Michael J. Barcelona

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.

An assessment of natural biotransformation of petroleum hydrocarbons and chlorinated solvents at an aquifer plume transect

Field biogeochemical characterization and laboratory microcosm studies were performed to assess the potential for future biotransformation of trichloroethylene (TCE) and toluene in a plume containing petroleum hydrocarbons and chlorinated solvents at the former Wurtsmith Air Force Base in Oscoda, MI. In situ terminal electron accepting processes (TEAPs), contaminant composition and microbial phylogeny were studied at a plume transect 100 m downgradient of the source. The presence of reduced electron acceptors, relevant microbial communities, and elevated dissolved methane and carbon dioxide concentrations at the transect, as well as downgradient accumulation of BTEX metabolites and dechlorination products, indicated that past or current reductive dechlorination at the transect was likely driven by BTEX biodegradation in the methanogenic zone. However, TCE and toluene mineralization in sediment-groundwater microcosms without added electron acceptors did not exceed 5% during 300 days of incubation and was nearly invariant with original sediment TEAP, even following amendments of nitrogen and phosphorus. Mineralization rates were on the order of 0.0015-0.03 mumol/g day. After 8 months, microcosms showed evidence of methanogenesis, but CH4 and CO2 production arose from the degradation of contaminants other than toluene. Cis-dichloroethylene was observed in only one methanogenic microcosm after more than 500 days. It appears likely that spatially and temporally dynamic redox zonation at the plume transect will prevent future sustained reductive dehalogenation of highly chlorinated solvents, for during the course of a year, the predominant TEAP at the highly contaminated water table shifted from methanogenesis to iron- and sulfate-reduction. It is recommended that biotransformation studies combine considerations of long-term, spatially relevant changes in redox zonation with laboratory-scale studies of electron donor utilization and cometabolic substrate transformation to yield a more accurate assessment of natural bioattenuation of specific pollutants in aquifers contaminated by undefined organic waste mixtures.

A Probabilistic Methodology to Assess the Risk of Groundwater Quality Degradation

An approach to assess the risk of groundwater quality degradation with regard to fixed standards, based on DisjunctiveKriging (DK) is presented. The DK allows one to evaluate the Conditional Probability (CP) of overriding a given threshold of concentration of a pollutant at a given time, and at a generic point in a consideredgroundwater system. The result of such investigation over the considered area can be plotted in form of maps of spatial risk. By repeating this analysis at different times, severalspatial riskmaps will be produced, one for each consideredtime. By means of non-parametric statistics, the temporal trendof the CPs can be evaluated at every point of the considered area. The trend index, assessed by means of a sort of classification of the trend values obtained as described above,can be superimposed on the most recent values of the spatialrisk (i.e.: the most recent values of probability). Consequentlya classification of the risk of groundwater quality degradationresults with which to weigh both the spatial distribution and thetemporal behaviour of the probability to exceed a given standardthreshold. The methodology has been applied to values of nitrateconcentration sampled in the monitoring well network of theModena plain, northern Italy. This area is characterised by intensive agricultural exploitation and hog breeding along withindustrial and civil developments. The influence of agriculture on groundwater results in a high nitrate pollution that limitsits use for potable purposes.

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.

Isotopic composition of fatty acids of extremely piezophilic bacteria from the Mariana Trench at 11,000 m

Two extremely piezophilic bacteria, DB21MT-2 and DB21MT-5, isolated from sediments of the Mariana Trench at 11,000 m, were grown in the laboratory under low-temperature (10 jC) and high-pressure (70 MPa) conditions. Phospholipid esterlinked fatty acids (PLFA) were analyzed by gas chromatography/mass spectrometry (GC/MS) and isotopic compositions of individual fatty acids were determined by gas chromatography isotope ratio mass spectrometry (GC-IRMS) in order to determine isotopic fractionation effects during biosynthesis. The piezophiles are characterized by fatty acids with carbon chains ranging from 14 to 22 carbons in length and by the presence of abundant polyunsaturated fatty acids (PUFA). Fatty acids from each culture exhibit a wide range of isotopic compositions (d 13 C vs. Pee Dee Belemnite (PDB)). With two exceptions (C15:0 and C17:1 from DB21MT-2), all fatty acids are depleted in 13 C relative to that of bulk carbon in the bacterial growth substrate. In addition, strain DB21MT-2 shows systematically higher (less negative) d 13 C values than strain DB21MT-5. The implications for isotope fractionation in fatty acid biosynthesis and the interpretation of isotopic composition of sedimentary fatty acids are discussed. It is suggested that the same type of microorganisms could have rather different d 13 C under the same growth conditions, and that sedimentary fatty acids with distinct d 13 C values do not necessarily have to originate from different organisms. Interpretation of d 13 C values of sedimentary biomarkers may be complicated by such large variations in d 13 Co f

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.

Geophysical Characterization, Redox Zonation, and Contaminant Distribution at a Groundwater/Surface Water Interface

Three transects along a groundwater/surface water interface were characterized for spatial distributions of chlorinated aliphatic hydrocarbons and geochemical conditions to evaluate the natural bioremediation potential of this environmental system. Partly on the basis of ground penetrating radar measurements, a conductive sediment layer was detected from the shore out to at least 300 m offshore which exhibited gradients in redox pairs and contaminant profiles. The cis-Dichloroethene and 1-chloroethene were predominant in the presence of elevated methane and ferrous iron concentrations and depressed sulfate and aquifer solids-bound iron concentrations. The shallow monitoring points were generally hypoxic to aerobic and exhibited values of specific conductance reflective of near-shore lake water, indicating reoxygenation of the contaminant plume due to wave infiltration. The barge transect yielded trace contaminant concentrations and showed evidence of sulfate reduction. These analyses contributed to the understanding of processes affecting contaminant fate and transport at near-shore mixing zones.

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.

Gypsum-organic interactions in natural seawater: Effect of organics on precipitation kinetics and crystal morphology☆

Abstract Organic compounds which are chloroform extractible from seawater affect gypsum precipitation by inhibiting the nucleation of crystals. The effect is selective towards calcium-populated crystal habit faces and results in the growth of more tabular, equidimensional crystals. Studies using model organic compounds suggest that the inhibition takes place through an adsorption mechanism and show that longer-chained n-fatty acids are more actively adsorbed. The specific adsorption mechanism (at Ca 2+ habit faces) is also supported by the fact that the free-acid (base-soluble) fraction of the chloroform extractibles from seawater is more active than the neutral fraction. The calcium ion complexing ability of model organic compounds is not correlatable with their inhibitory effects on gypsum crystal nucleation.