Summary of Study ST003577
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002207. The data can be accessed directly via it's Project DOI: 10.21228/M8BN6T This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003577 |
| Study Title | Multi ‘omics indicate depth-discrete partitioning of nitrogen metabolism in a toxic Planktothrix rubescens bloom in the winter water column |
| Study Type | Metabolomics |
| Study Summary | Limnological sampling efforts have traditionally focused on the summer water column of larger lakes. For example, hundreds of researchers visit prominent systems like the Laurentian Great Lakes to investigate summer cyanobacterial blooms. Concurrently, the recent advancement of meta-omics techniques has led to a surge in molecular studies within these systems. However, these approaches have created several knowledge gaps: smaller, regional lakes remain largely unstudied, the winter ecology of lakes is poorly understood, and meta-omics techniques often fail to make meaningful scientific contributions when used in isolation. To address these gaps, we conducted a combined metagenomic and metabolomic field study focused on a local community concern: the winter pink discoloration of Mead’s Quarry (Knoxville, TN). This case study was designed and executed by seven graduate students at the University of Tennessee-Knoxville enrolled in the course MICR 669: Advanced Techniques in Field Microbiology (Spring 2023). Utilizing a $3,000 budget, the team carried out in-depth meta-omics research. Our metagenomic analyses and phylogenetic confirmations identified a bloom of the pink cyanobacterium Planktothrix rubescens as the primary cause. Physiochemical measurements revealed that the bloom was concentrated in the light-limited (<1 μmol photons m⁻² s⁻¹) metalimnion of the cold (<14°C) water column. Additionally, microcystin-LR levels exceeded the U.S. EPA limit for recreational exposure (8 μg L⁻¹). Untargeted metabolomics indicated that P. rubescens communities were differentially partitioning nitrogen metabolism strategies by depth (0.5 m vs. 2.0 m). This study highlights the previously underappreciated roles of arginine biosynthesis and pyrimidine metabolism in the ecological success of P. rubescens. We propose a novel ecological hypothesis regarding the proliferation of this harmful cyanobacterial bloom former in the light-limited metalimnion. Overall, our findings suggest that meaningful meta-omics research can be conducted locally with limited funding as part of a course-based curriculum involving at least seven graduate students. Furthermore, we demonstrate that the synergistic use of multiple meta-omics techniques is feasible on a small scale while yielding significant scientific impact. |
| Institute | University of Tennessee |
| Department | Microbiology |
| Laboratory | Steve Wilhelm |
| Last Name | Abiodun |
| First Name | Blessing |
| Address | 652 Buehler Dabney Hall, Knoxville |
| babiodun@vols.utk.edu | |
| Phone | 8653349978 |
| Submit Date | 2024-10-31 |
| Num Groups | 3 |
| Total Subjects | 12 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-11-20 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002207 |
| Project DOI: | doi: 10.21228/M8BN6T |
| Project Title: | Multi-omics indicate depth-discrete partitioning of nitrogen metabolism in a toxic Planktothrix rubescens bloom in the winter water column |
| Project Type: | Multiomics characterization of Meads Quarry Water after Harmful Algae Bloom |
| Project Summary: | This project attempts to: 1. Identify the dominant cyanobacteria bloom former in a local lake in Knoxville (Meads Quarry) Tennessee using metagenomics. 2. Use a complementary technique of metabolomics to understand the metabolism within this cyanobacteria 3. Identify how different physiochemical parameters affect the metabolism of the cyanobacteria identified across various limniological stratification |
| Institute: | University of Tennessee |
| Department: | Microbiology |
| Laboratory: | Steve Wilhelm |
| Last Name: | Abiodun |
| First Name: | Blessing |
| Address: | 652 Buehler Dabney Hall, Knoxville |
| Email: | babiodun@vols.utk.edu |
| Phone: | 8653349978 |
Subject:
