Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA053797

Local Sample ID:	2N
Subject ID:	SU000952
Subject Type:	NMR based metabolomics of microbes
Subject Species:	Microbacterium sediminis
Taxonomy ID:	904291

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Subject:

Subject ID:	SU000952
Subject Type:	NMR based metabolomics of microbes
Subject Species:	Microbacterium sediminis
Taxonomy ID:	904291

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
2N	SA053797	FL010081	NT-S	Treatment

Collection:

Collection ID:	CO000946
Collection Summary:	A psychrotolerant strain of Microbacterium sediminis numbered as YLB-01 isolated from deep sea sediment of the Indian Ocean was used in this study. The strain can grow at temperatures ranging from 4 °C to 50 °C with an optimal growth temperature of 28 °C (Yu et al. 2013). Tryptone soy broth (TSB) medium, which contains 15 g/L tryptone, 5 g/L soy peptone, and 5 g/L NaCl, was used to cultivate the strain. The strain was maintained in glycerol tubes under -80 °C in a refrigerator and was activated using streaking inoculation on agar plates before use. For metabolomics research, a single colony was first inoculated from an agar plate to 32 test tubes containing 5 mL of TSB medium each and cultivated using a shaker under 28 °C for 12 hours. These seed cultures were then transferred to 150 mL Erlenmeyer flasks containing 100 mL of TSB medium each. In order to obtain a sufficient amount of cells, these cells were cultivated under normal temperature (28 °C) and atmospheric pressure (0.1 MPa) until logarithmic or stationary phase before transferred to lower temperature. Specifically, half of these samples were cultivated using a shaker under 28 °C for 18 hours to reach mid logarithmic phase (assigned as scenario L) and the other half were cultivated for 24 hours to reach stationary phase (assigned as scenario S) under the same condition. All samples were then transferred into 100 mL normal saline bags and put into a water-filled high-pressure chamber with the pressure set at 30 MPa. For half of the samples from a certain scenario (L or S) the temperature of the chamber was set at 28 °C. For the other half of the samples the temperature of the chamber was set at 4 °C. All samples were harvested after 7 days. The samples were grouped as NT-L, LT-L, NT-S, and LT-S based on their different cultivation conditions and sampling time
Sample Type:	cells

Treatment:

Treatment ID:	TR000966
Treatment Summary:	half of these samples were cultivated using a shaker under 28 °C for 18 hours to reach mid logarithmic phase (assigned as scenario L) and the other half were cultivated for 24 hours to reach stationary phase (assigned as scenario S) under the same condition. All samples were then transferred into 100 mL normal saline bags and put into a water-filled high-pressure chamber with the pressure set at 30 MPa. For half of the samples from a certain scenario (L or S) the temperature of the chamber was set at 28 °C. For the other half of the samples the temperature of the chamber was set at 4 °C. All samples were harvested after 7 days. The samples were grouped as NT-L, LT-L, NT-S, and LT-S based on their different cultivation conditions and sampling time

Treatment ID:

TR000966

Treatment Summary:

half of these samples were cultivated using a shaker under 28 °C for 18 hours to reach mid logarithmic phase (assigned as scenario L) and the other half were cultivated for 24 hours to reach stationary phase (assigned as scenario S) under the same condition. All samples were then transferred into 100 mL normal saline bags and put into a water-filled high-pressure chamber with the pressure set at 30 MPa. For half of the samples from a certain scenario (L or S) the temperature of the chamber was set at 28 °C. For the other half of the samples the temperature of the chamber was set at 4 °C. All samples were harvested after 7 days. The samples were grouped as NT-L, LT-L, NT-S, and LT-S based on their different cultivation conditions and sampling time

Sample Preparation:

