Ester-linked Fatty Acids Research Articles

Occurrence of fatty acids linked to non-phospholipid compounds in the polar fraction of a marine sedimentary extract from Carteau cove, France

A surface sediment from Carteau cove, located at the western part of the Gulf of Fos (France), was collected and analysed to determine the lipid composition of its most polar fraction. Analyses by high performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) of the polar fraction of the sediment extract revealed the presence of phospholipids (PL), with mainly phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE), but also the presence of non-phospholipid polar compounds (NPC). Phospholipids and non-phospholipid polar compounds (NPC) were isolated by preparative thin-layer chromatography and transmethylated. Phospholipid ester-linked fatty acids ranged from 12- to 20-carbon atoms and accounted for 34.8 μg g−1 of dry sediment whereas fatty acids derived from non-phospholipid polar compounds ranged from 12- to 28-carbon atoms and accounted for 39.1 μg g−1 of dry sediment. The occurrence of significant amounts of fatty acids derived from non-phospholipid polar compounds of a sediment polar fraction illustrates the importance of the preliminary analysis and purification of phospholipids to avoid an overestimation of the microbial biomass when determined with a current phospholipid ester-linked fatty acids (PLFA) analysis.

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

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 C14 to C19. 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.

Tubulin Anchoring to Glycolipid-enriched, Detergent-resistant Domains of the Neuronal Plasma Membrane

After incubation of intact living cultured rat cerebellar granule cells at 37 degrees C with a new GM1 ganglioside analog, carrying a diazirine group and labeled with (125)I in the ceramide moiety, followed by photoactivation, a relatively small number of radiolabeled proteins were detected in a membrane-enriched fraction. A protein of about 55 kDa with a pI of about 5 carried a large portion of the radioactivity even if incubation and cross-linking were performed at 4 degrees C and in the presence of inhibitors of endocytosis, suggesting that it is cross-linked at the plasma membrane. Immunoprecipitation and Western blotting experiments showed the positivity of this protein for tubulin. Trypsin treatment of intact cells ruled out the involvement of a plasma membrane surface tubulin. Release of radioactivity from cross-linked tubulin after KOH treatment (but not hydroxylamine treatment) suggested that the photoactivated ganglioside reacts with an ester-linked fatty acid anchor of tubulin. Low buoyancy, detergent-resistant membrane fractions, isolated from cells after incubation with the GM1 analogue and photoactivation, proved their enrichment in endogenous and radioactive GM1 ganglioside, sphingomyelin, cholesterol, signal transduction proteins, and tubulin. It is noteworthy that radioactive tubulin was also detected in this fraction, indicating the presence of tubulin molecules carrying a fatty acid anchor in detergent-resistant, ganglioside-enriched domains of the plasma membrane. Parallel experiments carried out with a phosphatidylcholine analogue, also carrying a diazirine group and labeled with (125)I in the fatty acid moiety, showed the specificity of tubulin interaction with GM1. Taken together, these results indicate that some tubulin molecules are associated with a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane. This novel feature of membrane domains can provide a key for a better understanding of their biological role.

00/00867 Behavior of organic matter from Callovian shales during low-temperature air oxidation

The phospholipid ester-linked fatty acids (PLFA) from members of the family Halomonadaceae and the genus Flavobacterium were analyzed in detail by capillary GC and GC-MS. Precise monounsaturated double bond position and geometry was determined by GC-MS analysis of the dimethyl disulphide adducts of the monounsaturated fatty acids. No clear distinction was observed between members of the two genera Halomonas and Deleya based upon PLFA composition. The major PLFAs detected in strains from the two genera were 18 : 1w7c, 16: 1w7c and 16 : 0. These three fatty acids accounted for greater than 95% of the total PLFA detected. The PLFA profiles of “Flavobacterium salegens” (ACAM 48) and “Flavobacterium gondwanense” (ACAM 44), proposed new species of moderately halophilic bacteria isolated from Organic Lake, Antarctica, were characterized by high abundances of i15 : 0, a15 : 0, 15 : 0, i15 : 1w10c, a15 : 1w10c and i16 : 0. The two species were distinguishable from each other based on their PLFA and hydroxy fatty acid profiles. The position of the double bond in the branched C15 monoununsaturated fatty acids is unusual, and may be a taxonomic feature of these bacteria. It may also be possible to use these signature fatty acids (i and a15 : 1w10c) in microbial ecology studies of antarctic saline lakes where the presence and relative importance of these bacteria is being investigated.

