Budding yeast undergo robust oscillations in oxygen consumption during continuous growth in a nutrient-limited environment. Using liquid chromatography-mass spectrometry and comprehensive 2D gas chromatography-mass spectrometry-based metabolite profiling methods, we have determined that the intracellular concentrations of many metabolites change periodically as a function of these metabolic cycles. These results reveal the logic of cellular metabolism during different phases of the life of a yeast cell. They may further indicate that oscillation in the abundance of key metabolites might help control the temporal regulation of cellular processes and the establishment of a cycle. Such oscillations in metabolic state might occur during the course of other biological cycles.gas chromatography-mass spectrometry ͉ liquid chromatography-mass spectrometry ͉ metabolic cycle ͉ metabolite profiling C ircadian rhythms are driven by biological clocks found in virtually all kingdoms of life (1, 2). Genome-wide expression studies in plants, flies, and mice have shown that many genes are expressed periodically as a function of the circadian cycle (3-6). It is now predictable from studies of oscillating gene expression that the circadian regulatory apparatus should differentially control metabolic state as a function of the circadian cycle (7,8). By contrast, direct and comprehensive measurements of changes in sentinel metabolites have not been performed as a function of the circadian cycle or of biological cycles of other temporal dimensions.The budding yeast Saccharomyces cerevisiae exhibits various modes of oscillatory behavior during continuous growth (9-12). We recently established a continuous culture system that reveals an Ϸ4-to 5-h yeast metabolic cycle (YMC) in which over half of the genome is periodically expressed as a function of robust recurring oscillations in oxygen consumption (12). Expression profiling studies have led to the prediction that a variety of cellular and metabolic processes are temporally orchestrated around three major phases of the YMC [oxidative (Ox), reductive/building (RB), and reductive/ charging (RC)] (12). Cells undergoing the YMC are furthermore synchronized with respect to the cell cycle, because the YMC strictly gates cell division to the RB phase when respiration decreases significantly (12).A prediction of temporal compartmentalization is that the concentrations of critical metabolites might exhibit periodic fluctuation and, in turn, play a reciprocal role in regulating the YMC (8). To determine whether cyclic changes in metabolic state might occur during the YMC, we used both liquid chromatography (LC)/ tandem mass spectrometry (LC-MS/MS) and comprehensive 2D gas chromatography (GC)/time-of-f light mass spectrometry (GCϫGC-TOFMS) to monitor the intracellular concentrations of Ϸ150 common metabolites at regularly spaced intervals throughout the YMC. The combined use of LC-MS-and GC-MS-based methods provides a means to evaluate the consistency of results for metabolites detected by both ...
Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry coupled with rapid chemometric analysis were used to identify chemical differences in metabolite extracts isolated from yeast cells either metabolizing glucose (repressed (R) cells) via fermentation or metabolizing ethanol by respiration (derepressed (DR) cells). Principal component analysis (PCA) followed by parallel factor analysis (PARAFAC) in concert with the LECO ChromaTOF software located and identified the differences in composition between the two types of cell extracts and provided a reliable ratio of the metabolite concentrations. In this report, we demonstrate the analytical method developed to provide relatively rapid analysis of three selective mass channels (m/z 73, 205, 387), although in principle all collected mass channels could be analyzed. Twenty-six metabolites that differentiate repressed cells from derepressed cells were identified. The DR/R ratio of metabolite concentrations ranged from 0.02 for glucose to 67 for trehalose. The average biological variation of the sample extracts was 31%. This analysis demonstrates the utility and benefit of using PCA combined with PARAFAC and ChromaTOF software on extremely complex samples to derive useful information from complex three-dimensional chromatographic data objectively and relatively rapidly.
The first extensive study of yeast metabolite GC x GC-TOFMS data from cells grown under fermenting, R, and respiring, DR, conditions is reported. In this study, recently developed chemometric software for use with three-dimensional instrumentation data was implemented, using a statistically-based Fisher ratio method. The Fisher ratio method is fully automated and will rapidly reduce the data to pinpoint two-dimensional chromatographic peaks differentiating sample types while utilizing all the mass channels. The effect of lowering the Fisher ratio threshold on peak identification was studied. At the lowest threshold (just above the noise level), 73 metabolite peaks were identified, nearly three-fold greater than the number of previously reported metabolite peaks identified (26). In addition to the 73 identified metabolites, 81 unknown metabolites were also located. A Parallel Factor Analysis graphical user interface (PARAFAC GUI) was applied to selected mass channels to obtain a concentration ratio, for each metabolite under the two growth conditions. Of the 73 known metabolites identified by the Fisher ratio method, 54 were statistically changing to the 95% confidence limit between the DR and R conditions according to the rigorous Student's t-test. PARAFAC determined the concentration ratio and provided a fully-deconvoluted (i.e. mathematically resolved) mass spectrum for each of the metabolites. The combination of the Fisher ratio method with the PARAFAC GUI provides high-throughput software for discovery-based metabolomics research, and is novel for GC x GC-TOFMS data due to the use of the entire data set in the analysis (640 MB x 70 runs, double precision floating point).
