Phospholipid Ester-linked Fatty Acid Profiles Research Articles

A reanalysis of phospholipid fatty acids as ecological biomarkers for methanotrophic bacteria

Aerobic methane-oxidizing bacteria (MB) are the primary terrestrial sinks for the greenhouse gas methane. A distinct characteristic of MB is the presence of specific phospholipid ester-linked fatty acids (PLFA) in their membranes that differentiate them from each other and also from all other organisms. These distinct PLFA patterns facilitate microbial ecology studies. For example, the assimilation of C from methane into PLFA can be traced in environmental samples using stable isotope ((13)C) probing (SIP), which links the activity of MB to their community composition in situ. However, the phylogenetic resolution of this method is low because of a lack of PLFA profiles from cultured MB species. In this study, PLFA profiles of 22 alphaproteobacterial (type II) MB were analysed after growth on methane, methanol or both substrates together. Growth on different substrates did not affect the PLFA profiles of the investigated strains. A number of Methylocystis strains contained novel C18:2 fatty acids (omega 7c,12c and omega 6c,12c) that can be used as diagnostic biomarkers. The detection of these novel PLFA, combined with the analyses of multiple type II strains, increased the phylogenetic resolution of PLFA analysis substantially. Multivariate analysis of the expanded MB PLFA database identified species groups that closely reflected phylogenies based on 16S rRNA and pmoA gene sequences. The PLFA database therefore provides a robust framework for linking identity to activity in MB communities with a higher resolution than was previously possible.

Soil microbial community composition and land use history in cultivated and grassland ecosystems of coastal California

Phospholipid ester-linked fatty acid (PLFA) profiles were used to evaluate soil microbial community composition for 9 land use types in two coastal valleys in California. These included irrigated and non-irrigated agricultural sites, non-native annual grasslands and relict, never-tilled or old field perennial grasslands. All 42 sites were on loams or sandy loams of similar soil taxa derived from granitic and alluvial material. We hypothesized that land use history and its associated management inputs and practices may produce a unique soil environment, for which microbes with specific environmental requirements may be selected and supported. We investigated the relationship between soil physical and chemical characteristics, management factors, and vegetation type with microbial community composition. Higher values of total soil C, N, and microbial biomass (total PLFA) and lower values of soil pH occurred in the grassland than cultivated soils. The correspondence analysis (CA) of the PLFA profiles and the canonical correspondence analysis (CCA) of PLFA profiles, soil characteristics, and site and management factors showed distinct groupings for land use types. A given land use type could thus be identified by soil microbial community composition as well as similar soil characteristics and management factors. Differences in soil microbial community composition were highly associated with total PLFA, a measure of soil microbial biomass, suggesting that labile soil organic matter affects microbial composition. Management inputs, such as fertilizer, herbicide, and irrigation, also were associated with the distinctive microbial community composition of the different cultivated land use types.

Effects of petroleum hydrocarbons on the phospholipid fatty acid composition of a consortium composed of marine hydrocarbon-degrading bacteria

Phospholipid ester-linked fatty acids (PLFAs) of a bacteria consortium, reconstituted in vitro from ten marine hydrocarbon-degrading bacteria, were studied. Culture of the consortium with ammonium acetate as the sole carbon source mainly yielded even-numbered PLFAs composed of straight chain saturated (ca. 26%) and monounsaturated (ca. 71%) fatty acids. Growth of the consortium on Blend Arabian Light petroleum (BAL 250) resulted in a complex PLFA profile with the appearance of (1) odd-numbered straight chain PLFAs, mainly 15:0 and 17:0, (2) iso- and anteiso-PLFAs with 15–17 carbon atoms, (3) 10-, 11-, 12- and 13-monomethylated PLFAs with 16–19 carbon atoms and (4) odd-numbered monounsaturated PLFAs, mainly 17:1 Δ9Z and 19:1 Δ10. When petroleum grown cells were transferred into a medium containing ammonium acetate, the consortium exhibited mainly even-numbered PLFAs: this demonstrated the ability of bacterial strains to restore their PLFA compositions after exposure to petroleum hydrocarbons. It, therefore, appears that the PLFA compositions of these bacteria are strongly influenced by the carbon sources so that several potential biomarkers of hydrocarbon-degrading bacterial activities could be recognized. In addition, an hydrocarbon-degrading activity index (HDAI) was derived from the characteristic PLFA profiles of the consortium grown on ammonium acetate and petroleum hydrocarbons. The HDAI may prove to be a tool revealing the development of hydrocarbon-degrading strains in oil-contaminated sediments.