The Lipid Component of Lipoproteins from Borrelia burgdorferi: Structural Analysis, Antigenicity, and Presentation via Human Dendritic Cells

The spirochaetal bacteria Borrelia burgdorferi (Bb) is the tick-borne causative agent of lyme disease. The major membrane immunogens of Bb are outer surface proteins. The lipid component of these lipoproteins is relevant for the immunogenicity of Bb-lipoproteins. To characterize the antigenic properties, the native lipid component of lipoproteins was isolated and the detailed molecular structure was analyzed. The molecular structure of the lipoprotein–lipid component turned out to be S(propane-2′,-3′diol)-3-thio-2-aminopropanic acid (S-glyceryl-cysteine) with one ester-linked fatty acid, one acetyl group, and one N-terminal amide-bound fatty acid. Fatty acid analysis of the lipid component indicated a heterogeneous composition comprising C16:0, C18:0, C18:1, C18:2, and C 20:0. The antigenicity was tested with in vitro bioassays using human blood-derived dendritic cells (DCs) as antigen-presenting cells and autologous Bb-specific T-cells. We found that human DCs present the lipid component of Bb-lipoproteins via MHC class II inducing an antigen-specific T-cell immune response in vitro.

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

Ground-water chemistry and the stable C isotope composition (δ 13C 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 13C 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 13C in δ 13C 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 13C 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 13C of dissolved inorganic C.

Radioactive fingerprinting of microorganisms that oxidize atmospheric methane in different soils.

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with (14)CH(4) followed by analysis of radiolabelled phospholipid ester-linked fatty acids ((14)C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The (14)C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1omega8c (up to 9.0% of the total (14)C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1omega9, 18:1omega7, and 18:0). The (14)C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH(4). The (14)C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the (14)C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.

Rapid response of soil microbial communities from conventional, low input, and organic farming systems to a wet/dry cycle

Soil microbial communities may be strongly influenced by agricultural practices which change the soil environment. One such practice is the use of organic amendments and cover crops which increase carbon availability to microorganisms. Another is irrigation which, in California’s hot, rain-free growing season, can cause severe wet/dry cycles. We investigated (i) long-term differences in amounts of organic inputs using soils from organic, low input, and conventional farming systems, and (ii) differences in severity of soil drying following irrigation, using soil from two depths, 0–3 and 3–15 cm. All soils were air-dried and re-wetted, and we measured short-term changes in microbial biomass carbon (MBC), dissolved organic carbon (DOC), respiration, and phospholipid ester-linked fatty acid (PLFA) composition before and for 27 h after re-wetting. Respiration rates were fit to a two-first-order-component model. Carbon respired from the more slowly utilized C pool of the two-component model, MBC, and DOC increased with increasing amounts of organic inputs, and PLFA composition of the organic and conventional soils clearly differed in their mole percentages of numerous fatty acids when analyzed by principal components analysis and redundancy analysis. Despite these differences, the response of microbial communities in the three farming systems to soil drying and re-wetting was similar. For example, the relative increase in MBC following soil re-wetting did not differ among the farming system soils. In contrast, the relative increase in MBC after re-wetting was greater, and the respiratory response to soil re-wetting was more rapid in the surface (0–3 cm) than deeper (3–15 cm) layer. Higher ratios of cyclopropyl fatty acids to their precursors suggested greater stress to bacteria in the deep than surface layer, and these ratios declined more rapidly after re-wetting in the deep than surface layer. This study suggested that adaptation to wet/dry cycles by surface microorganisms had occurred during the 3-month growing season, leading to changes in both microbial process rates and community composition.

Fatty acid composition of Mesorhizobium huakuii lipopolysaccharides. Identification of 27-oxooctacosanoic acid

Lipopolysaccharides of three Mesorhizobium huakuii strains carried a number of amide-linked 3-hydroxylated fatty acids including: 3-OH-12:0, 3-OH- i-13:0, 3-OH-20:0, 3-OH- i-21:0, 3-OH-22:0, 3-OH-23:0 and unsaturated 3-OH-22:1. The first three of the above mentioned acids are the main amide-linked fatty acids in the LPS preparations. The main ester-bound fatty acids comprise 16:0, i-17:0, 18:0, 20:0 and 27-OH-28:0. Among minor constituents of lipid A 25-OH-26:0 and 29-OH-30:0 together with some non-polar fatty acids were found. Additionally, the presence of 4-oxo-20:0, 4-oxo- i-21:0 and 4-oxo-22:0 amide-bound fatty acids as well as the 27-oxo-28:0 ester-linked fatty acid were proved. To our knowledge oxo fatty acids are rare constituents of lipopolysaccharides and 27-oxo-28:0 was found for the first time in the LPS preparations from members of Rhizobiaceae.

