Quantitative Bioreactor Monitoring of Intracellular Bacterial Metabolites in Clostridium autoethanogenum Using Liquid Chromatography–Isotope Dilution Mass Spectrometry

Abstract

We report a liquid chromatography–isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly 13C-labeled internal standards (U-13C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% 13C–glucose as a carbon source. The U-13C IS mixture combined with 12C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with R 2 ≥ 0.99, limits of detection were ≤1.0 μM, limits of quantification were ≤10 μM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid “cycle” were the most affected pathways associated with the pH shift in the bioreactor fermentation of C. autoethanogenum and the concomitant changes in ethanol production.

The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum

“…These observations, and the ability to rapidly alter the direction of alcohol metabolism upon changes in CO availability (Figure 2C), support the theory that acetogenic/solventogenic metabolism is strongly controlled by environmental parameters (e.g. redox pressure, pH, substrate and/or product concentrations; Abubackar et al, 2012;Abubackar, Fernández-Naveira, et al, 2016;Mahamkali et al, 2020;Richter et al, 2016;Safo et al, 2021). Ethanol could be consumed as a sole substrate by C. autoethanogenum in the presence of CO 2 , yet the consumption of ethanol halts before reaching complete depletion (Figure S1).…”

Full-Length NAD+-I Riboswitches Bind a Single Cofactor but Cannot Discriminate against Adenosine Triphosphate

“…23 In Gram-negative Clostridium autoethanogenum undergoing acetogenesis and solventogenesis, ATP and NAD + concentrations were measured at concentrations of 118−134 μM and 1.6−1.7 mM, respectively. 24 These ATP and NAD + concentrations suggest that both effectors will saturate the nadA riboswitch binding site based on the K D values of 11.0 ± 3.5 μM for ATP and 31.5 ± 1.5 μM for NAD + measured here (Table 1). Lack of strong binding specificity by the nadA riboswitch and others, such as the azaaromatic, cobalamin, and ykkC-2C riboswitches noted above, could signify that lax ligand preference is more common than realized previously.…”

“…In mid-log phase Gram-positive E. coli , ATP levels are 9.6 mM, whereas NAD + is 2.6 mM . In Gram-negative Clostridium autoethanogenum undergoing acetogenesis and solventogenesis, ATP and NAD + concentrations were measured at concentrations of 118–134 μM and 1.6–1.7 mM, respectively . These ATP and NAD + concentrations suggest that both effectors will saturate the nadA riboswitch binding site based on the K D values of 11.0 ± 3.5 μM for ATP and 31.5 ± 1.5 μM for NAD + measured here (Table ).…”

“…Specifically, the need to distinguish between one-NAD + -per-riboswitch versus two-NAD + -per-riboswitch binding modes . A second prediction is that the full-length riboswitch should discriminate against the γ-phosphate of ATP but favor NAD + recognition because these nucleotides are present at concentrations of 0.12–9.6 mM and 1.6–2.6 mM in growing bacteria. , …”

“…22 A second prediction is that the full-length riboswitch should discriminate against the γ-phosphate of ATP but favor NAD + recognition because these nucleotides are present at concentrations of 0.12−9.6 mM and 1.6−2.6 mM in growing bacteria. 23,24 Here, we build upon previous biochemical and structural analyses of the nadA motif with the goal of quantifying cofactor binding stoichiometry, affinity, and specificity for the fulllength NAD + -I riboswitch, which has not been studied appreciably in previous investigations. 9,11,22 Accordingly, we measured riboswitch interactions with NAD + , NADH, ADP, ATP, and the dinucleotide analogue AP 2 -ribavirin.…”