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.

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 community structure analysis of euxinic sediments using phospholipid fatty acid biomarkers

Microbial community structure in sediments, as determined by the phospholipid ester-linked fatty acid (PLFA) analysis, collected from Kojima Lake during summer and winter was described with reference to the environmental quality of the lake. A characteristic feature of the study area was the persistence of anoxia in summer and winter in the deeper area, and the sediments were under the reduced condition with high levels of sulfide, chemical oxygen demand, and low values of oxidation-reduction potential. PLFA profiles of the sediment were dominated by the saturated, branched and monounsaturated fatty acids. Small amounts (less than 5% of the total PLFA) of polyunsaturated fatty acids (biomarker fatty acids of microeukaryotes) and long chain fatty acids were detected in sediments. Total PLFA concentrations in sediments were higher in summer than in winter. Microbial community structure in the lake sediments was predominated by the prokaryotes, as evidenced from the larger amounts of bacterial biomarker fatty acids in the range of C10 to C20. The presence of signature fatty acids of sulfate reducing bacteria in sediments was consistent with the high levels of sulfide in the sediment and anoxic condition in the study area. PLFA showed significant differences in PLFA profiles between shallow and deeper areas, indicating the differences in the lipid contributing communities. However, no such a significant difference was observed between summer and winter.

Characterization of Microbial Community Structure in the Surface Sediment of Osaka Bay, Japan, by Phospholipid Fatty Acid Analysis

Twenty-eight sediment samples collected from Osaka Bay, Japan, were analyzed for phospholipid ester-linked fatty acids (PLFA) to determine regional differences in microbial community structure of the bay. The abundance of three major groups of C(10) to C(19) PLFA (saturated, branched, and monounsaturated PLFA), which accounted for 84 to 97% of the total PLFA, indicated the predominance of prokaryotes in the sediment. The distribution of six clusters obtained by similarity analysis in the bay revealed a marked regional distribution in the PLFA profiles. Total PLFA concentrations (0.56 to 2.97 mug/g [dry weight] of the sediment) in sediments also showed marked variation among the stations, with higher concentrations of total PLFA in the central part of the bay. The biomass, calculated on the basis of total PLFA concentration, ranged from 0.25 x 10 to 1.35 x 10 cells per g (dry weight) of the sediment. The relative dominance of microbial groups in sediments was described by using the reported bacterial biomarker fatty acids. Very small amounts of the characteristic PLFA of microeukaryotes in sediments indicated the restricted distribution of microeukaryotes. By examining the distribution of clusters and groups of microorganisms in the bay, there were two characteristics of the distribution pattern: (i) the predominance of anaerobic bacteria and gram-positive prokaryotes, characterized by the high proportions of branched PLFA in the eastern and northeastern sides of the bay, where the reported concentrations of pollutants were also high, and (ii) the predominance of aerobic prokaryotes and eukaryotes, except for a few stations, in the western and southwestern sides of the bay, as evidenced by the large amounts of monounsaturated PLFA. Such significant regional differences in microbial community structure of the bay indicate shifts in microbial community structure.

Distribution of phospholipid ester-linked fatty acid biomarkers for bacteria in the sediment of Ise Bay, Japan

