Summary of Study ST003583
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 PR002213. The data can be accessed directly via it's Project DOI: 10.21228/M8K531 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 | ST003583 |
| Study Title | Unraveling metabolism underpinning biomass composition shift in algae under simulated outdoor conditions using 13C fluxomics |
| Study Type | Algae |
| Study Summary | Algae have been widely investigated due to their promise as sustainable hosts for production of biofuels and biochemicals, and amenability to large scale outdoor cultivation. Algae biomass compositional quality is a key determinant of its value since valuable components such as carbohydrates and lipids can be converted to biofuels and other chemicals. The carbohydrate and lipid fraction of biomass are typically enriched during later stages of cultivation when nutrients such as nitrogen and phosphorous are depleted. In this work, we present a 13C labeling study to unravel the metabolic changes underpinning the transition from high protein content biomass under nitrogen replete status to a carbohydrate and lipid rich biomass under nitrogen depleted state in Scenedesmus UTEX 393. UTEX 393 tends to first accumulate carbohydrates and subsequently lipids, under outdoor cultivation conditions. Our work provides a unique insight into algal metabolism as we perform this study in a custom-built reactor equipped to match raceway pond operation. This is crucial to bridge the gap between ‘ideal’ laboratory conditions and ‘real-world’ cultivation conditions. We show that the transition to carbohydrates is characterized by increased flux diverted to starch instead of replenishing the CBB cycle for CO2 fixation whereas the subsequent transition to lipids is fueled by NADPH produced by PEP carboxylase-ME cycle. The transition to lipid rich biomass is activated by first lowering the ATP/NADPH demand. Exogenous addition of malic acid showed a nearly two-fold lipid content under continuous illumination, however this improvement was significantly diminished under diel environmental conditions indicating that further work on diel flux dynamics is warranted. Our results will have a significant impact in further developing fluxomics methods for algae under real world conditions and guiding metabolic engineering strategies to control the flux of carbon to biofuels and biochemicals using a sustainable algae platform. |
| Institute | National Renewable Energy Lab |
| Department | Biosciences |
| Laboratory | Laurens Lab |
| Last Name | Laurens |
| First Name | Lieve |
| Address | 15013 Denver West Paekway, Golden, CO 80401 |
| lieve.laurens@nrel.gov | |
| Phone | +1 720-273-6534 |
| Submit Date | 2024-11-13 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-12-01 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002213 |
| Project DOI: | doi: 10.21228/M8K531 |
| Project Title: | Unraveling metabolism underpinning biomass composition shift in algae under simulated outdoor conditions using 13C fluxomics |
| Project Type: | Life Sciences |
| Project Summary: | Algae have been widely investigated due to their promise as sustainable hosts for production of biofuels and biochemicals, and amenability to large scale outdoor cultivation. Algae biomass compositional quality is a key determinant of its value since valuable components such as carbohydrates and lipids can be converted to biofuels and other chemicals. The carbohydrate and lipid fraction of biomass are typically enriched during later stages of cultivation when nutrients such as nitrogen and phosphorous are depleted. In this work, we present a 13C labeling study to unravel the metabolic changes underpinning the transition from high protein content biomass under nitrogen replete status to a carbohydrate and lipid rich biomass under nitrogen depleted state in Scenedesmus UTEX 393. UTEX 393 tends to first accumulate carbohydrates and subsequently lipids, under outdoor cultivation conditions. Our work provides a unique insight into algal metabolism as we perform this study in a custom-built reactor equipped to match raceway pond operation. This is