scholarly journals Substrate-dependent CO2 fixation in heterotrophic bacteria revealed by stable isotope labelling

2020 ◽  
Vol 96 (6) ◽  
Author(s):  
Marina Spona-Friedl ◽  
Alexander Braun ◽  
Claudia Huber ◽  
Wolfgang Eisenreich ◽  
Christian Griebler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Heterotrophic Bacteria ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Organic Substrate ◽  
Gas Chromatography Mass Spectrometry ◽  
Logarithmic Phase ◽  
Natural Environments ◽  
Organic Substrates ◽  
Stable Isotope Labelling

ABSTRACT Virtually all heterotrophs incorporate carbon dioxide by anaplerotic fixation. Little explored, however, is the interdependency of pathways and rates of CO2fixation on the concurrent usage of organic substrate(s). Potentially, this could reveal which substrates out of a pool of dissolved organic carbon are utilised by environmental microorganisms. To explore this possibility, Bacillus subtilis W23 was grown in a minimal medium with normalised amounts of either glucose, lactate or malate as only organic substrates, each together with 1 g/L NaH13CO3. Incorporation of H13CO3− was traced by elemental analysis-isotope ratio mass spectrometry of biomass and gas chromatography-mass spectrometry of protein-derived amino acids. Until the late logarithmic phase, 13C incorporation into the tricarboxylic acid cycle increased with time and occurred via [4–13C]oxaloacetate formed by carboxylation of pyruvate. The levels of 13C incorporation were highest for growth on glucose and lowest on malate. Incorporation of 13C into gluconeogenesis products was mainly detected in the lactate and malate experiment, whereas glucose down-regulated this path. A proof-of-principle study with a natural groundwater community confirmed the ability to determine incorporation from H13CO3− by natural communities leading to specific labelling patterns. This underlines the potential of the labelling approach to characterise carbon sources of heterotrophic microorganisms in their natural environments.

scholarly journals Probing rapid carbon fixation in fast-growing seaweed Ulva meridionalis using stable isotope 13C-labelling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shuntaro Tsubaki ◽  
Hiroshi Nishimura ◽  
Tomoya Imai ◽  
Ayumu Onda ◽  
Masanori Hiraoka
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Carbon Fixation ◽  
High Growth ◽  
Gas Chromatography Mass Spectrometry ◽  
Ion Cyclotron Resonance ◽  
Stable Isotope Labelling ◽  
Ft Icr Ms ◽  
Ft Icr ◽  
C Nmr

AbstractThe high growth rate of Ulva seaweeds makes it a potential algal biomass resource. In particular, Ulva meridionalis grows up to fourfold a day. Here, we demonstrated strong carbon fixation by U. meridionalis using 13C stable isotope labelling and traced the 13C flux through sugar metabolites with isotope-ratio mass spectrometry (IR-MS), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), 13C-nuclear magnetic resonance spectrometry (13C-NMR), and gas chromatography-mass spectrometry (GC–MS). U. meridionalis was first cultured in 13C-labelled enriched artificial seawater for 0–12 h, and the algae were collected every 4 h. U. meridionalis grew 1.8-fold (dry weight), and the 13C ratio reached 40% in 12 h, whereas 13C incorporation hardly occurred under darkness. At the beginning of the light period, 13C was incorporated into nucleic diphosphate (NDP) sugars in 4 h, and 13C labelled peaks were identified using FT-ICR-MS spectra. Using semiquantitative 13C-NMR measurements and GC–MS, 13C was detected in starch and matrix polysaccharides after the formation of NDP sugars. Moreover, the 14:10 light:dark regime resulted into 85% of 13C labelling was achieved after 72 h of cultivation. The rapid 13C uptake by U. meridionalis shows its strong carbon fixation capacity as a promising seaweed biomass feedstock.

