Ester-linked Fatty Acids Research Articles

A Multi-biological Assay Approach to Assess Microbial Diversity in Arsenic (As) Contaminated Soils

ABSTRACTMicrobial genetic, structural and functional diversity was assessed in response to arsenic (As) pollution in a former gold mine soil. Ester-linked fatty acid methyl ester (EL-FAME), quantitative PCR (qPCR), denaturating gradient gel electrophoresis (DGGE), enzyme activities and MicroResp techniques were used.Multivariate analysis showed that As bioavailability in soil was an important driver affecting microbial diversity. Microbial biomass assessed by EL-FAMEs and qPCR generally decreased under higher bioavailable As, as well as enzyme activities and C substrate utilization. Conversely, actinomycetes and fungal biomass increased along with total As content suggesting the selection of more resistant species.

Abundance and diversity of sedimentary bacterial communities in a coastal productive setting in the Western Irish Sea

The bacterial community composition and biomass abundance from a depositional mud belt in the western Irish Sea and regional sands were investigated by phospholipid ester-linked fatty acid profiling, denaturing gradient gel electrophoresis and barcoded pyrosequencing of 16S rRNA genes. The study area varied by water depth (12–111m), organic carbon content (0.09–1.57% TOC), grain size, hydrographic regime (well-mixed vs. stratified), and water column phytodetrital input (represented by algal polyunsaturated PLFA). The relative abundance of bacterial-derived PLFA (sum of methyl-branched, cyclopropyl and odd-carbon number PLFA) was positively correlated with fine-grained sediment, and was highest in the depositional mud belt. A strong association between bacterial biomass and eukaryote primary production was suggested based on observed positive correlations with total nitrogen and algal polyunsaturated fatty acids. In addition, 16S rRNA genes affiliated to the classes Clostridia and Flavobacteria represented a major proportion of total 16S rRNA gene sequences. This suggests that benthic bacterial communities are also important degraders of phytodetrital organic matter and closely coupled to water column productivity in the western Irish Sea.

Comparison of biochemical and microscopic methods for quantification of arbuscular mycorrhizal fungi in soil and roots

Arbuscular mycorrhizal fungi (AMF) are well-known plant symbionts which provide enhanced phosphorus uptake as well as other benefits to their host plants. Quantification of mycorrhizal density and root colonization has traditionally been performed by root staining and microscopic examination methods, which are time-consuming, laborious, and difficult to reproduce between laboratories. A number of biochemical markers for estimating mycorrhizal hyphae and spores have been published. In this study we grew maize plants in three different soils in a replicated greenhouse experiment and compared the results from two microscopic methods, spore density and root colonization, to the results from three lipid biomarker methods: neutral lipid fatty acid, phospholipid fatty acid, and ester-linked fatty acid analysis. Ester-linked fatty acid analysis gave consistent results for both spore density and root colonization, but neutral lipid fatty acid analysis had the highest correlation to AMF spore counts. Phospholipid fatty acid analysis was not correlated to spore density and did not reproducibly correlate to root colonization.

TNF-α and Th2 Cytokines Induce Atopic Dermatitis–Like Features on Epidermal Differentiation Proteins and Stratum Corneum Lipids in Human Skin Equivalents

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are upregulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (tumor necrosis factor alpha (TNF-α)) cytokines on epidermal morphogenesis, proliferation, differentiation, and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31, and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes, and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.

Two Lipid Signals Guide Fruiting Body Development of Myxococcus xanthus

ABSTRACTMyxococcus xanthus produces several extracellular signals that guide fruiting body morphogenesis and spore differentiation. Mutants defective in producing a signal may be rescued by codevelopment with wild-type cells or cell fractions containing the signal. In this paper, we identify two molecules that rescue development of the E signal-deficient mutant LS1191 at physiological concentrations, iso15:0 branched-chain fatty acid (FA) and 1-iso15:0-alkyl-2,3-di-iso15:0-acyl glycerol (TG1), a development-specific monoalkyl-diacylglycerol. The physiological concentrations of the bioactive lipids were determined by mass spectrometry from developing wild-type cells using chemically synthesized standards. Synthetic TG1 restored fruiting body morphogenesis and sporulation and activated the expression of the developmentally regulated gene with locus tag MXAN_2146 at physiological concentrations, unlike its nearly identical tri-iso15:0 triacylglycerol (TAG) counterpart, which has an ester linkage instead of an ether linkage. iso15:0 FA restored development at physiological concentrations, unlike palmitic acid, a straight-chain fatty acid. The addition of either lipid stimulates cell shortening, with an 87% decline in membrane surface area, concomitantly with the production of lipid bodies at each cell pole and in the center of the cell. We suggest that cells produce triacylglycerol from membrane phospholipids. Bioactive lipids may be released by programmed cell death (PCD), which claims up to 80% of developing cells, since cells undergoing PCD produce lipid bodies before lysing.