Surface sediments collected from 40 stations in Ise Bay, which is one of the most polluted bays in Japan, were subjected to phospholipid ester-linked fatty acid (PLFA) analysis. Thirty-five fatty acids were identified in the sediments; they included saturated, unsaturated and branched fatty acids. The major fatty acids included the even-numbered straight chain fatty acid 16:0 (20%), branched chain fatty acids i15:0 (8%) and a15:0 (11%),and monounsaturated fatty acids 16:1d9c (11%) and 18:1d11 (10%). Fatty acids which are common in bacterial membranes were found, and low amounts of longer chain fatty acids were detected in relatively constant amounts in the bay. One of the characteristic features of the PLFA analysis in Ise Bay is the virtual absence of polyunsaturated fatty acids in the sediments, except for 18:2. The PLFA profiles indicate that the microbial community structure is characterized by the absence of polyunsaturated fatty acids typical of microeukaryotes and high proportions of fatty acid biomarkers of prokaryotes in sediments. A wide distribution of aerobic bacteria. Gram-positive bacteria, anaerobic bacteria and sulfate-reducing bacteria in the microbial community structure was indicated by the presence of biomarker fatty acids. Similarity analysis of the PLFA profiles in sediments of all the stations showed that they were similar at the 90% level. The results of Tukey's test showed that a majority of the fatty acids in sediments were not significantly enriched in the bay. The absence of significant variation in the PLFA profiles in sediments revealed that the microbial community structure is similar throughout the bay, and this uniformity was attributed to the reported pollution and eutrophication in Ise Bay. Further, the significant PLFA patterns, with a high proportion of prokaryotic biomarker fatty acids and an absence of microeukaryotic biomarkers, indicate that PLFA analysis could be used as a measure of pollution in sediments.

Determination of microbial biomass and its community structure from the distribution of phospholipid ester-linked fatty acids in sediments of Hiroshima Bay and its adjacent bays

Phospholipid ester-linked fatty acid (PLFA) composition in sediment samples collected from 20 sampling stations of Hiroshima Bay and its adjacent bays are reported. Thirty-one individual fatty acids were identified in the sediments which comprised of saturated, monounsaturated, polyunsaturated and branched fatty acids. A majority of these fatty acids are characteristic of aerobic and anaerobic bacteria especially sulphate-reducing bacteria. PLFA profiles in the sediments varied among the stations indicating the differences in the contributing microbial communities. The calculated biomass also varied from 0·3 × 10 7 to 2·6 × 10 7 cells g −1 dry weight sediment. The shift in microbial community structure within the study area is understood from the seven clusters obtained by the similarity analysis of PLFA composition. The results of environmental parameters revealed the existence of both oxic and anoxic conditions in the study area and the PLFA composition in sediments is discussed in relation to these environmental parameters. Low concentrations of polyunsaturated fatty acids (PUFA) determined in sediments, characteristic of microeukaryotes, could be attributed to the existing environmental conditions and pollution in the study area. Small quantities of long chain fatty acids in sediments indicated that the terrestrial input is low.

Variation in Microbial Biomass and Community Structure in Sediments of Eutrophic Bays as Determined by Phospholipid Ester-Linked Fatty Acids

The distribution of phospholipid ester-linked fatty acids (PLFA) in sediments of eutrophic bays (Hiroshima Bay and Aki Nada) was studied to quantify the microbial biomass, community structure, and nutritional status. A total of 63 fatty acids in the range of C(10) to C(24) were determined. They consist of saturated fatty acids, branched fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, and variation was revealed in the relative proportions of these fatty acids in sediments. On the basis of the PLFA concentration in sediments, the calculated microbial biomass showed variation (mean +/- standard deviation = 0.70 x 10 +/- 0.53 x 10 cells per g [dry weight] of sediment) in the eutrophic bays. In sediments, a higher amount of biomass was observed in the coastal area of Hiroshima Bay than that observed in the rest of the bay and adjacent Aki Nada. The microbial community structure of the present study area, as characterized by the PLFA profiles, showed very low percentages of polyunsaturated fatty acids and long-chain fatty acids characteristic of microeukary-otes and terrestrial input, respectively, and high percentages of fatty acids characteristic of bacteria. The distribution of PLFA profiles also showed the relative contribution of both aerobic and anaerobic bacteria, especially sulfate-reducing bacteria, in the study area. The relative proportions of PLFA revealed distinctive differences among the stations of the study area, as is evidenced from six clusters obtained for the PLFA profiles. The results of Tukey's honestly significant difference test further confirmed that the sediments in the coastal area of Hiroshima Bay were significantly enriched by a number of fatty acids when compared with other areas investigated where relatively few fatty acids were present in significant quantities. No marked variation in environmental parameters in the surface- and bottom-water samples was observed, indicating the absence of any water movement in the study area. Furthermore, low redox potential and the levels of sulfide in the sediment revealed the reduced condition of the sediment. The existing environmental conditions and pollution of the study area were attributed to the observed microbial community structure in the sediments.