crucial to bridge the gap between ‘ideal’ laboratory conditions and ‘real-world’ cultivation conditions. We show that the transition to carbohydrates is characterized by increased flux diverted to starch instead of replenishing the CBB cycle for CO2 fixation whereas the subsequent transition to lipids is fueled by NADPH produced by PEP carboxylase-ME cycle. The transition to lipid rich biomass is activated by first lowering the ATP/NADPH demand. Exogenous addition of malic acid showed a nearly two-fold lipid content under continuous illumination, however this improvement was significantly diminished under diel environmental conditions indicating that further work on diel flux dynamics is warranted. Our results will have a significant impact in further developing fluxomics methods for algae under real world conditions and guiding metabolic engineering strategies to control the flux of carbon to biofuels and biochemicals using a sustainable algae platform. |
| Institute: | National Renewable Energy Lab |
| Department: | Biosciences |
| Laboratory: | Laurens Lab |
| Last Name: | Laurens |
| First Name: | Lieve |
| Address: | 15013 Denver West Parkway, Golden, Colorado, 80401, USA |
| Email: | lieve.laurens@nrel.gov |
| Phone: | +1 720-273-6534 |
| Funding Source: | This work was financially supported by U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The National Renewable Energy Laboratory (NREL) is operated for the US DOE under Contract No. DE-AC36-08GO28308. The views expressed in this article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. |
| Contributors: | Deshpande, A., Cawthon, B., Loob, J., Van Wychen, S., and Laurens, L.M.L |
Subject:
| Subject ID: | SU003712 |
| Subject Type: | Plant |
| Subject Species: | Scenedesmus obliquus UTEX 393 |
| Species Group: | Algae |
Factors:
Subject type: Plant; Subject species: Scenedesmus obliquus UTEX 393 (Factor headings shown in green)
| mb_sample_id | local_sample_id | Time (s) |
|---|---|---|
| SA391325 | A_0 | 0 |
| SA391331 | B_0 | 0 |
| SA391337 | D_0 | 0 |
| SA391326 | A_20M | 1200 |
| SA391332 | B_20M | 1200 |
| SA391338 | D_20M | 1200 |
| SA391327 | A_30S | 30 |
| SA391333 | B_30S | 30 |
| SA391339 | D_30S | 30 |
| SA391328 | A_5M | 300 |
| SA391334 | B_5M | 300 |
| SA391340 | D_5M | 300 |
| SA391329 | A_60M | 3600 |
| SA391335 | B_60M | 3600 |
| SA391341 | D_60M | 3600 |
| SA391330 | A_1M | 60 |
| SA391336 | B_1M | 60 |
| SA391342 | D_1M | 60 |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO003705 |
| Collection Summary: | Transient 13C labeling was performed at midday on days 2, 4, and 9 to represent states before, during, and after nitrogen depletion conditions in the SAGE #3 bioreactor system. Each experiment (before, during, and after) was performed separately due to the destructive nature of sampling. The entire labeling experiment occurred from 30 min before the midday light peak to 30 mins after. CO2 supply via pH feedback was stopped 5 minutes before 13C label introduction via a bolus addition of 11.76 mM NaH13CO3 (Cambridge Isotope Laboratories, Andover, MA) and 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffer (Sigma Aldrich, St. Louis) to each reactor. NaH13CO3 was preferred to 13CO2 to avoid gas-liquid mass transfer limitations and achieve rapid equilibrium. The buffer is needed to maintain culture pH at ~7.5. Six timepoints were chosen for metabolite sampling: 0 (unlabeled), 30 s, 1 min, 5 min, 20 min, and 1 hr and each of the six reactors represented a separate timepoint. These time points were chosen to capture the complete labeling dynamics of the rapidly labeling Calvin Benson Bassham (CBB) cycle metabolites as well as relatively slower labeling metabolites. Depending on culture density, 60-300 mL of culture was sampled using a 300 mL syringe, rapidly filtered via a fast filtration setup using 9 cm Fisherbrand glass fiber filters (Catalog No.09-804-90A) and quenched in liquid nitrogen bath. The whole process of drawing the sample to quenching consistently took ~15 s and was performed by a team of two researchers. The remaining culture was used for analysis of biomass composition, total nitrogen, and ash free dry weight (AFDW) in accordance with NREL laboratory analytical procedures. |