Effect of Long Term Fertilization on Microbial Biomass, Enzyme Activities, and Community Structure in Rice Paddy Soil

The effects of long-term fertilization on soil biological properties and microbial community structure in the plough layer in a rice paddy soil in southern Korea were investigated in relation to the continuous application of chemical fertilizers (NPK), straw based compost (Compost), combination these two (NPK + Compost) for last 40 years. No fertilization plot (Control) was installed for comparison. Though fertilization significantly improved rice productivity over control, the long-term fertilization of NPK and compost combination was more effective on increasing rice productivity and soil nutrient status than single application of compost or chemical fertilizer. All fertilization treatments had shown significant improvement in soil microbial properties, however, continuous compost fertilization markedly increased soil enzyme and microbial activities as compared to sole chemical fertilization. Results of microbial community structure, evaluated by EL-FAME (ester-linked fatty acid methyl esters) method, revealed big difference among Control, NPK, and Compost. However, both Compost and Compost+NPK treatments belonged to the same cluster after statistical analysis. The combined application of chemical fertilizer and organic amendments could be more rational strategy to improve soil nutrient status and promote soil microbial communities than the single chemical fertilizer or compost application.

Agronomic application of olive mill wastewaters with rock phosphate increase the 10Me18:0 fatty acid marker of actinomycetes and change rhizosphere microbial functional groups under long-term field conditions

The objectives of this study were to determine the long-term effects of agronomic application of olive mill wastewaters (OMW) with rock phosphate (RP) in a field of olive trees on the soil microbial community structures. Ester-linked fatty acid methyl ester (EL-FAME) profiles were used for characterizing the numerically dominant portion of soil microbial communities. EL-FAME analysis showed significant shifts of specific groups of fatty acids after application of OMW with RP. In particular, the FAME 10Me18:0 indicative of actinomycetes increased and the FAME 18:1ω9 and 18:2ω6 commonly found in fungi decreased. The soil microbial cells reacted to the addition of OWM and RP with an increase in the percentage of cy17:0 and cy19:0 cyclopropane fatty acids. Other changes in FAME composition included the increase of saturated/unsaturated ratio. It seems that alterations of fatty acid composition, after agronomic application of OMW and RP, are responsible for the substantial maintenance of cell membrane integrity, physiological functions and growth.

Microbial responses to carbon and nitrogen supplementation in an Antarctic dry valley soil

AbstractThe soils of the McMurdo Dry Valleys are exposed to extremely dry and cold conditions. Nevertheless, they contain active biological communities that contribute to the biogeochemical processes. We have used ester-linked fatty acid (ELFA) analysis to investigate the effects of additions of carbon and nitrogen in glucose and ammonium chloride, respectively, on the soil microbial community in a field experiment lasting three years in the Garwood Valley. In the control treatment, the total ELFA concentration was small by comparison with temperate soils, but very large when expressed relative to the soil organic carbon concentration, indicating efficient conversion of soil organic carbon into microbial biomass and rapid turnover of soil organic carbon. The ELFA concentrations increased significantly in response to carbon additions, indicating that carbon supply was the main constraint to microbial activity. The large ELFA concentrations relative to soil organic carbon and the increases in ELFA response to organic carbon addition are both interpreted as evidence for the soil microbial community containing organisms with efficient scavenging mechanisms for carbon. The diversity of the ELFA profiles declined in response to organic carbon addition, suggesting the responses were driven by a portion of the community increasing in dominance whilst others declined.