68 Flux analysis of aerobic glycolysis in bovine blastocysts and CT1 cells

2019 ◽  
Vol 31 (1) ◽  
pp. 159
Author(s):  
J. Chung ◽  
R. Clifford ◽  
G. Sriram ◽  
C. Keefer
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Metabolic Flux ◽  
Aerobic Glycolysis ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Flux Analysis ◽  
Metabolic Modelling ◽  
Chromatography Mass Spectrometry

Embryo quality and maternal recognition are crucial for successful initiation of bovine pregnancy. Previous studies have proposed that better quality embryos use aerobic glycolysis to meet a high demand for biomass components. While hexoses are the principal carbon sources that provide energy to glycolysis, little is known about partitioning of hexoses into metabolic pathways or alteration of partitioning when different hexoses are simultaneously available. Specific metabolic utilisation of 13C-labelled substrates can be quantified by gas chromatography-mass spectrometry, an excellent noninvasive approach for studying cellular metabolism. To assess hexose flux through central metabolism, bovine blastocysts and CT1 cells (a bovine trophectoderm cell line) were cultured in SOF-based media supplemented with combinations of 50% uniformly labelled (U) and 50% naturally abundant (NA) glucose (Glc) or fructose (Fru) (U−13C Glc+NA Glc, U−13C Fru+NA Fru, U−13C Glc+NA Fru, and U−13C Fru+NA Glc), such that total hexose concentration was 1.5mM. Metabolites in spent media from 24-h cultures of single or 5 blastocysts (40-μL drops; 5% CO2, 5% O2, 90% N2) and 1-, 2-, 3-, 6-, 8-, and 24-h incubations of CT1 cells (150 μL; ~3×104 cells per well; 5% CO2, 95% air) were extracted with a MeOH-CHCl3 reagent, derivatized, and analysed by gas chromatography-mass spectrometry. Measurement of mass isotopomer distributions of metabolites, chiefly pyruvate, lactate, and amino acids, followed by correction for natural abundances and metabolic modelling, revealed several insights. For instance, five Day 7 or Day 8 blastocysts (Day 0=fertilization) supplied with U−13C Glc+NA Fru displayed 13C enrichments of 80.3%±1.4% for pyruvate and 71.6%±2.8% for lactate, whereas when supplied with U−13C Fru+NA Glc, they displayed lower 13C enrichments of 5.7%±2.4% for pyruvate and 2.8%±0.4% lactate (mean±standard deviation, n=3 to 4). Metabolic modelling revealed that when Glc and Fru are simultaneously available, the blastocysts used 2.5±0.2 moles of Fru per 100 moles of Glc used. Furthermore, 13C enrichment of pyruvate was 42.0±0.6% when U−13C Glc+NA Glc was supplied and 37.8±2.7% when U−13C Fru+NA Fru was supplied. Lactate enrichments followed a similar trend. This indicates that, individually, Glc and Fru were utilised majorly through aerobic glycolysis with some involvement of the pentose phosphate pathway. Alanine was negligibly labelled in all of the experiments, suggesting either a low TCA flux or that alanine is diluted by extra- or intracellular amino or fatty acids. Single blastocysts and CT1 cells showed a similar labelling pattern when hexoses were available. Following Glc depletion at 8h in CT1 cultures, the 13C enrichments of alanine and citrate in the media increased, suggesting a sharp alteration of metabolic state. These findings demonstrate that metabolic flux can be comprehensively analysed for single bovine blastocysts and CT1 cell metabolism models that of the blastocyst. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23237 from the USDA National Institute of Food and Agriculture.

Nitrification as a source of soluble organic substrate in biological treatment

1994 ◽  
Vol 30 (6) ◽  
pp. 1-8 ◽  
Author(s):  
Bruce E. Rittmann ◽  
John M. Regan ◽  
David A. Stahl
Keyword(s):  
Kinetic Analysis ◽  
Organic Compounds ◽  
Biological Treatment ◽  
Heterotrophic Bacteria ◽  
Theoretical Work ◽  
Organic Substrate ◽  
Nitrifying Bacteria ◽  
Organic Substrates ◽  
Soluble Microbial Products ◽  
Microbial Products

In complex, multispecies populations, exchange of substrates can be an important beneficial interaction. Prior experimental and theoretical work has led to the hypothesis that the formation of soluble microbial products (SMP) by nitrifying bacteria can provide a supplementary organic substrate for heterotrophic bacteria, thereby augmenting their accumulation and stability, especially when inputs of organic substrates are low. In this study, chemostat experiments carried out with a NO2−-oxidizing strain (Nitrobacter sp.) and an NH4+-oxidizing strain (Nitrosomonas europaea) demonstrated that both nitrifiers produce SMP that can support heterotrophic bacteria. The first evidence was the presence of significant concentrations of soluble COD in the chemostat effluent, even though the influent was free of organic compounds. Second, a small heterotrophic population was maintained, apparently through utilization of the nitrifier-produced SMP. A preliminary kinetic analysis suggested that SMP kinetic parameters can be adapted from parameters measured for heterotrophs.