Fatty acid composition ofMesorhizobium huakuiilipopolysaccharides. Identification of 27-oxooctacosanoic acid

Lipopolysaccharides of three Mesorhizobium huakuii strains carried a number of amide-linked 3-hydroxylated fatty acids including: 3-OH-12:0, 3-OH-i-13:0, 3-OH-20:0, 3-OH-i-21:0, 3-OH-22:0, 3-OH-23:0 and unsaturated 3-OH-22:1. The first three of the above mentioned acids are the main amide-linked fatty acids in the LPS preparations. The main ester-bound fatty acids comprise 16:0, i-17:0, 18:0, 20:0 and 27-OH-28:0. Among minor constituents of lipid A 25-OH-26:0 and 29-OH-30:0 together with some non-polar fatty acids were found. Additionally, the presence of 4-oxo-20:0, 4-oxo-i-21:0 and 4-oxo-22:0 amide-bound fatty acids as well as the 27-oxo-28:0 ester-linked fatty acid were proved. To our knowledge oxo fatty acids are rare constituents of lipopolysaccharides and 27-oxo-28:0 was found for the first time in the LPS preparations from members of Rhizobiaceae.

Characterization of methanotrophic bacterial populations in soils showing atmospheric methane uptake.

The global methane cycle includes both terrestrial and atmospheric processes and may contribute to feedback regulation of the climate. Most oxic soils are a net sink for methane, and these soils consume approximately 20 to 60 Tg of methane per year. The soil sink for atmospheric methane is microbially mediated and sensitive to disturbance. A decrease in the capacity of this sink may have contributed to the approximately 1%. year(-1) increase in the atmospheric methane level in this century. The organisms responsible for methane uptake by soils (the atmospheric methane sink) are not known, and factors that influence the activity of these organisms are poorly understood. In this study the soil methane-oxidizing population was characterized by both labelling soil microbiota with (14)CH(4) and analyzing a total soil monooxygenase gene library. Comparative analyses of [(14)C]phospholipid ester-linked fatty acid profiles performed with representative methane-oxidizing bacteria revealed that the soil sink for atmospheric methane consists of an unknown group of methanotrophic bacteria that exhibit some similarity to type II methanotrophs. An analysis of monooxygenase gene libraries from the same soil samples indicated that an unknown group of bacteria belonging to the alpha subclass of the class Proteobacteria was present; these organisms were only distantly related to extant methane-oxidizing strains. Studies on factors that affect the activity, population dynamics, and contribution to global methane flux of "atmospheric methane oxidizers" should be greatly facilitated by use of biomarkers identified in this study.

Microbial Diversity in Nankai Trough Sediments at a Depth of 3,843 m

Dense populations of bivalves, primarily Calyptogena sp., were observed at cold seeps of the Nankai Trough. Bacterial input to the sediment was estimated through determination of phospholipid ester-linked fatty acid (PLFA) and DNA profiles. Results indicated a bacterial biomass of 109 cells (g dry wt)-1 while individual fatty acid profiles revealed a predominance of monounsaturated fatty acids, mainly 18:1 isomers. The presence of these fatty acids can be interpreted to reflect a response to low temperature and a predominance of psychrophilic bacteria. DNA fragments encoding bacterial ribosomal RNA small-subunit sequences (16S rDNA) were amplified by the polymerase chain reaction method using DNA extracted directly from the sediment samples. From the sequencing results, at least 19 kinds of bacterial 16S rDNAs related to mostly the Proteobacteria and a few gram-positive bacteria were identified. These results suggest that the bacterial community in the Nankai Trough sediments consists of mainly bacteria belonging to the Proteobacteria γ, e, and δ subdivisions. Bacteria belonging to the e and δ subdivisions, which are known to include epibiont and sulfate reducing bacteria, respectively, were mostly detected in the sediment obtained from inside the area of the Calyptogena community, and the δ-Proteobacteria may function to supply reduced sulfur to bacterial endosymbionts of Calyptogena.

