIKEN Advanced Science Institute. 微生物のプランクトンコミュニティからの代謝物抽出のための方法が示されています。コミュニティ全体のサンプリングが特別に用意されたフィルター上にろ過することにより達成される。凍結乾燥後、水性の水溶性代謝物が抽出されます。このアプローチは、自然または実験的な微生物群集のトランスオミクス研究への環境メタボロミクスの応用が可?...