Sampleprep ID:	SP000959
Sampleprep Summary:	All 100 mL of the fermentation broth in a flask was harvested and poured into a 250 mL centrifuge bottle and centrifuged at 6000g and 4 °C for 5 min. The supernatant was discarded and the cell pellets were quenched using 100 mL of a buffer composed of 3:2 methanol/water and 0.85% (wt./vol.) NaCl at -40 °C. The resuspended mixture was again centrifuged at 6000g and 4 °C for 5 min. The cell pellets were then washed using cold PBS for 3 times. The mixture was resuspended and transferred into a 5 mL Eppendorf tube during the 3rd wash and then centrifuged at 6000g and 4 °C for 5 min. The cell pellets were kept under -80 °C until use. Intracellular metabolites were extracted using a procedure adopted from Ye et al. (Ye et al. 2012). The frozen samples were homogenized in 600 μL of cold 1:1 acetonitrile/water buffer. To destroy the bacterial cells, the samples were further sonicated on wet ice for 180 cycles with each cycle consisting of 2 s pulses and 3 s stops. The supernatant was collected by centrifugation at 12000 g for 10 min at 4 °C. The remaining solid residues were further extracted using the same extract solution and intensively homogenized via vortexing. The second supernatant was collected after centrifugation and pooled with the first one. The combined supernatants from the two extractions were lyophilized, and stored at -80 °C. Immediately before 1H NMR measurements were taken, the extract powder was redisclosed in 550 µL phosphate buffer (50 mM K2HPO4/NaH2PO4, 10% D2O, 1mM 3-(Trimethylsilyl) propionate-2,2,3,3-d4 acid sodium salt (TSP), pH7.4). Subsequently, all the samples were vortexed and centrifuged at 12000 g for 15 min at 4 °C to remove any insoluble components. Finally, aliquots of the supernatant were transferred into 5 mm NMR tubes (Beckonert et al. 2007).

Sampleprep ID:

SP000959

Sampleprep Summary:

All 100 mL of the fermentation broth in a flask was harvested and poured into a 250 mL centrifuge bottle and centrifuged at 6000g and 4 °C for 5 min. The supernatant was discarded and the cell pellets were quenched using 100 mL of a buffer composed of 3:2 methanol/water and 0.85% (wt./vol.) NaCl at -40 °C. The resuspended mixture was again centrifuged at 6000g and 4 °C for 5 min. The cell pellets were then washed using cold PBS for 3 times. The mixture was resuspended and transferred into a 5 mL Eppendorf tube during the 3rd wash and then centrifuged at 6000g and 4 °C for 5 min. The cell pellets were kept under -80 °C until use. Intracellular metabolites were extracted using a procedure adopted from Ye et al. (Ye et al. 2012). The frozen samples were homogenized in 600 μL of cold 1:1 acetonitrile/water buffer. To destroy the bacterial cells, the samples were further sonicated on wet ice for 180 cycles with each cycle consisting of 2 s pulses and 3 s stops. The supernatant was collected by centrifugation at 12000 g for 10 min at 4 °C. The remaining solid residues were further extracted using the same extract solution and intensively homogenized via vortexing. The second supernatant was collected after centrifugation and pooled with the first one. The combined supernatants from the two extractions were lyophilized, and stored at -80 °C. Immediately before 1H NMR measurements were taken, the extract powder was redisclosed in 550 µL phosphate buffer (50 mM K2HPO4/NaH2PO4, 10% D2O, 1mM 3-(Trimethylsilyl) propionate-2,2,3,3-d4 acid sodium salt (TSP), pH7.4). Subsequently, all the samples were vortexed and centrifuged at 12000 g for 15 min at 4 °C to remove any insoluble components. Finally, aliquots of the supernatant were transferred into 5 mm NMR tubes (Beckonert et al. 2007).

Analysis:

MB Sample ID:	SA053797
Analysis ID:	AN001484
Analysis Type:	NMR
Num Factors:	4

NMR:

NMR ID:	NM000115
Analysis ID:	AN001484
Instrument Name:	Bruker Avance III 600 MHz spectrometer
Instrument Type:	FT-NMR
NMR Experiment Type:	1D 1H
Spectrometer Frequency:	600 MHz