Warming constrains bacterial community responses to nutrient inputs in a southern, but not northern, maritime Antarctic soil

We investigated the effects of increased soil temperature, water and nutrient availability on soil bacterial communities at Wynn Knolls on Signy Island (60 °S) in the northern maritime Antarctic and at Mars Oasis (71 °S) in the southern maritime Antarctic. After 10–12 months, analyses of the concentrations of ester linked fatty acids (ELFAs) in soil indicated that bacterial communities responded positively to single applications of substrates at both locations, with 20% and 49% increases in total Gram positive and Gram negative bacterial markers, respectively, in response to the application of tryptic soy broth (TSB; a complex substrate containing organic carbon and nitrogen, plus other nutrient elements) at Wynn Knolls, and 120% and 44% increases in Gram positive bacterial markers at Mars Oasis in response to the application of TSB and the amino acid glycine (a relatively simple source of organic carbon and nitrogen), respectively. Responses to the warming treatment were not detected at Wynn Knolls, where open top chambers (OTCs) increased mean monthly soil temperatures by up to 0.7 °C, but at Mars Oasis, where OTCs increased monthly soil temperatures by up to 2.4 °C, warming led to 41% and 46% reductions in the concentrations of Gram positive bacterial markers in soil to which glycine and TSB had been applied, respectively. Warming also led to 55% and 51% reductions in the ratio of Gram positive to Gram negative markers in soils at Mars Oasis to which glycine and TSB had been applied. These data suggest that warming may constrain the responses of bacterial communities to carbon and nitrogen inputs arising from dead plant matter entering maritime Antarctic soils in future decades.

Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference

Application of polymerase chain reaction (PCR) techniques has developed significantly from a qualitative technology to include powerful quantitative technologies, including real-time PCR, which are regularly used for detection and quantification of nucleic acids in many settings, including community analysis where culture-based techniques are not suitable. Many applications of real-time PCR involve absolute quantification which is susceptible to inaccuracies caused by losses during DNA extraction or inhibition caused by co-extracted compounds. We present here an improvement to this approach involving the addition of an artificial internal standard, prior to nucleic acid extraction. The standard was generated by in-situ mutagenesis from an E. coli template to ensure it both did not amplify with bacterial primers used for quantification and was short enough to minimise possible interference with other analyses. By estimating gene target copies by relative abundance, this approach accounts for both loss during extraction and inhibition effects. We present a novel application of relative real time PCR, using the internal standard as a reference, allowing accurate estimation of total bacterial populations both within and across a wide range of soils and demonstrate its improvement over absolute quantification by comparison of both approaches to ester linked fatty acid analysis of the same soils.

Ornithine lipids and their structural modifications: from A to E and beyond

Ornithine lipids (OLs) are phosphorus-free membrane lipids that are widespread in eubacteria, but absent from archaea and eukaryotes. They contain a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of the amino acid ornithine. A second fatty acyl group is ester-linked to the 3-hydroxy position of the first fatty acid. About 25% of the bacterial species whose genomes have been sequenced are predicted to have the capacity to form OLs. Distinct OL hydroxylations have been described in the ester-linked fatty acid, the amide-linked fatty acid, and the ornithine moiety. These modifications often seem to form part of a bacterial stress response to changing environmental conditions, allowing the bacteria to adjust membrane properties by simply modifying already existing membrane lipids without the need to synthesize new lipids.

Fungal and bacterial denitrification are differently affected by long-term pH amendment and cultivation of arable soil

Bacteria are considered as playing a predominant role in the production of nitrous oxide (N2O) in arable soil. Despite the knowledge that fungi are able to denitrify their contribution to denitrifier N2O production from arable soil is uncertain. Here, we assess the capability of fungi and bacteria to contribute to N2O emission from arable soil by measuring potential denitrification rates (PDR) as N2O production, after application of selective inhibitors aimed at distinguishing between fungal and bacterial denitrification, and related PDR to characteristics of the soil microbial community. Soil was sampled from a long-term crop rotation maintained since 1961 at seven different pH levels, ranging in 0.5 increments from pH 4.5 to 7.5, and along a cultivation gradient from freshly ploughed soil to three years under ley grass. Over both pH and cultivation gradients, bacteria contributed up to 54% and fungi contributed to 18% of the PDR. Residual N2O production that was not targeted by the selective inhibitors and hence could not be attributed to fungi or bacteria might be due to pre-synthesised enzymes or resistant organisms. The PDR of the bacterial community responded positively to increase in soil pH with the lowest PDR at pH 4.2 and the highest around pH 5.9. In contrast, fungal denitrification was not influenced by soil pH. Changes in ester linked fatty acids (ELFA) concentrations showed that whilst total bacterial biomass decreased with increasing pH fungal biomass was not significantly influenced by pH, driving an increase in the ratio of fungal–bacterial biomass. Both fungal biomass and bacterial biomass, and the PDR from the control treatment (no inhibitor application) across the pH gradient were greatest under long-term ley. Concentrations of fatty acids a15:0, 16:1ω7 and 17:1ω8 of microbial origin were positively correlated with the proportion of denitrification activity that was repressed by bacterial inhibitors. This suggests that there is a relationship between organisms that possess the fatty acids a15:0, 16:1ω7 and 17:1ω8, and the function of denitrification. Our results demonstrate that both fungal and bacterial denitrification were occurring in this arable soil. That management for pH and cultivation had differing effects on the potential contribution of fungal and bacterial denitrification to N2O production has implications for the development of appropriate management practices for mitigation of this greenhouse gas.