Degradation of phthalate and Di-(2-Ethylhexyl)phthalate by indigenous and inoculated microorganisms in sludge-amended soil

The metabolism of phthalic acid (PA) and di-(2-ethylhexyl)phthalate (DEHP) in sludge-amended agricultural soil was studied with radiotracer techniques. The initial rates of metabolism of PA and DEHP (4.1 nmol/g [dry weight]) were estimated to be 731.8 and 25.6 pmol/g (dry weight) per day, respectively. Indigenous microorganisms assimilated 28 and 17% of the carbon in [14C]PA and [14C]DEHP, respectively, into microbial biomass. The rates of DEHP metabolism were much greater in sludge assays without soil than in assays with sludge-amended soil. Mineralization of [14C]DEHP to 14CO2 increased fourfold after inoculation of sludge and soil samples with DEHP-degrading strain SDE 2. The elevated mineralization potential was maintained for more than 27 days. Experiments performed with strain SDE 2 suggested that the bioavailability and mineralization of DEHP decreased substantially in the presence of soil and sludge components. The microorganisms metabolizing PA and DEHP in sludge and sludge-amended soil were characterized by substrate-specific radiolabelling, followed by analysis of 14C-labelled phospholipid ester-linked fatty acids (14C-PLFAs). This assay provided a radioactive fingerprint of the organisms actively metabolizing [14C]PA and [14C]DEHP. The 14C-PLFA fingerprints showed that organisms with different PLFA compositions metabolized PA and DEHP in sludge-amended soil. In contrast, microorganisms with comparable 14C-PLFA fingerprints were found to dominate DEHP metabolism in sludge and sludge-amended soil. Our results suggested that indigenous sludge microorganisms dominated DEHP degradation in sludge-amended soil. Mineralization of DEHP and PA followed complex kinetics that could not be described by simple first-order equations. The initial mineralization activity was described by an exponential function; this was followed by a second phase that was described best by a fractional power function. In the initial phase, the half times for PA and DEHP in sludge-amended soil were 2 and 58 days, respectively. In the late phase of incubation, the apparent half times for PA and DEHP increased to 15 and 147 days, respectively. In the second phase (after more than 28 days), the half time for DEHP was 2.9 times longer in sludge-amended soil assays than in sludge assays without soil. Experiments with radiolabelled DEHP degraders suggested that a significant fraction of the 14CO2 produced in long-term degradation assays may have originated from turnover of labelled microbial biomass rather than mineralization of [14C]PA or [14C]DEHP. It was estimated that a significant amount of DEHP with poor biodegradability and extractability remains in sludge-amended soil for extended periods of time despite the presence of microorganisms capable of degrading the compound (e.g., more than 40% of the DEHP added is not mineralized after 1 year).

Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, six heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from sea water/sediment samples collected near the isle of Borkum (North Sea), using Mihagol-S (C14,15-n-alkanes) as principal carbon source. These Gram-negative, aerobic, rod-shaped bacteria use a limited number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and pyruvate and its methyl ether. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both extracellular and cell-bound surface-active glucose lipids which reduced the surface tension of water from 72 to 29 mN m-1 (16). This novel class of glycolipids was found to be produced only by these strains. The 16S rRNA gene sequence analysis showed that these strains are all members of the gamma-subclass of the Proteobacteria. Their phospholipids ester-linked fatty acid composition was shown to be similar to that of members of the genus Halmonas, although they did not demonstrate a close phylogenetic relationship to any previously described species. On the basis of the information summarized above, a new genus and species, Alcanivorax borkumensis, is described to include these bacteria. Strain SK2T is the type strain of A. borkumensis.

Identification of a Novel Heptoglycan of α1→2-Linkedd-glycero-d-manno-Heptopyranose: CHEMICAL AND ANTIGENIC STRUCTURE OF LIPOPOLYSACCHARIDES from KLEBSIELLA PNEUMONIAE SSP. PNEUMONIAE ROUGH STRAIN R20 (O1−:K20−)

