Measuring non-steady-state metabolic fluxes in starch-converting faecal microbiota in vitro
- 1 November 2010
- journal article
- Published by Brill in Beneficial Microbes
- Vol. 1 (4), 391-406
- https://doi.org/10.3920/bm2010.0038
Abstract
This paper explores human gut bacterial metabolism of starch using a combined analytical and computational modelling approach for metabolite and flux analysis. Non-steady-state isotopic labelling experiments were performed with human faecal microbiota in a well-established in vitro model of the human colon. After culture stabilisation, [U-13C] starch was added and samples were taken at regular intervals. Metabolite concentrations and 13C isotopomeric distributions were measured amongst other things for acetate, propionate and butyrate by mass spectrometry and NMR. The vast majority of metabolic flux analysis methods based on isotopomer analysis published to date are not applicable to metabolic non-steady-state experiments. We therefore developed a new ordinary differential equation-based representation of a metabolic model of human faecal microbiota to determine eleven metabolic parameters that characterised the metabolic flux distribution in the isotope labelling experiment. The feasibility of the model parameter quantification was demonstrated on noisy in silico data using a downhill simplex optimisation, matching simulated labelling patterns of isotopically labelled metabolites with measured metabolite and isotope labelling data. Using the experimental data, we determined an increasing net label influx from starch during the experiment from 94±1 µmol/l/min to 133±3 µmol/l/min. Only about 12% of the total carbon flux from starch reached propionate. Propionate production mainly proceeded via succinate with a small contribution via acrylate. The remaining flux from starch yielded acetate (35%) and butyrate (53%). Interpretation of 13C NMR multiplet signals further revealed that butyrate, valerate and caproate were mainly synthesised via cross-feeding, using acetate as a co-substrate. This study demonstrates for the first time that the experimental design and the analysis of the results by computational modelling allows the determination of time-resolved effects of nutrition on the flux distribution within human faecal microbiota in metabolic non-steady-state.This publication has 16 references indexed in Scilit:
- Computational quantification of metabolic fluxes from a single isotope snapshot: application to an animal biopsyBioinformatics, 2010
- Determination of metabolic fluxes in a non-steady-state systemPhytochemistry, 2007
- Application of liquid chromatography–mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusionsJournal of Chromatography B, 2007
- Elementary metabolite units (EMU): A novel framework for modeling isotopic distributionsMetabolic Engineering, 2007
- Beyond diversity: functional microbiomics of the human colonTrends in Microbiology, 2006
- Host-Bacterial Mutualism in the Human IntestineScience, 2005
- The Effect of Lactulose on the Composition of the Intestinal Microbiota and Short-chain Fatty Acid Production in Human Volunteers and a Computer-controlled Model of the Proximal Large IntestineMicrobial Ecology in Health & Disease, 2003
- In Vivo Quantification of Parallel and Bidirectional Fluxes in the Anaplerosis of Corynebacterium glutamicumOnline Journal of Public Health Informatics, 2000
- 13C-NMR study of glucose and pyruvate catabolism in four acetogenic species isolated from the human colonFEMS Microbiology Letters, 1997
- Dynamic determination of the thermal characteristics of fermentation tanksBiotechnology & Bioengineering, 1968