Natural attenuation of contaminated marine sediments from an old floating dock Part II: Changes of sediment microbial community structure and its relationship with environmental variables

Changes of microbial community structure and its relationship with various environmental variables in surface marine sediments were examined for a one-year period after the removal of an old floating dock in Hong Kong SAR, South China. Temporal variations in the microbial community structure were clearly revealed by principal component analysis (PCA) of the microbial ester-linked fatty acid methyl ester (EL-FAME) profiles. The most obvious shift in microbial community structure was detected 6months after the removal of the dock, although no significant decline in the levels of pollutants could be detected. As determined by EL-FAME profiles, the microbial diversity recovered and the predominance of gram-negative bacteria was gradually replaced by gram-positive bacteria and fungi in the impacted stations. With redundancy analysis (RDA), the concentration of total polycyclic aromatic hydrocarbons (PAHs) was found to be the second important determinant of microbial community structure, next to Time. The relative abundance of 18:1ω9c and hydroxyl fatty acids enriched in the PAH hot spots, whereas 16:1ω9 and 18:1ω9t were negatively correlated to total PAH concentration. The significant relationships observed between microbial EL-FAME profiles and pollutants, exampled by PAHs in the present study, suggested the potential of microbial community analysis in the assessment of the natural attenuation process in contaminated environments.

Response of Soil Microbial Communities to Different Cultivation Systems in Controlled Horticultural Land

Ester-linked fatty acid methyl ester (EL-FAME) profiles were used to describe differences in soil microbial communities influenced by conventional farming system (CFS), and organic farming system (OFS) in controlled horticultural land. Soil physicochemical properties and soil microbial communities were determined in the experimental fields. Higher organic matter content in OFS reduced soil bulk density which in turn increased the soil porosity. Generally, soil chemical properties in OFS were higher than those of CFS, but EC value in OFS was significantly lower than that of CFS. With the exception of Fe content, other macronutrient contents and pH in both farming system decreased with the soil depth. Soil microbial biomass of OFS was approximately 1.3 times in topsoil and 1.8 times in subsoil higher than those of CFS. Lower ratios of cy17:0 to <TEX>$16:1{\omega}7c$</TEX> and cy19:0 to <TEX>$18:1{\omega}7c$</TEX> were found in the CFS soils than the OFS soils, indicating that microbial stress decreased. The ratio of MUFA to SFA was higher in OFS due to organic input to the soil. In principal components analysis (PCA), the first variable accounted for 54.3%, while the second for 27.3%, respectively. The PC1 of the PCA separated the samples from CFS and OFS, while the PC2 of the PCA separated the samples from topsoil and subsoil. EL-FAMEs with the positive eigenvector coefficients for PC1 were cy17: 0 to <TEX>$16:1{\omega}7c$</TEX> ratio, cy19:0 to <TEX>$18:1{\omega}7c$</TEX> ratio, soil pH, soil organic matter, and soil <TEX>$NO_3$</TEX>-N content. Our findings suggest that the shifting cy19:0 to <TEX>$18:1{\omega}7c$</TEX> ratio should be considered as potential factors responsible for the clear microbial community differentiation observed between different cultivation systems and soil depth in controlled horticultural land.

Microbial community dynamics and biodegradation of polycyclic aromatic hydrocarbons in polluted marine sediments in Hong Kong

Dynamics of microbial community and biodegradation of polycyclic aromatic hydrocarbons (PAHs) in polluted marine sediments, artificially spiked with a mixture of PAHs (fluorene, phenanthrene, fluoranthene and pyrene), were examined for a period of 60days. Microbial communities were characterised by bacterial counts, ester-linked fatty acid methyl ester (EL-FAME) analysis and denaturing gradient gel electrophoresis (DGGE). A noted reduction in species diversity occurred only in the high PAH level treatment at onset. Both EL-FAME and DGGE demonstrated a marked shift in microbial community, in all the PAH level treatments, afterwards, with increases in the number of fatty acid degraders, the relative abundance of fatty acid biomarkers for gram-negative bacteria and a decrease in species diversity. The shift was also accompanied by the significant decrease in PAH concentrations. By the end of the experiment, diversity indices, based on both approaches, recovered when PAH concentrations declined to their background levels, except in the high PAH level treatment.

