Wenna Guan

Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry

Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work.

Separation and Quantification of Water-Soluble Cellular Metabolites in Clostridium thermocellum using Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry

A new protocol for metabolomic studies was developed by combining liquid chromatography-tandem mass spectrometry and isotope dilution mass spectrometry with universal 13C labeled internal standards from Escherichia Coli. The multiple reaction monitoring mode of mass spectrometry was used for quantification. Forty-five water-soluble intracellular metabolites, including 20 amino acids, 16 organic acids (primarily from the tricarboxylic acid cycle), and 9 cofactors, were measured and 34 of them were successfully quantified using the 13C-labeled internal standards. The limit of detection, limit of quantification, precision, and linearity of the methods were evaluated. The methods were applied to the quantitative analysis of intracellular metabolites extracted from wild-type and ethanol-adapted strains of Clostridium thermocellum cultivated with and without ethanol stress, and all 34 metabolites including all 9 cofactors were successfully quantified. Further multivariate data analyses of the metabolic differences between wild-type and ethanol-adapted strains were performed on the quantitative data, which can help elucidate the metabolic mechanism behind ethanol adaptation in C. thermocellum. Supplemental materials are available for this article. Go to the publishers online edition of Analytical Letters to view the supplemental file.

Efficacy of head space solid-phase microextraction coupled to gas chromatography-mass spectrometry method for determination of the trace extracellular hydrocarbons of cyanobacteria

Hydrocarbons are widespread in cyanobacteria, and the biochemical synthetic pathways were recently identified. Intracellular fatty alka(e)nes of cyanobacteria have been detected by liquid-liquid extraction (LLE) coupled to gas chromatography-mass spectrometry (GC/MS). However, whether fatty alka(e)nes can be released to cyanobacterial culture media remains to be clarified. This work develops a sensitive method for analyzing the trace level of extracellular hydrocarbons in cyanobacterial culture media by head space solid-phase microextraction (HS-SPME) coupled to GC/MS. Headspace (HS) extraction mode using polydimethylsiloxane fiber to extract for 30min at 50°C was employed as the optimal extraction conditions. Five cyanobacterial fatty alka(e)nes analogs including pentadecene (C15:1), pentadecane (C15:0), heptadecene (C17:1), heptadecane (C17:0), nonadecane (C19:0) were analyzed, and the data obtained from HS-SPME-GC/MS method were quantified using internal standard peak area comparisons. Limits of detection (LOD), limits of quantitation (LOQ), linear dynamic range, precisions (RSD) and recovery for the analysis of extracellular fatty alka(e)nes of cyanobacteria by HS-SPME-GC/MS were evaluated. The LODs limits of detection (S/N = 3) varied from 10 to 21 ng L-1. The correlation coefficients (r) of the calibration curves ranged from 0.9873 to 0.9977 with a linearity from 0.1 to 50 μg L-1. The RSD values were ranging from 7.8 to 14.0% and from 4.0 to 8.8% at 1.0 μg L-1 and 10.0 μg L-1 standard solutions, respectively. Comparative analysis of extracellular fatty alka(e)nes in the culture media of model cyanobacteria Synechocystis sp. PCC 6803 demonstrated that sensitivity of HS-SPME-GC/MS method was significantly higher than LLE method. Finally, we found that heptadecane can be released into the culture media of Synechocystis sp. PCC 6803 at the later growth period.