| Sample Type: | Algae |
Treatment:
| Treatment ID: | TR003721 |
| Treatment Summary: | Depending on culture density, 60-300 mL of culture was sampled using a 300 mL syringe, rapidly filtered via a fast filtration setup using 9 cm Fisherbrand glass fiber filters (Catalog No.09-804-90A) and quenched in liquid nitrogen bath. The whole process of drawing the sample to quenching consistently took ~15 s and was performed by a team of two researchers. The remaining culture was used for analysis of biomass composition, total nitrogen, and ash free dry weight (AFDW) in accordance with NREL laboratory analytical procedures |
Sample Preparation:
| Sampleprep ID: | SP003719 |
| Sampleprep Summary: | Biomass filters stored at -80°C were kept on dry ice prior to metabolite extraction. Filters were then transferred to 15 mL centrifuge tubes on dry ice and extraction was performed by adding 2 mL of methanol at -20°C followed by maceration until a pulp was formed. Subsequently, 2 mL of chloroform at 4°C was added and the pulp macerated. Once extraction was complete with the methanol:chloroform mixture, 5 mL of chloroform and 1 mL of 0.05% ammonium hydroxide (pH ~10.4) was added for phase separation. The extracts were vortexed gently followed by centrifugation at 4°C at 2500 g. The clear layer on top was removed, filtered using a syringe filter, diluted in acetonitrile (ACN) (3:1 ACN: extract) and finally transferred to a vial prior to injection. |
| Processing Storage Conditions: | Described in summary |
| Extraction Method: | Methanol-Chloroform-Water |
| Extract Storage: | -80℃ |
Chromatography:
| Chromatography ID: | CH004471 |
| Chromatography Summary: | The metabolites were separated using hydrophilic interaction chromatography (HILIC) using a BEH Amide column (1.7 µm, 2.1 mm X 150 mm, ACQUITY Premier BEH Amide, Waters Corporation) via a gradient method. Solvent A comprised of 20 mM ammonium acetate and 15 mM ammonium hydroxide in 97% 18.2 mΩ-cm water and 3% acetonitrile whereas solvent B comprised of 20 mM ammonium acetate and 15 mM ammonium hydroxide in 95% acetonitrile and 5% 18.2 mΩ-cm water. All the chemicals used were LC-MS grade. The chromatography method used a constant flow rate of 0.2 mL/min and a linear gradient to enable separation of a wide range of metabolites followed by a column regeneration step as follows: 90% solvent B for 1 min followed by a linear gradient down to 75% solvent B for 23 min, a linear gradient down to 45% solvent B in 2 min, a linear gradient down to 25% B in 4 min, followed by a step to the starting composition of 90% B and hold for 6 min for column regeneration for a total run time of 36 min. The column temperature was maintained at 25°C and injection volume was set to 20 µL. Metabolite extracts were diluted such that the final injection solvent was 75% acetonitrile. Data was collected using a Thermo Scientific Q-Exactive mass spectrometer in negative ion mode. The scan ranged from 75 to 1000 m/z with a resolution of 140,000. AGC target was set to 3e6 while maximum IT was 200 ms. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY Premier BEH Amide (150 x 2.1 mm, 1.7µm) |
| Column Temperature: | 25°C |
| Flow Gradient: | 90% solvent B for 1 min followed by a linear gradient down to 75% solvent B for 23 min, a linear gradient down to 45% solvent B in 2 min, a linear gradient down to 25% B in 4 min, followed by a step to the starting composition of 90% B and hold for 6 min for column regeneration for a total run time of 36 min |
| Flow Rate: | 0.2 mL/min |
| Internal Standard: | 1-13C Leucine |
| Solvent A: | 97% Water (18.2 mΩ-cm)/3% Acetonitrile; 20 mM ammonium acetate; 15 mM ammonium hydroxide |
| Solvent B: | 95% Acetonitrile/5% Water (18.2 mΩ-cm); 20 mM ammonium acetate; 15 mM ammonium hydroxide |
| Randomization Order: | Samples were randomized |
| Chromatography Type: | HILIC |
Analysis:
| Analysis ID: | AN005886 |
| Analysis Type: | MS |
| Chromatography ID: | CH004471 |
| Num Factors: | 6 |
| Num Metabolites: | 188 |
| Units: | Normalized Area Count |