In a preliminary investigation (Süsskind, M., Müller-Loennies, S., Nimmich, W., Brade, H., and Holst, O. (1995) Carbohydr. Res. 269, C1-C7), we identified after deacylation of lipopolysaccharides (LPS) from Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1(-):K20(-)) as a major fraction the oligosaccharide,-structure; see text- where Kdo was 3-deoxy-D-manno-oct-2-ulopyranosonic acid and Hepp was manno-heptopyranose. The presence of the threo-hex-4-enuronopyranosyl residue indicated a substituent at O-4 of the second GalA residue linked to O-3 of the second L,D-Hep residue, which had been eliminated by treatment with hot alkali. We now report the complete structure of lipopolysaccharide, which was elucidated by additional characterization of isolated core oligosaccharides and analysis of the lipid A. The substituent at O-4 of the second GalpA is D-GlcpN, which in a fraction of the LPS is substituted at O-6 by three or four residues of D-glycero-D-manno-heptopyranose (D,D-Hepp). The complete carbohydrate backbone of the LPS is as follows, -structure; see text- (L-glycero-D-manno-heptopyranose; L,D-Hepp), where all hexoses possess the D-configuration. Sugars marked with an asterisk are present in nonstoichiometric amounts. The structure is unique with regard to the presence of an alpha1-->2-linked D-glycero-D-manno-heptoglycan (oligosaccharide), which has not been described to date, and does not contain phosphate substituents in the core region. Fatty acid analysis of lipid A identified (R)-3-hydroxytetradecanoic acid as sole amide-linked fatty acid and (R)-3-hydroxytetradecanoic acid, tetradecanoic acid, small amounts of 2-hydroxytetradecanoic acid, hexadecanoic acid, and traces of dodecanoic acid as ester-linked fatty acids, substituting the carbohydrate backbone D-GlcpN4Pbeta1-->6D-GlcpNalpha1P. The nonreducing GlcN carries four fatty acids, present as two 3-O-tetradecanoyltetradecanoic acid residues, one of which is amide-linked and the other ester-linked to O-3'. The reducing GlcN is substituted in a nature fraction of lipid A by two residues of (R)-3-hydroxytetradecanoic acid, one in amide and the other in ester linkage at O-3. Two minor fractions of lipid A were identified; in one, the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing GlcN is esterified with hexadecanoic acid, resulting in 3-O-hexadecanoyltetradecanoic acid, and in the second, one of the 3-O-tetradecanoyltetradecanoic acid residues at the nonreducing GlcN is replaced by 3-O-dodecanoyltetradecanoic acid. Thus, the complete structure of LPS is as shown in Fig. 1. After immunization of BALB/c mice, two monoclonal antibodies were obtained that were shown to be specific for the core of LPS from K. pneumoniae ssp. pneumoniae, since they did not react with LPS or whole-cell lysates of a variety of other Gram-negative species. Both monoclonal antibodies could be inhibited by LPS but not by isolated oligosaccharides and are thus considered to recognize a conformational epitope in the core region.

Direct fingerprinting of metabolically active bacteria in environmental samples by substrate specific radiolabelling and lipid analysis

Substrate specific radio assays were used for enumeration and fingerprinting of microorganisms in environmental samples. Direct fingerprinting was based on incorporation of 14C-labelled substrates into microbial lipids. A radioactive fingerprint was obtained by subsequent radio analysis of whole sample phospholipid ester-linked fatty acids ( 14C-PLFA fingerprint). This approach provided a 14C-PLFA fingerprint of the organism actively metabolizing the added 14C-labelled substrate. Labelled and unlabelled PLFAs were analysed as methyl ester derivatives by gas–liquid chromatography with flame ionization detection. The presence of 14C-PLFAs were determined by collection of 14CO 2 produced after combustion of the fatty acids. Additional analysis of the microbial community was carried out by analysis of the radioactivity assimilated into poly-β-hydroxyalkanoates relative to that assimilated into total phospholipids ( 14C-PHA/ 14C-PL ratio). The number of organisms involved in the degradation of a 14C-labelled substrate was estimated using a 14C-most-probable-number technique. These different 14C-based methods were evaluated by studying [ 14C]methane oxidation in agricultural soil, and [ 14C]phenanthrene degradation in activated sludge and marine sediment. The radio assays resulted in distinct fingerprints of the bacterial populations capable of degrading the different radiolabelled substrates. Manipulation of the incubation conditions (e.g., oxygen status) resulted in significant changes in population specific metabolic activity and labelling pattern. Phenotypically related microorganisms appeared to dominate [ 14C]phenanthrene degradation in activated sludge and marine sediment under oxic conditions. Anaerobic [ 14C]phenanthrene degraders in activated sludge produced a very different 14C-PLFA fingerprint. In methane enriched agricultural soil, aerobic methane oxidation was dominated by organisms most similar to the Type I methanotrophic bacteria. Several of the findings obtained by the 14C-PLFA analysis could not have been established on the basis of conventional PLFAs analysis alone. The results suggest that variations of this simple 14C-fingerprinting method may be applicable to studies of substrate metabolism in mixed microbial communities. Direct fingerprinting based on substrate specific radiolabelling may also aid in phenotypic characterization of heterotrophic microorganisms without the need for enrichment or cultivation.