Membrane fatty acids as phenotypic markers in the polyphasic taxonomy of methylotrophs within the Proteobacteria

A polyphasic approach to bacterial taxonomy attempts to integrate phylogenetic relationships with phenotypic marker analysis. This study describes the application of membrane fatty acids as a phenotypic marker for methylotrophs. Detailed phospholipid, ester-linked fatty acid (PLFA) profiles are reported for 17 methylotrophic eubacterial strains. These profiles included verification of double bond positions and geometries, both critical features for this analysis. Multivariate cluster analysis was used to indicate groupings of these strains along with literature values of both methylotrophs and non-methylotrophs based on the PLFA phenotype. Like many phenotypic characteristics, PLFA profiles were influenced by environmental conditions. The instabilities displayed, however, were predictable from physiological studies including increased trans/cis and cyclopropyl/cis ratios. Cluster analysis of PLFA profiles generated by separate investigators with different culture conditions indicated reproducibility by strain and species. The PLFA phenotype relationships compare favourably with phylogenetic associations based on 16S rRNA data for methylotrophs and will continue to be a valuable phenotypic marker for Proteobacteria taxonomy.

The Phospholipid Ester-linked Fatty Acid Composition of Thermophilic Bacteria

Summary The phospholipid ester-linked fatty acids (PLFA) of thermophilic (optimum growth temperature 58°C and 68°C) bacteria were analyzed in detail by capillary GC and GC-MS. The bacteria were isolated from the non-volcanically but naturally heated waters of the Australian Artesian Basin, volcanically heated waters of New Zealand and the United States and artificially heated waters of a sugar mill. In general, as has been the case of the PLFA profiles of the few thermophilic eubacteria studied to date, a similar preponderance of iso and anteiso saturated branched fatty acids was observed in many of the isolates examined. However, exceptions to this rule were also observed. Notably four thermophilic anaerobic isolates were characterised by the presence of high levels of saturated normal fatty acids (53–89% of total PLFA), with evencarbon numbered fatty acids predominating, and one isolate contained high levels (40%) of monounsaturated fatty acids (predominantly il5 : 1 w 8c and il7 : 1 w 7c). An unusual fatty acid il7 : 1 w 11c was present in T. aquaticus and is reported for the first time.

The Phospholipid Ester-linked Fatty Acid Composition of Members of the Family Halomonadaceae and Genus Flavobacterium: A Chemotaxonomic Guide

Summary 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 : 1 w 7c, 16: 1 w 7c 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 : 1 w 10c, a15 : 1 w 10c 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 C 15 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 : 1 w 10c) in microbial ecology studies of antarctic saline lakes where the presence and relative importance of these bacteria is being investigated.

Microbial Biomass, Activity, and Community Structure of Water and Particulates Retrieved by Backflow from a Waterflood Injection Well

Oil field injection water was allowed to back flow from two wells at the Packard drill site in Los Angeles, Calif., and was sampled at various times to obtain information about the biomass, potential activity, and community structure of the microbiota in the reservoir formation and in the injection water. Biomass was greatest in water samples that came from the zone near the injection site and dropped off sharply in subsequent samples, which were assumed to come from zones farther away from the well. Samples obtained from near the well also had visible exopolysaccharide blankets, as seen in scanning electron microscopic preparations. In one of the wells that was sampled, rates of glucose or acetate incorporation into microbial lipids correlated with biomass; but in the other well, activities correlated with the sampling time (volume of water that back flowed). Transmission electron micrographs showed a diverse, gram-negative bacterial population in a variety of physiological states. The analysis of the phospholipid ester-linked fatty acid profiles of the samples revealed consistently large proportions of 18:1omega7c fatty acids, indicating the presence of many anaerobes, facultative organisms, or both. Proportions of cyclopropyl fatty acids and ratios of trans/cis monoenoic compounds increased with the volume of water that back flowed (analogous with the distance into the formation), while the ratio of unsaturated/saturated compounds decreased, possibly indicating higher levels of stress or starvation in the microbial communities farthest from the injection well. Greater than 90% of the total biomass was trapped on glass fiber filters, indicating that the microbiota were largely attached to particles or were clumped. These sampling techniques and analytical methods may prove useful in monitoring for problems with microbes (e.g., plugging) in waterflood operations and in the preparation of water injection wells for enhanced oil recovery by the use of microbes.