| Subject ID: | SU003706 |
| Subject Type: | Water sample |
Factors:
Subject type: Water sample; Subject species: - (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Depth |
|---|---|---|---|
| SA389890 | D1_R1 | Water | 0.5m |
| SA389891 | D1_R2 | Water | 0.5m |
| SA389892 | D1_R3 | Water | 0.5m |
| SA389893 | D1_R4 | Water | 0.5m |
| SA389894 | D2_R1 | Water | 2.0m |
| SA389895 | D2_R2 | Water | 2.0m |
| SA389896 | D2_R3 | Water | 2.0m |
| SA389897 | D2_R4 | Water | 2.0m |
| SA389898 | D3_R1 | Water | 6.0m |
| SA389899 | D3_R2 | Water | 6.0m |
| SA389900 | D3_R3 | Water | 6.0m |
| SA389901 | D3_R4 | Water | 6.0m |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO003699 |
| Collection Summary: | Water sampling for metabolomics analysis was conducted using a standardized filtration method to ensure clean and precise sample collection. We prepared materials, including a 0.2 µm filter, syringes, swinnex filter holders, and pre-labeled cryovials. After rinsing the sampling tripod and ensuring the collection bucket was thoroughly mixed, we collected water samples. Each sample was filtered by assembling the swinnex with the filter and pushing 120 mL through the apparatus, recording the total volume filtered until saturation. After filtration, we carefully removed the filter, folded it to preserve the cellular material, and placed it into a pre-labeled cryovial. The samples were then stored in liquid nitrogen, and subsequently at -80°C, until extraction. This process minimized contamination and optimized sample integrity for downstream metabolomics analysis. |
| Sample Type: | Water |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003715 |
| Treatment Summary: | No treatment |
Sample Preparation:
| Sampleprep ID: | SP003713 |
| Sampleprep Summary: | The flash-frozen filters were placed in empty petri dishes and allowed to thaw gradually at 4°C. Once thawed, the filters were unfolded with the cell side facing down, and metabolites were extracted following established protocols (Lu et al., 2010; Rabinowitz & Kimball, 2007). Specifically, 1.5 mL of an extraction solvent (40% methanol, 20% water, 40% acetonitrile, and 0.1 M formic acid) was added to fully immerse each filter. The petri dishes were then chilled at -20°C for 20 minutes to enhance metabolite release. After chilling, the extraction solvent containing the released metabolites was transferred to microcentrifuge tubes. The filters were turned cell-side up, and any remaining cells were extracted with an additional 400 µL of solvent, which was also transferred to the tubes. The tubes were vortexed and centrifuged at 15,000 rpm for 5 minutes at 4°C to separate the metabolite-rich supernatant from the pellet. The supernatants were carefully transferred to new microcentrifuge tubes and dried under nitrogen gas. Once dried, the samples were reconstituted in 300 µL of LC-MS grade water, vortexed, and centrifuged again at 15,000 rpm for 5 minutes at 4°C to ensure uniformity. The final aliquots were then placed into autosampler vials for UHPLC-HRMS analysis. |
| Processing Storage Conditions: | 4℃ |
| Extract Storage: | 4℃ |
Combined analysis:
| Analysis ID | AN005873 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Phenomenex Synergi Hydro-RP (100 x 2mm,2.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH004461 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Phenomenex Synergi Hydro-RP (100 x 2mm,2.5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 0 min, 0% B; 2.5 min, 0% B; 5 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; 25 min, 0% B |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 97% water/3% methanol; 11mM tributylamine; 15 mM acetic acid |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005593 |
| Analysis ID: | AN005873 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw mass spectral files were converted to mzML files using a package from ProteoWizard, msConverter. All mzML files were imported into an open-source software, metabolomics analysis and visualization engine (El-MAVEN) where metabolites were manually identified using an in-house library based on exact mass (±5 ppm) and retention time (±2 min) |
| Ion Mode: | NEGATIVE |