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. Microbial 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 C 12–C 20 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 analysis, varied with depth, and between locations at similar depths, indicating considerable microbial heterogeneity 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.

Phospholipid fatty acid profiles in selected members of soil microbial communities

Fatty acids derived from phospholipids and lipopolysaccharides were investigated from 33 taxonomically different organisms (bacteria, fungi and plant cells) known a priori to inhabit soil (except E. coli). The extended extraction procedure used, liberated non-ester-linked fatty acids in addition to ester-linked fatty acids, hydroxy substituted fatty acids in three different fractions. The amount of non-ester-linked fatty acids was as high as 70% of the total phospholipid fatty acids in some fungi and varied considerably in different organisms. The cis vaccenic acid constituted about 50% of phospholipid fatty acids in selected bacteria belonging to the alpha subclass of Proteobacteria. These fatty acids were not found in other selected organisms. A large amounts of branched chain fatty acids were found in various organisms. If the branching are localised on positions other than iso and anteiso they were strong indicators for gram positive bacteria. The cyclopropyl fatty acids are mainly localized in gram negative bacteria. The beta hydroxy fatty acid of the outer membrane are widespread among bacterial taxa and fungi. These fatty acids are not recommended to use as “signature” fatty acids for gram negative bacteria.

Quantitative Sampling of Indoor Air Biomass by Signature Lipid Biomarker Analysis: Feasibility Studies in a Model System

Standard methods for characterizing the microbial content of indoor air rely on detection of viable microbes that are collected in water or impacted onto growth substrata. Viable counts consistently underestimate microbial numbers in environmental samples by 90–99.9%. Assays of biochemical components characteristic of all cells provide an assessment method independent of the ability to culture the organisms. This article provides evidence that lipid analysis provides quantitative recovery of known volumes of culturable bacteria. Monocultures of Escherichia coli, Bacillus subtilis, and Legionella bozemanii and two mixtures of these organisms were deposited onto glass fiber filters using an air test stand constructed as a modification of the ASTM 1215 standard. Filter deposited biomass was determined by three methods: (1) viable counts of bacteria sampled using an impinger, (2) phospholipid ester-linked fatty acid (PLFA) analysis, and (3) hydroxy fatty acid (OH FA) analysis. PLFA and OH FA were quantified by gas chromatography-mass spectrometry. Bacterial counts determined as colony forming units/filter following impinger sampling ranged between 25.6% (B. subtilis) to 18.5% (L. bozemanii) of the counts determined by PLFA analysis. Sampling efficiency and mechanical and dehydration stresses to the bacteria during aerosolization could have caused the decreased culturable viable counts in comparison to PLFA analysis of viable cells. Signature lipid analysis provides insight into the community composition. Analysis of the glass filters after aerosolization showed that capture was not selective, and monocultures and mixtures gave the expected signature lipid patterns enabling differentiation between species.

Quantitative Sampling of Indoor Air Biomass by Signature Lipid Biomarker Analysis: Feasibility Studies in a Model System

Standard methods for characterizing the microbial content of indoor air rely on detection of viable microbes that are collected in water or impacted onto growth substrata. Viable counts consistently underestimate microbial numbers in environmental samples by 90–99.9%. Assays of biochemical components characteristic of all cells provide an assessment method independent of the ability to culture the organisms. This article provides evidence that lipid analysis provides quantitative recovery of known volumes of culturable bacteria. Monocultures of Escherichia coli, Bacillus subtilis, and Legionella bozemanii and two mixtures of these organisms were deposited onto glass fiber filters using an air test stand constructed as a modification of the ASTM 1215 standard. Filter deposited biomass was determined by three methods: (1) viable counts of bacteria sampled using an impinger, (2) phospholipid ester-linked fatty acid (PLFA) analysis, and (3) hydroxy fatty acid (OH FA) analysis. PLFA and OH FA were quantified b...