Polar metabolites resulting from petroleum biodegradation are measured in groundwater samples as TPHd unless a silica gel cleanup (SGC) is used on the sample extract to isolate hydrocarbons. Even though the metabolites can be the vast majority of the dissolved organics present in groundwater, SGC has been inconsistently applied because of regulatory concern about the nature and toxicity of the metabolites. A two‐step approach was used to identify polar compounds that were measured as TPHd in groundwater extracts at five sites with biodegrading fuel sources. First, gas chromatography with mass spectrometry (GC‐MS) was used to identify and quantify 57 individual target polar metabolites. Only one of these compounds—dodecanoic acid, which has low potential human toxicity—was detected. Second, nontargeted analysis was used to identify as many polar metabolites as possible using both GC‐MS and GC×GC‐MS. The nontargeted analysis revealed that the mixture of polar metabolites identified in groundwater source areas at these five sites is composed of approximately equal average percentages of organic acids, alcohols and ketones, with few phenols and aldehydes. The mixture identified in downgradient areas at these five sites is dominated by acids, with fewer alcohols, far fewer ketones, and very few aldehydes and phenols. A ranking system consistent with systems used by USEPA and the United Nations was developed for evaluating the potential chronic oral toxicity to humans of the different classes of identified polar metabolites. The vast majority of the identified polar metabolites have a “Low” toxicity profile, and the mixture of identified polar metabolites present in groundwater extracts at these five sites is unlikely to present a significant risk to human health.
We examined the chemical composition and properties of several diesel fuels and blendstocks derived from Fischer–Tropsch (FT) synthesis, hydroisomerization of lipids, and fermentation of sugar via the terpenoid metabolic pathway. Comprehensive two-dimensional gas chromatographic analysis with nonpolar and polar columns, 13C NMR, GC-MS, and elemental analysis were used to assess fuel chemistry. Performance properties included density, heat of combustion, cetane number, and cloud point, as well as other properties. The fuels consisted almost entirely of normal and iso-paraffins. Three samples contained residual oxygen below 0.1 mass %. All of the renewable and synthetic diesel fuels have significantly lower density than is typical for a petroleum-derived diesel fuel. As a result, they have slightly higher net heat of combustion on a mass basis (2%–3% higher), but lower heat of combustion on a volume basis (3%–7% lower). Two critical diesel performance properties, cetane number and cloud point, were correlated with iso-paraffin content and chain length. The results confirm that properties of hydroisomerized fats and oils, as well as FT diesel, can be tuned by increasing the degree of isomerization to lower cloud point which also lowers the cetane number. In spite of this trade-off between cloud point, and cetane number, the cetane numbers were still over 70 for fuels with cloud points as low as −27 °C. The terpenoid biofuel exhibited a cloud point below −70 °C and a cetane number of 58.
A need exists for compact sensor systems capable of in situ monitoring of groundwater for accidental releases of fuel and oil. The work reported here addresses this need, using shear horizontal surface acoustic wave (SH-SAW) sensors, which function effectively in liquid environments. To achieve enhanced sensitivity and partial selectivity for hydrocarbons, the devices are coated with thin chemically sensitive polymer films. Various polymer materials are investigated with the goal of identifying a set of coatings suitable for a sensor array. The system is tested with compounds indicative of fuel and oil releases, in particular, the BTEX compounds (benzene, toluene, ethylbenzene, and xylenes), in the low milligrams/liters to high micrograms/liters concentration range. Particular emphasis is placed on detection of benzene, a known carcinogen. It was observed that within the above concentration range, responses to multiple analytes in a mixture are additive, and there is a characteristic response time for each coating/analyte pair, which is largely independent of concentration. With the use of both the steady-state and transient-response information of SH-SAW sensor devices coated with three different polymer materials, poly(ethyl acrylate), poly(epichlorohydrin), and poly(isobutylene), a response pattern was obtained for benzene that is easily distinguishable from those of the other BTEX compounds. The time courses of the responses to binary analyte mixtures were modeled accurately using dual-exponential fits, yielding a characteristic concentration-independent time constant for each analyte/coating pair. Benzene concentration was quantified in the aqueous phase in the presence of the other BTEX compounds.
This paper summarizes the results of a 5-y research study of the nature and toxicity of petroleum biodegradation metabolites in groundwater at fuel release sites that are quantified as diesel-range "Total Petroleum Hydrocarbons" (TPH; also known as TPHd, diesel-range organics (DRO), etc.), unless a silica gel cleanup (SGC) step is used on the sample extract prior to the TPH analysis. This issue is important for site risk management in regulatory jurisdictions that use TPH as a metric; the presence of these metabolites may preclude site closure even if all other factors can be considered "low-risk." Previous work has shown that up to 100% of the extractable organics in groundwater at petroleum release sites can be biodegradation metabolites. The metabolites can be separated from the hydrocarbons by incorporating an SGC step; however, regulatory agency acceptance of SGC has been inconsistent because of questions about the nature and toxicity of the metabolites. The present study was conducted to answer these specific questions. Groundwater samples collected from source and downgradient wells at fuel release sites were extracted and subjected to targeted gas chromatography-mass spectrometry (GC-MS) and nontargeted two-dimensional gas chromatography with time-of-flight mass spectrometry (GCÂGC-MS) analyses, and the metabolites identified in each sample were classified according to molecular structural classes and assigned an oral reference dose (RfD)-based toxicity ranking. Our work demonstrates that the metabolites identified in groundwater at biodegrading fuel release sites are in classes ranked as low toxicity to humans and are not expected to pose significant risk to human health. The identified metabolites naturally attenuate in a predictable manner, with an overall trend to an increasingly higher proportion of organic acids and esters, and a lower human toxicity profile, and a life cycle that is consistent with the low-risk natural attenuation paradigm adopted by many regulatory agencies for petroleum release sites. Integr Environ Assess Manag 2017;13:714-727. C
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