scholarly journals Genealogy Profiling through Strain Improvement by Using Metabolic Network Analysis: Metabolic Flux Genealogy of Several Generations of Lysine-Producing Corynebacteria

2002 ◽  
Vol 68 (12) ◽  
pp. 5843-5859 ◽  
Author(s):  
Christoph Wittmann ◽  
Elmar Heinzle
Keyword(s):  
Mass Spectrometry ◽  
Network Analysis ◽  
Metabolic Network ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Strain Improvement ◽  
Gas Chromatography Mass Spectrometry ◽  
Limiting Factor ◽  
Strain Optimization ◽  
Metabolic Network Analysis

ABSTRACT A comprehensive approach of metabolite balancing, 13C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C3 carboxylation and C4 decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C3 carboxylation and C4 decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.

scholarly journals Heptanoate as a Neural Fuel: Energetic and Neurotransmitter Precursors in Normal and Glucose Transporter I-Deficient (G1D) Brain

2012 ◽  
Vol 33 (2) ◽  
pp. 175-182 ◽  
Author(s):  
Isaac Marin-Valencia ◽  
Levi B Good ◽  
Qian Ma ◽  
Craig R Malloy ◽  
Juan M Pascual
Keyword(s):  
Mass Spectrometry ◽  
Glucose Transporter ◽  
Tricarboxylic Acid Cycle ◽  
Gas Chromatography Mass Spectrometry ◽  
Resonance Spectroscopy ◽  
Type I ◽  
Acetyl Coa ◽  
Chromatography Mass Spectrometry ◽  
Neurotransmitter Precursors ◽  
The Brain

It has been postulated that triheptanoin can ameliorate seizures by supplying the tricarboxylic acid cycle with both acetyl-CoA for energy production and propionyl-CoA to replenish cycle intermediates. These potential effects may also be important in other disorders associated with impaired glucose metabolism because glucose supplies, in addition to acetyl-CoA, pyruvate, which fulfills biosynthetic demands via carboxylation. In patients with glucose transporter type I deficiency (G1D), ketogenic diet fat (a source only of acetyl-CoA) reduces seizures, but other symptoms persist, providing the motivation for studying heptanoate metabolism. In this work, metabolism of infused [5,6,7-13C3]heptanoate was examined in the normal mouse brain and in G1D by 13C-nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). In both groups, plasma glucose was enriched in 13C, confirming gluconeogenesis from heptanoate. Acetyl-CoA and glutamine levels became significantly higher in the brain of G1D mice relative to normal mice. In addition, brain glutamine concentration and 13C enrichment were also greater when compared with glutamate in both animal groups, suggesting that heptanoate and/or C5 ketones are primarily metabolized by glia. These results enlighten the mechanism of heptanoate metabolism in the normal and glucosedeficient brain and encourage further studies to elucidate its potential antiepileptic effects in disorders of energy metabolism.

scholarly journals The Polycyclic Aromatic Hydrocarbon (PAH) degradation activities and genome analysis of a novel strain Stenotrophomonas sp. Pemsol isolated from Mexico

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8102
Author(s):  
Temidayo O. Elufisan ◽  
Isabel C. Rodríguez-Luna ◽  
Omotayo Opemipo Oyedara ◽  
Alejandro Sánchez-Varela ◽  
Armando Hernández-Mendoza ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Crude Oil ◽  
Contaminated Soil ◽  
Antimicrobial Agents ◽  
Carbon Sources ◽  
Genomic Islands ◽  
Gas Chromatography Mass Spectrometry ◽  
Chromatography Mass Spectrometry ◽  
Wide Range ◽  
Polycyclic Aromatic

