Jiasong Fang

Structural determination and quantitative analysis of bacterial phospholipids using liquid chromatography/electrospray ionization/mass spectrometry

Abstract This report presents a comprehensive spectral analysis of common bacterial phospholipids using electrospray/mass spectrometry (ESI/MS) under both negative and positive ionization conditions. Phospholipids under positive ionization yield sodium-adduct molecular ions which are most useful to determine the molecular mass of the compounds. Nitrogen-containing phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine yield abundant protonated ions, which are absent in the mass spectra of nitrogen-free phospholipids, (e.g. phosphatidic acid, phosphatidylinositol, and phosphatidylglycerol). Phospholipids generally fragment at the glycerol-phosphate bond, resulting in diglyceride ions and ions of the polar head groups of the compounds. Negative ESI/MS spectra provide salient information on the regiochemistry of the phospholipids. Fatty acid composition and distribution can be clearly assigned based on the intensity ratio of sn−2/sn−1 fragment ions. Interfacing liquid chromatography with ESI/MS enables the preseparation and quantitative determination of phospholipids with high sensitivity (picomoles/μl). This is an order of magnitude higher than that of previously published methods. Results of phospholipid analyses of a Type II methylotrophic bacterium Methylosinus trichosporium (strain OB3b) showed that the determination of intact phospholipids is promising in bacterial identification.

Biogeochemical evidence for microbial community change in a jet fuel hydrocarbons-contaminated aquifer

A glacio-fluvial aquifer located at Wurtsmith Air Force Base, MI, had been contaminated with JP-4 fuel hydrocarbons released after the crash of a tanker aircraft in October of 1988. Mi- crobial biomass and community structure, associated with the aquifer sediments, were characterized based on the analysis of phospholipid ester-linked fatty acids (PLFA). Fatty acids ranged from C12- C20 in carbon chain length, including saturated, monounsaturated, branched, and cyclopropyl fatty acids. Polyunsaturated fatty acids were virtually absent from the aquifer sediments. These findings suggested that bacteria were dominant organisms within the aquifer microbial communities. The total microbial biomass and community composition in aquifer sediments, as determined by PLFA analy- sis, varied with depth, and between locations at similar depths, indicating considerable microbial het- erogeneity in the subsurface. PLFA patterns in the hydrocarbon-contaminated anaerobic zones indicated higher microbial biomass and metabolically more diverse microbial communities than those in aerobic zones. # 1998 Elsevier Science Ltd. All rights reserved

A direct comparison between fatty acid analysis and intact phospholipid profiling for microbial identification

Abstract Two chemical methods for characterization of micro organisms were compared: phospholipid ester-linked fatty acid (PLFA) analysis by gas chromatography/mass spectrometry, and intact phospholipid profiling (IPP) using liquid chromatography/electrospray ionization/mass spectrometry. Both methods were tested on five reference pseudomonad strains: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. PLFA detected eight major fatty acids in these pseudomonads, ranging in chain length from C 14 to C 19 . IPP detected 16 phospholipids in three different classes: phosphatidylglycerol, phosphatidylethanolamine and phosphatidyl-dimethylethanolamine. Factor analysis of the data showed that IPP is superior to the PLFA technique in microbial differentiation and identification.

Phospholipid compositional changes of five pseudomonad archetypes grown with and without toluene

Abstract Bacterial physiological responses to toluene exposure were investigated in five reference pseudomonad strains that express different toluene degradation pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. The intact phospholipids of these archetypes, grown with and without toluene, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. All strains showed significant changes in phospholipid content and composition as an adaptive response to toluene exposure, as well as considerable diversity in response mechanisms. For example, the phospholipid content of toluene-grown PKO1, F1, and KR1 were 10.9–34.7% of that found in succinate-grown strains, while the phospholipid content of mt-2 and G4 increased by 56% and 94%, respectively, when grown on toluene. In addition, PKO1, F1, and mt-2 responded to the presence of toluene by synthesizing more phosphatidylglycerol, whereas G4 and KR1 synthesized phospholipids with polyunsaturated fatty acids (C18:2) on one or both of the sn-2 positions. These changes in phospholipid composition and concentration probably reflect the sensitivity and degree of tolerance of these strains to toluene, and suggest that different mechanisms are utilized by dissimilar bacteria to maintain optimal lipid ordering in the presence of such environmental pollutants.

