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MB Sample ID: SA188158
| Local Sample ID: | 200612_Smp_GBTFate2MT0-pos_C_pos_CYANO |
| Subject ID: | SU002089 |
| Subject Type: | Other organism |
| Subject Species: | Natural mixed marine microbial community |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002089 |
| Subject Type: | Other organism |
| Subject Species: | Natural mixed marine microbial community |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 200612_Smp_GBTFate2MT0-pos_C_pos_CYANO | SA188158 | FL023170 | Sample | Sample type |
| 200612_Smp_GBTFate2MT0-pos_C_pos_CYANO | SA188158 | FL023170 | 1 | Dilution factor |
| 200612_Smp_GBTFate2MT0-pos_C_pos_CYANO | SA188158 | FL023170 | 2 | Experiment number |
| 200612_Smp_GBTFate2MT0-pos_C_pos_CYANO | SA188158 | FL023170 | - | Timepoint |
Collection:
| Collection ID: | CO002082 |
| Collection Summary: | Samples were collected aboard the R/V Kilo Moana in April, 2019. Experiments were conducted at two stations: station 4 at 41°40.85’ N and 158°3.01’ W, and station 5 at 37°0.21’ N and 158°0.20’ W, respectively. Water for samples of the in situ conditions and for the incubation experiments were collected with Niskin bottles attached to the CTD from 15 m water depth, which was within the surface mixed layer, in the morning. |
| Sample Type: | Suspended Marine Particulate Matter |
| Collection Method: | CTD Niskin Bottle |
| Collection Location: | North Pacific |
| Volumeoramount Collected: | 2L-10L |
Treatment:
| Treatment ID: | TR002101 |
| Treatment Summary: | In order to determine the kinetics of GBT (glycine betaine) uptake, whole seawater was spiked with varying concentrations of 13C5, 15N1-GBT and incubated for 25–42 minutes. Water was collected into 2 L bottles around 8:00 am local time for both experiments. In order to minimize the biological transformation of 13C5, 15N1-GBT into other molecules and to limit the induction of enzymatic activity, the incubation time with 13C5, 15N1-GBT was kept short. The short incubation time necessitated that samples be spiked throughout the course of the day since only six samples could be processed at a time. Before and after the addition of 13C5, 15N1-GBT, bottles were kept in flow-through incubators with blue shading to be at in situ temperature and approximately mixed layer light conditions. Samples were spiked to have final concentrations of 0, 2, 5, 10, 50, 200, or 2000 nM 13C5, 15N1-GBT. Triplicates of each 13C5, 15N1-GBT concentration were processed for both experiments, with replicates spread throughout the sampling period. After incubation with the spiked molecule, seawater was filtered onto 47 mm diameter, 0.2 µm pore size PTFE (Omnipore) filters using a peristaltic pump, polycarbonate filter holder, and Masterflex PharMed BPT tubing (Cole-Parmer). Filtering time was 10–42 minutes with an average time of 22 minutes. Experimental blanks were collected for each spike concentration during the northern experiment by collecting filtrate from one replicate of each treatment and re-filtering filtrate onto a new filter. This provided a measure of dissolved organic compounds adsorbed onto the filter during processing. Filters were frozen in liquid nitrogen immediately after filtration and stored at -80 °C until the filters were extracted using the metabolite extraction method described in Boysen et al. (2018). For the GBT fate experiments, two time course incubation experiments were performed, one at each of the stations described above. For each experiment, 2 L bottles were filled with seawater collected with the CTD from 15 m depth (within the mixed layer) at approximately 6:00 am local time. All 2 L bottles were spiked with 500 nM 13C5, 15N1-GBT and incubated in temperature-controlled incubators at 10 °C and 14 °C for the north and south experiments, respectively. Samples for the initial timepoint (T0) were filtered directly after being spiked, resulting in actual incubation times of approximately 20 minutes. At each time point triplicate 2 L bottles were sampled for analysis of bacterial and picophytoplankton abundance and biomass via flow cytometry, metabolites, and total hydrolyzable amino acids. Timepoints sampled in the north experiment were 0, 4.5, 9, 12.5, 36, 50, and 98 hours. Timepoints sampled in the south experiment were 0, 6, 13, 25, 51, and 100 hours. For all samples, incubation time was calculated by taking the difference between the time the bottle was spiked and the midpoint time of sample filtration. |
| Treatment: | Isotopically labeled glycine betaine additions to natural seawater |
| Treatment Compound: | 13C5, 15N1 GBT (glycine betaine labeled with 5 carbon-13 atoms and one nitrogen-15 atom) |
| Treatment Dosevolume: | 0, 2, 5, 10, 50, 200, 2000 nM; 500nM |
| Treatment Doseduration: | 25-42 minutes; 0, 4.5, 6, 9,12.5, 13, 25, 36, 50, 51, 98, 100 hours |
Sample Preparation:
| Sampleprep ID: | SP002095 |
| Sampleprep Summary: | Polar and nonpolar metabolites were extracted using a modified Bligh−Dyer extraction using 1:1 methanol/water (aqueous phase) and dichloromethane (organic phase). Methodological blanks were extracted and analyzed along with each sample set. Each sample was aliquotted into two vials and isotope-labeled internal standards were added either before or after the extraction to one vial for all samples, blanks, and pooled samples. To evaluate the effect of obscuring variation due to different matrix strengths and analytical drift, pooled samples were run at both full and half concentration (diluted with water) at least three times throughout a sample set. |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Bligh-Dyer |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003271 | AN003272 | AN003273 | AN003274 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | HILIC | HILIC | Reversed phase |
| Chromatography system | Waters Acquity I-Class | Waters Acquity I-Class | Waters Acquity I-Class | Waters Acquity I-Class |
| Column | SeQuant ZIC-HILIC (150 x 2.1mm,5um) | SeQuant ZIC-HILIC (150 x 2.1mm,5um) | SeQuant ZIC-HILIC (150 x 2.1mm,5um) | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Triple quadrupole | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Waters Xevo-TQ-S | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | POSITIVE | NEGATIVE | POSITIVE |
| Units | nmol/L | Peak area | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH002415 |
| Chromatography Summary: | See protocol wkumler_20211201_100602_PR_CH_CH_Ingalls_Lab_LC_Methods.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002416 |
| Chromatography Summary: | See protocol wkumler_20211201_100602_PR_CH_CH_Ingalls_Lab_LC_Methods.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003043 |
| Analysis ID: | AN003271 |
| Instrument Name: | Waters Xevo-TQ-S |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | See protocol PR_MS_Ingalls_Lab_MS_Methods_TQS.txt |
| Ion Mode: | POSITIVE |
| MS ID: | MS003044 |
| Analysis ID: | AN003272 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See protocol PR_MS_Ingalls_Lab_MS_Methods_GBT.txt |
| Ion Mode: | POSITIVE |
| MS ID: | MS003045 |
| Analysis ID: | AN003273 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See protocol PR_MS_Ingalls_Lab_MS_Methods_GBT.txt |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003046 |
| Analysis ID: | AN003274 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See protocol PR_MS_Ingalls_Lab_MS_Methods_GBT.txt |
| Ion Mode: | POSITIVE |