Background Stenotrophomonas are ubiquitous gram-negative bacteria, which can survive in a wide range of environments. They can use many substances for their growth and are known to be intrinsically resistant to many antimicrobial agents. They have been tested for biotechnological applications, bioremediation, and production of antimicrobial agents. Method Stenotrophomonas sp. Pemsol was isolated from a crude oil contaminated soil. The capability of this isolate to tolerate and degrade polycyclic aromatic hydrocarbons (PAH) such as anthraquinone, biphenyl, naphthalene, phenanthrene, phenanthridine, and xylene was evaluated in Bushnell Hass medium containing PAHs as the sole carbon sources. The metabolites formed after 30-day degradation of naphthalene by Pemsol were analyzed using Fourier Transform Infra-red Spectroscopic (FTIR), Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS). The genome of Pemsol was also sequenced and analyzed. Results Anthraquinone, biphenyl, naphthalene, phenanthrene, and phenanthridine except xylene can be used as sole carbon sources for Pemsol’s growth in Bushnell Hass medium. The degradation of naphthalene at a concentration of 1 mg/mL within 30 days was tested. A newly formed catechol peak and the disappearance of naphthalene peak detected on the UPLC-MS, and GC-MS analyses spectra respectively confirmed the complete degradation of naphthalene. Pemsol does not produce biosurfactant and neither bio-emulsify PAHs. The whole genome was sequenced and assembled into one scaffold with a length of 4,373,402 bp. A total of 145 genes involved in the degradation of PAHs were found in its genome, some of which are Pemsol-specific as compared with other 11 Stenotrophomonas genomes. Most specific genes are located on the genomic islands. Stenotrophomonas sp. Pemsol’s possession of few genes that are associated with bio-emulsification gives the genetic basis for its inability to bio-emulsify PAH. A possible degradation pathway for naphthalene in Pemsol was proposed following the analysis of Pemsol’s genome. ANI and GGDH analysis indicated that Pemsol is likely a new species of Stenotrophomonas. It is the first report on a complete genome sequence analysis of a PAH-degrading Stenotrophomonas. Stenotrophomonas sp. Pemsol possesses features that make it a good bacterium for genetic engineering and will be an excellent tool for the remediation of crude oil or PAH-contaminated soil.

scholarly journals Revealing the ocean metabolome with mass spectrometry

2019 ◽  
Author(s):  
E. Maggie Sogin ◽  
Erik Puskas ◽  
Nicole Dubilier ◽  
Manuel Liebeke
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Heterotrophic Bacteria ◽  
Biotic Interactions ◽  
Gas Chromatography Mass Spectrometry ◽  
Mangrove Sediment ◽  
Marine Microorganisms ◽  
The North ◽  
Marine Habitats ◽  
Chromatography Mass Spectrometry

AbstractAll life exchanges molecules with its environment. While these metabolites are commonly measured in terrestrial and limnic ecosystems, the presence of salt in marine habitats has hampered quantitative analyses of the ocean metabolome. To overcome these limitations, we developed SeaMet, a gas chromatography-mass spectrometry (GC-MS) method that detects hundreds of metabolites down to nano-molar concentrations in less than one milliliter of seawater. Using a set of metabolites dissolved in artificial seawater to benchmark our method, we show metabolite signal detection increased on average across ions by 324 fold in comparison to standard GC-MS methods. Our observed signal improvement occurred across tested metabolite classes and provides reproducible and quantifiable results. To showcase the capabilities of our method, we used SeaMet to explore the production and consumption of metabolites during culture of a heterotrophic bacteria that is widespread in the North Sea. Our approach revealed successional uptake of amino acids, while sugars were not consumed, and highlight the power of metabolomics in providing insights into nutrient uptake and energy conservation in marine microorganisms. We also applied SeaMet to explore the in situ metabolome of coral reef and mangrove sediment porewaters. Despite the fact that these ecosystems occur in nutrient-poor waters, we uncovered a remarkable diversity and abundance of sugars and fatty acids, compounds predicted to be rapidly consumed by marine microorganisms. Our method advances marine metabolomics by enabling the unbiased, and quantitative analysis of marine metabolites, and will help provide new insights into carbon cycle dynamics and ocean biogeochemistry.SignificanceMetabolites are the chemical currency of cellular metabolism across all domains of life. However, describing metabolites that occur in the oceans is lagging behind similar studies conducted on land. The central challenge in marine metabolomics is that salt prevents the comprehensive analysis of metabolites in seawater. We developed a method, SeaMet, that overcomes the limitations of salt on metabolite detection. SeaMet provides a time and cost efficient method, using gas chromatography-mass spectrometry, for the reproducible identification and quantification of a broad range of marine compounds. Considering the oceans contain the largest organic carbon pool on Earth, describing the marine metabolome is critical for understanding the drivers behind element cycles, biotic interactions, ecosystem function, and atmospheric CO2 storage.