Stable carbon isotope biogeochemistry of a shallow sand aquifer contaminated with fuel hydrocarbons

Abstract Ground-water chemistry and the stable C isotope composition (δ 13 C DIC ) of dissolved inorganic C (DIC) were measured in a sand aquifer contaminated with JP–4 fuel hydrocarbons. Results show that ground water in the upgradient zone was characterized by DIC content of 14–20 mg C/L and δ 13 C DIC values of −11.3‰ to −13.0‰. The contaminant source zone was characterized by an increase in DIC content (12.5 mg C/L to 54 mg C/L), Ca, and alkalinity, with a significant depletion of 13 C in δ 13 C DIC (−11.9‰ to −19.2‰). The source zone of the contaminant plume was also characterized by elevated levels of aromatic hydrocarbons (0 μg/L to 1490 μg/L) and microbial metabolites (aromatic acids, 0 μg/L to 2277 μg/L), non-detectable dissolved O 2 , NO 3 and SO 4 . Phospholipid ester-linked fatty acid analyses suggest the presence of viable SO 4 -reducing bacteria in ground water at the time of sampling. The ground-water chemistry and stable C isotope composition of ground-water DIC are interpreted using a chemical reaction model involving rainwater recharge, contributions of CO 2 from soil gas and biodegradation of hydrocarbons, and carbonate dissolution. The major-ion chemistry and δ 13 C DIC were reconciled, and the model predictions were in good agreement with field measurements. It was concluded that stable C isotope measurements, combined with other biogeochemical measures can be a useful tool to monitor the dominant terminal electron-accepting processes in contaminated aquifers and to identify mineralogical, hydrological, and microbiological factors that affect δ 13 C of dissolved inorganic C.

Coupled oxidation of aromatic hydrocarbons by horseradish peroxidase and hydrogen peroxide.

The oxidation capability of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) coupled oxidation of aromatic hydrocarbons (o-xylene-d10 and naphthalene-d8) was investigated. Batch experiments were conducted using horseradish peroxidase prepared in potassium phosphate buffer in the presence of H2O2. The oxidation of aromatic hydrocarbon was tested as a function of HRP at a fixed concentration of H2O2, and as a function of the concentration of H2O2 at a constant HRP activity (4000 units/ml). The mass removal of o-xylene-d10 and naphthalene-ds increased with increasing HRP enzymatic activity, and up to 54% and 51% of mass removal were observed for o-xylene-d10 and naphthalene-d8, respectively. Increasing the concentration of H2O2 resulted in increased mass removal of aromatic hydrocarbons.

Determination of organic acids in ground water by liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry

ABSTRACT Current methods of determining organic acids in ground water are labor-intensive, time-consuming and require a large volume of sample (100 milliliter to 1.0 liter). This paper reports a new method developed to determine aliphatic, alicyclic, and aromatic acids in ground water using liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC/APCI/MS). This method was shown to be fast (less than 1 hour), effective, and reproducible, requiring only 1.0 mL of ground-water sample. Ground water was pH-adjusted, filtered through 0.45 μm filters and directly injected into the LC. A binary solvent system consisting of 40 mM of aqueous ammonium acetate and methanol and a C18 column were used for chromatographical separation. The APCI was operated under negative ionization mode. Selected ion monitoring (SIM) was used for detection and quantitation of the analytes. This method was applied to the analysis of organic acids in ground-water samples collected from an aquifer contaminated ...

Phospholipids of Five Pseudomonad Archetypes for Different Toluene Degradation Pathways

Abstract Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was used to determine intact phospholipid profiles for five reference pseudomonad strains harboring different (aerobic) toluene catabolic pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. These five strains contained a predominant pool of phosphatidylethanolamines. Other phospholipids identified include phosphatidylglycerol, phosphatidylserine, phosphatidylmethylethanolamine, and phosphatidyldimethylethanolamine. There was a clear separation in phospholipid profiles that allows for the differentiation between the Pseudomonas and Burkholderia genera. Factor analysis of the phospholipid profiles showed that B. cepacia G4, P. putida mt-2, and B. pickettii PKO1 were clearly separated, while P. putida F1 and P. mendocina KR1 were clustered as a group. These results suggest that intact phospholipid profiling could be used to evaluate the relative abundance of specific degraders in bioreactors or in aquifer material. Nevertheless, the usefulness of this technique for taxonomic characterization of such complex samples remains to be demonstrated because of potential confounding effects of overlapping profiles and potential changes in phospholipid composition due to different growth conditions.