Soil microbial community composition and diversity in Panax quinquefolius rhizosphere

The objective of this work was to analyze the soil microbial community diversity and structure in Panax quinquefolius rhizosphere and elucidated the rules of the ecology shift. Two community-based microbiological measurements, community level physiological profiling (CLPP) using Biolog sole C source utilization tests and phospholipid ester-linked fatty acid (PLFA) profiles, were used to evaluate soil microbial community function and composition of different Panax quinquefolius cropping soils. The properties, pH, N, P, K and organic matter were analyzed also. There were many differences in soil properties such as lower pH (5.82) in Jilin soil but higher pH (8.27) in Beijing soil and there were significant higher content of organic matter, available N, P, K in Jilin soil than those in Beijing soil. The analysis of soil microbial ecology showed a similar result in AWCD, Biolog and PLFA pattern. According to the PC1 score in principal analysis of Biolog and PLFA, the soils in 2, 3 and 4 years cultivated P. quinquefolius rhizophere were distinguished from the control and 1 year soils. The result indicated that the metabolism function reduced and the structure shifted in rhizophere soil microbial community after P. quinquefolius cropping. These may be the primary reason of the disease occur severely in P. quinquefolius field.

Olive mill wastewater effects on the microbial communities as studied in the field of olive trees by analysis of fatty acid signatures

The aim of this work was to study the effects of spreading olive mill wastewater (OMW) on the soil surface of an olive grove on the soil microbial communities. Analyses of ester-linked fatty acid methyl esters (EL-FAME) were used to assess variations in the soil microbial community structure following land spreading of OMW. Our data provide evidence that agronomic application of OMW has important effects on soil microbial community. Bacteria were relatively more reduced by these treatments than fungi and actinomycetes as revealed by an increased index of fungal/bacterial FAME and actinomycetes/bacterial FAME. Specific FAME markers indicated a significant reduction in the Gram-positive bacteria. However, the relative proportion of the Gram-negative bacteria was not significantly different after agronomic application of OMW. The ratios of cyclopropyl/monoenoic precursors decreased and the total monounsaturated/total saturated fatty acids increased in the OMW amended soils, suggesting that the microbes inhabiting the control soil are more carbon limited than the OMW amended soils. The changes in the FAME pattern of the soil organisms possibly were related (i) to an altered substrate quantity, that is the availability of substrates after the treatments, (ii) the complex nature of OMW which also contains high molecular-mass recalcitrant polyphenols.

As simple as ACB – new insights into the role of acyl‐CoA‐binding proteins in Arabidopsis

As simple as ACB – new insights into the role of acyl‐CoA‐binding proteins in Arabidopsis

Resilience of microorganisms and aggregation of a sandy calcareous soil to amendment with organic and synthetic fertilizer

Marginal coastal soils are dependent on appropriate land management to prevent soil erosion, as a result of low soil stability combined with exposure to strong winds. An example of such an area is the machair, a fixed dune system utilized for agriculture in the northwest of Scotland, UK. The separate and combined effects of synthetic NPK fertilizer and a traditional soil conditioner (kelp, a seaweed) on soil structure formation, stabilization and biological parameters were studied on a cropped field on the machair. Soil physical properties examined included water retention at 10 kPa matric suction, water stable aggregates (WSA) >1 mm, aggregate stability, and biological properties including ester-linked fatty acid (ELFA) analysis and β-glucosidase activity for microbial biomass and activity, respectively. Significant treatment effects were few and inconsistent between sampling times, but included kelp and/or NPK fertilizer reducing aggregation, water retention, microbial biomass and activity relative to the unamended control treatment. Furthermore, seasonal variation, which could be attributed to changes in soil water content, was stronger than variation in response to fertilizer treatments. Principal components analysis of the ELFA data showed that ploughing promoted fungal biomass relative to bacteria, and confirmed both the absence of consistent synthetic and organic fertilizer effects and the sensitivity of microbial biomass to season. Overall, the study demonstrated the resilience of a calcareous sandy soil to amendment with fertilizer.

Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from Marinobacter aquaeolei VT8

Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium Marinobacter aquaeolei VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in Escherichia coli as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP(+). The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed.