Summary of Study ST004539

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 PR002859. The data can be accessed directly via it's Project DOI: 10.21228/M83V8G This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004539
Study Title	Extracellular short-chain fatty acid level alteration
Study Summary	We investigated metabolic interactions between Bacteroides thetaiotaomicron (DSMZ 2079; BT) and Collinsella aerofaciens (DSMZ 3979; CA) by coculturing the two species under deoxycholic acid (DCA) stress alongside untreated controls. Cultures were grown in five replicates anaerobically in Brain Heart Infusion–supplemented (BHIS) medium at 37°C for 6 h with shaking, using Hungate tubes supplemented with 300 µM DCA where indicated. Across these conditions, we quantified extracellular amino acids, short-chain fatty acids, vitamin B6, and both extracellular and intracellular DCA derivatives to capture key metabolic responses to bile acid stress and microbial interactions. This section focuses on changes in extracellular short-chain fatty acid profiles, providing an overview of how bile acid stress and coculture influence short-chain fatty acid metabolism within this two-member community. In this experiment, C. aerofaciens consistently produced ornithine and citrulline, with citrulline detected at higher concentrations. These patterns likely relate to the arginine deiminase (ADI) pathway, which interconverts arginine and citrulline while generating ATP and ammonium. Under hypoxic conditions, this pathway may support C. aerofaciens energy production, while ammonium release could help buffer environmental acidification caused by B. thetaiotaomicron’s substantial acetate output CA: Collinsella aerofaciens; BT: Bacteroides thetaiotaomicron; Co: Coculture of the two species
Institute	Helmholtz Centre for Environmental Research
Last Name	Yan
First Name	Wang
Address	Permoserstraße 15
Email	yan.wang@ufz.de
Phone	+49 341 60251532
Submit Date	2026-01-07
Study Comments	Raw LC–MS acquisition files are uploaded for the study samples. 20241216_SCFA_BTnCA_DCA_YW.wiff and 20241218_SCFA_BTnCA_DCA_YW-1v50.wiff contain the full LC–MS batches, including all study samples measured at two dilution levels. BHI blanks are included within the 2 batch files.
Analysis Type Detail	LC-MS
Release Date	2026-01-19
Release Version	1

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Project:

Project ID:	PR002859
Project DOI:	doi: 10.21228/M83V8G
Project Title:	Coculture of Collinsella aerofaciens and Bacteroides thetaiotaomicron under bile acid stress reveals vitamin B6 exchange
Project Summary:	Background: Although the bile acid-mediated microbiome-host interaction is known to shape both the composition and functionality of the gut microbiome, the mechanisms by which bile acid stress influences specific microbial metabolic interactions remain poorly understood. To address this gap, we examine the metabolic interplay between two key gut microbes. Bacteroides thetaiotaomicron, one of the most abundant species, possesses a broad enzymatic repertoire for polysaccharide degradation, while Collinsella aerofaciens is associated with liver-related diseases and plays a role in modifying primary bile acids. Results: In anaerobic coculture, C. aerofaciens mitigated the inhibitory effects of deoxycholic acid (DCA) on B. thetaiotaomicron by absorbing DCA from the medium and partially converting it into glycine-conjugated derivatives. Proteomic analysis showed that DCA broadly disrupted amino acid and vitamin metabolism pathways, particularly in B. thetaiotaomicron. In contrast, coculture led to a general upregulation of these pathways in C. aerofaciens, with a marked activation of vitamin B6 metabolism. Additionally, C. aerofaciens exhibited increased production of citrulline and ornithine in coculture. Conclusions: C. aerofaciens alleviates DCA toxicity on B. thetaiotaomicron through absorption, while promoting amino acid and vitamin metabolism, including the vitamin B6 synthesis pathway, during coculture. These results suggest that microbial interactions can enhance resistance to bile acid stress and may influence gut microbiome resilience, with potential relevance for liver- and bile acid–related disorders.
Institute:	Helmholtz Centre for Environmental Research
Department:	Department of Molecular Toxicology
Laboratory:	Functional Metabolomics
Last Name:	Wang
First Name:	Yan
Address:	Permoserstraße 15, 04318, Leipzig, Germany
Email:	yan.wang@ufz.de
Phone:	+49 341 60251532

Subject:

Subject ID:	SU004718
Subject Type:	Bacteria
Subject Species:	Bacteroides thetaiotaomicron,Collinsella aerofaciens
Taxonomy ID:	818,74426
Genotype Strain:	Bacteroides thetaiotaomicron (DSMZ 2079; BT) and Collinsella aerofaciens (DSMZ 3979; CA)

Factors:

Subject type: Bacteria; Subject species: Bacteroides thetaiotaomicron,Collinsella aerofaciens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment	Replicate
SA537972	1_BT_B	BT	Control	1
SA537973	2_BT_B	BT	Control	2
SA537974	3_BT_B	BT	Control	3
SA537975	4_BT_B	BT	Control	4
SA537976	5_BT_B	BT	Control	5
SA537977	6_BT_DCA	BT	final concentration 300µM DCA	1
SA537978	7_BT_DCA	BT	final concentration 300µM DCA	2
SA537979	8_BT_DCA	BT	final concentration 300µM DCA	3
SA537980	9_BT_DCA	BT	final concentration 300µM DCA	4
SA537981	10_BT_DCA	BT	final concentration 300µM DCA	5
SA537982	11_CA_B	CA	Control	1
SA537983	12_CA_B	CA	Control	2
SA537984	13_CA_B	CA	Control	3
SA537985	14_CA_B	CA	Control	4
SA537986	15_CA_B	CA	Control	5
SA537987	16_CA_DCA	CA	final concentration 300µM DCA	1
SA537988	17_CA_DCA	CA	final concentration 300µM DCA	2
SA537989	18_CA_DCA	CA	final concentration 300µM DCA	3
SA537990	19_CA_DCA	CA	final concentration 300µM DCA	4
SA537991	20_CA_DCA	CA	final concentration 300µM DCA	5
SA537992	21_CO_B	Co	Control	1
SA537993	22_CO_B	Co	Control	2
SA537994	23_CO_B	Co	Control	3
SA537995	24_CO_B	Co	Control	4
SA537996	25_CO_B	Co	Control	5
SA537997	26_CO_DCA	Co	final concentration 300µM DCA	1
SA537998	27_CO_DCA	Co	final concentration 300µM DCA	2
SA537999	28_CO_DCA	Co	final concentration 300µM DCA	3
SA538000	29_CO_DCA	Co	final concentration 300µM DCA	4
SA538001	30_CO_DCA	Co	final concentration 300µM DCA	5
SA538002	B1	NA	Blank	1
SA538003	B2	NA	Blank	2
SA538004	B3	NA	Blank	3

Showing results 1 to 33 of 33

Showing results 1 to 33 of 33

Collection:

Collection ID:	CO004711
Collection Summary:	After 6 hours of incubation, 2.5 mL of bacterial suspension was harvested. 2 mL of that was centrifuged at 4°C for 10 minutes to separate the supernatant and pellet. Both fractions were immediately frozen at –80°C for further omics analysis.
Sample Type:	Bacterial culture supernatant

Treatment:

Treatment ID:	TR004727
Treatment Summary:	Hungate anaerobic culture tubes containing BHIS medium were supplemented with DCA to a final concentration of 300 µM and flushed with pure nitrogen gas. Strains were then inoculated into the tubes in five replicates each. Cultures were monitored at 600 nm using a Nanocolour® UV/VIS II spectrophotometer (Macherey-Nagel). After 6 hours of incubation, 2.5 mL of bacterial suspension was harvested. 2 mL of that was centrifuged at 4°C for 10 minutes to separate the supernatant and pellet. Both fractions were immediately frozen at –80°C for further omics analysis.

Treatment ID:

TR004727

Treatment Summary:

Hungate anaerobic culture tubes containing BHIS medium were supplemented with DCA to a final concentration of 300 µM and flushed with pure nitrogen gas. Strains were then inoculated into the tubes in five replicates each. Cultures were monitored at 600 nm using a Nanocolour® UV/VIS II spectrophotometer (Macherey-Nagel). After 6 hours of incubation, 2.5 mL of bacterial suspension was harvested. 2 mL of that was centrifuged at 4°C for 10 minutes to separate the supernatant and pellet. Both fractions were immediately frozen at –80°C for further omics analysis.

Sample Preparation:

Sampleprep ID:	SP004724
Sampleprep Summary:	For short-chain fatty acid analysis, 20 µL of cell culture supernatant was mixed with 20 µL acetonitrile, 20 µL 200 mM 3-nitrophenylhydrazine hydrochloride (3-NPH), and 20 µL 120 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide and subsequently incubated in the Thermo Mix at 40°C for 30 min, shaking at 300 rpm. The derivatized samples were then stored at -20°C until measurement and diluted with 10% acetonitrile.

Sampleprep ID:

SP004724

Sampleprep Summary:

For short-chain fatty acid analysis, 20 µL of cell culture supernatant was mixed with 20 µL acetonitrile, 20 µL 200 mM 3-nitrophenylhydrazine hydrochloride (3-NPH), and 20 µL 120 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide and subsequently incubated in the Thermo Mix at 40°C for 30 min, shaking at 300 rpm. The derivatized samples were then stored at -20°C until measurement and diluted with 10% acetonitrile.

Combined analysis:

Analysis ID	AN007624
Chromatography ID	CH005782
MS ID	MS007321
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 µm)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	NEGATIVE
Units	µM

Chromatography:

Chromatography ID:	CH005782
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 µm)
Column Temperature:	40°C
Flow Gradient:	2 minutes at 15% B, a 15-minute gradient from 15% to 50% B, 1 minute at 100% B, and 3 minutes re-equilibration at 15% B.
Flow Rate:	0.35 mL/min
Solvent A:	100% Water; 0.01% formic acid
Solvent B:	100% Acetonitrile; 0.01% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS007321
Analysis ID:	AN007624
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	For identification and quantification, a scheduled MRM method was used, with specific transitions for every metabolite. Data acquisition and peak integration were performed in Analyst® software. The experiment duration was 21 min with a cycle time of 1.0 s, corresponding to 1260 cycles, and an MRM detection window of 45 s; no scheduled ionization delay was applied. The target scan time per sMRM experiment was set to 1.0 s to ensure sufficient dwell time and sensitivity across all monitored transitions. The ion source was operated with a curtain gas of 35 psi, an IonSpray voltage of −4.2 kV, and a source temperature of 450°C. Ion source gas 1 and gas 2 were both set to 50 psi.
Ion Mode:	NEGATIVE
Analysis Protocol File:	SCFA_method.pdf