Sensitive characterization of microbial ubiquinones from biofilms by electrospray/mass spectrometry.

Utilizing high-performance liquid chromatography/electrospray/tandem mass spectrometric analysis of the neutral lipid extract of microbial cells and biofilm communities, respiratory ubiquinone (UQ) (1-methyl-2-isoprenyl-3,4-dimethoxyparabenzoquinone) isoprenologues can be separated isocratically in minutes and assayed with a limit of quantification (LOQ) of 9 p.p.b. (11.1 fmol UQ9 microL(-1)). This corresponds to about 1.29 x 10(7) cells of Pseudomonas putida. Highest sensitivity is achieved using flow-injection analysis with multiple reaction monitoring wherein ammoniated molecular ions of specific isoprenologues pass through quadrupole one, are collisionally dissociated in quadrupole two and identified from the product ion fragment at m/z 197.1 in quadrupole three. This assay has a repeatability of between 6% and 10% over three orders of magnitude (r2 = 0.996). Quinone profiling based on dominant isoprenologue patterns provides taxonomic insights. Detection of prominent UQ isoprenologues indicates presence of microeukaryotes and alpha Proteobacteria with UQ10, obligatory aerobic Gram-negative bacteria with UQ4-14, facultative Gram-negative (and some gamma Proteobacteria growing in microniches with oxygen or to a much lesser extent nitrate as a terminal electron acceptor with UQ8, and other gamma Proteobacteria with UQ9. High sensitivity is essential as the phospholipid fatty acid (PLFA) to UQ molar ratios are 130 or greater. Previous studies have established that recovery of sediment communities with high PLFA/UQ ratios corresponded to areas of aerobic metabolism, an important consideration in bioremediation or nuclide mobilization.

Electrospray ionization/mass spectrometry compatible reversed-phase separation of phospholipids: piperidine as a post column modifier for negative ion detection.

An electrospray ionization (ESI) compatible separation of phospholipids (PL), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), was performed on a C18 column by reversed phase High Performance Liquid Chromatography (HPLC) with minimal ESI suppression. The mobile phase, used isocratically, consisted of methanol and water. ESI was used to efficiently transfer the ions present in solution to the gas phase for mass spectrometric (MS) detection. Formation of negative ions was reinforced by incorporating piperidine post column. Limits of detection (LOD) and limits of quantitation (LOQ) were experimentally determined to be 20 and 60 fmol/microl, respectively, when acquiring data in the selected ion monitoring (SIM) mode monitoring three ions with a single quadrupole MS. When acquiring data from m/z 110-900 in the scanning mode, the LOD and LOQ were experimentally determined to be 1 pmol/microl and 3 pmol/microl. When acquiring product ion spectra for m/z 747, the LOD and LOQ were experimentally determined to be 446 attomol/microl and 1.3 fmol/microl, respectively.

Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis.

Sphingomonas spp possess unique abilities to degrade refractory contaminants and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amplification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingomonas aromaticivorans B0695R in three texturally distinct soil types, showing detection limits between 1.3-2.2 x 10(3) CFU g(-1) dry soil. A sphingolipid extraction protocol was also developed to monitor Sphingomonas populations in soil quantitatively. The detection limit of the assay was 20 pmol g(-1) dry soil, equivalent to about 3 x 10(5) cells g(-1) dry soil. Survival of S. aromaticivorans B0695R was monitored in the three different soils by antibiotic selective plate counting, PCR and sphingolipid analysis. All three approaches showed that the B0695R cells persisted in the low biomass Sequatchie sub-soil at about 3-5 x 10(7)cells g(-1) dry soil. In comparison to the plate counting assay, both the PCR and sphingolipid analysis detected a significantly higher level of B0695R cells in the clay soil and Sequatchie top-soil, indicating the possibility of the presence of viable but non-culturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas population in the environment.

Microbial characterization of a JP-4 fuel-contaminated site using a combined lipid biomarker/polymerase chain reaction--denaturing gradient gel electrophoresis (PCR-DGGE)-based approach.

The impact of pollution on soil microbial communities and subsequent bioremediation can be measured quantitatively in situ using direct, non-culture-dependent techniques. Such techniques have advantages over culture-based methods, which often account for less than 1% of the extant microbial community. In 1988, a JP-4 fuel spill contaminated the glacio-fluvial aquifer at Wurtsmith Air Force Base, Michigan, USA. In this study, lipid biomarker characterization of the bacterial and eukaryotic communities was combined with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the eubacterial community to evaluate correlation between contaminant (JP-4 fuel) concentration and community structure shifts. Vadose, capillary fringe and saturated zone samples were taken from cores within and up- and down-gradient from the contaminant plume. Lipid biomarker analysis indicated that samples from within the plume contained increased biomass, with large proportions of typically gram-negative bacteria. Outside the plume, lipid profiles indicated low-biomass microbial communities compared with those within the initial spill site. 16S rDNA sequences derived from DGGE profiles from within the initial spill site suggested dominance of the eubacterial community by a limited number of phylogenetically diverse organisms. Used in tandem with pollutant quantification, these molecular techniques should facilitate significant improvements over current assessment procedures for the determination of remediation end-points.