scholarly journals Metabolic Reprogramming and Risk Stratification of Hepatocellular Carcinoma Studied by Using Gas Chromatography–Mass Spectrometry-Based Metabolomics

Cancers ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 231
Author(s):  
Chengnan Fang ◽  
Hui Wang ◽  
Zhikun Lin ◽  
Xinyu Liu ◽  
Liwei Dong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatocellular Carcinoma ◽  
Gas Chromatography ◽  
Risk Stratification ◽  
Tricarboxylic Acid Cycle ◽  
Nonnegative Matrix ◽  
Metabolic Reprogramming ◽  
Gas Chromatography Mass Spectrometry ◽  
Chromatography Mass Spectrometry ◽  
Tumor Tissues

Hepatocellular carcinoma (HCC) displays a high degree of metabolic and phenotypic heterogeneity and has dismal prognosis in most patients. Here, a gas chromatography–mass spectrometry (GC-MS)-based nontargeted metabolomics method was applied to analyze the metabolic profiling of 130 pairs of hepatocellular tumor tissues and matched adjacent noncancerous tissues from HCC patients. A total of 81 differential metabolites were identified by paired nonparametric test with false discovery rate correction to compare tumor tissues with adjacent noncancerous tissues. Results demonstrated that the metabolic reprogramming of HCC was mainly characterized by highly active glycolysis, enhanced fatty acid metabolism and inhibited tricarboxylic acid cycle, which satisfied the energy and biomass demands for tumor initiation and progression, meanwhile reducing apoptosis by counteracting oxidative stress. Risk stratification was performed based on the differential metabolites between tumor and adjacent noncancerous tissues by using nonnegative matrix factorization clustering. Three metabolic clusters displaying different characteristics were identified, and the cluster with higher levels of free fatty acids (FFAs) in tumors showed a worse prognosis. Finally, a metabolite classifier composed of six FFAs was further verified in a dependent sample set to have potential to define the patients with poor prognosis. Together, our results offered insights into the molecular pathological characteristics of HCC.

scholarly journals Compartmentation of acetyl CoA studied by analysis of tricarboxylic acid cycle acids and 3-hydroxybutyrate in bile of rats given [2,2,2-2H3]ethanol

1990 ◽  
Vol 265 (2) ◽  
pp. 569-574 ◽  
Author(s):  
C Norsten ◽  
T Cronholm
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Ketone Bodies ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Gas Chromatography Mass Spectrometry ◽  
Acetyl Coa ◽  
Acid Cycle ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Rat Bile

Acetate, 3-hydroxybutyrate, pyruvate, lactate, citrate, 2-oxoglutarate, succinate, fumarate and malate were analysed in rat bile by gas chromatography and gas chromatography/mass spectrometry of their O-melthyloxime-t-butyldimethylsilyl derivatives. The concentration of acetate increased to about 1.8 mmol/l after administration of [2,2,2-2H3]ethanol. Acetate was formed from ethanol to an extent of about 82% and retained all of the 2H at C-2, whereas 15% of the 2H had been lost in the tricarboxylic acid cycle intermediates and 24% in 3-hydroxybutyrate. Thus the exchange of 2H for 1H takes place after formation of acetyl CoA. For citrate and 3-hydroxybutyrate, 41% and 11% respectively was formed from [2,2,2-2H3]ethanol. These results indicate that different pools of acetyl CoA are used for the synthesis of ketone bodies and citrate, with the latter being derived from ethanol to a much larger extent. Smaller fractions of 2-oxoglutarate (16%) and succinate (5%) were derived from [2,2,2--2H3]ethanol, indicating significant contributions from amino acids.

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