Microbial community structure and biomass in developing drinking water biofilms

Traditional techniques to study microbes, such as culturable counts, microbial biomass, or microbial activity, do not give information on the microbial ecology of drinking water systems. The aim of this study was to analyze whether the microbial community structure and biomass differed in biofilms collected from two Finnish drinking water distribution systems (A and B) receiving conventionally treated (coagulation, filtration, disinfection) surface water. Phospholipid fatty acid methyl esters (PLFAs) and lipopolysaccharide 3-hydroxy fatty acid methyl esters (LPS 3-OH-FAs) were analyzed from biofilms as a function of water residence time and development time. The microbial communities were rather stabile through the distribution systems, as water residence time had minor effects on PLFA profiles. In distribution system A, the microbial community structure in biofilms, which had developed in 6 weeks, was more complex than those grown for 23 or 40 weeks. The microbial communities between the studied distribution systems differed, possibly reflecting the differences in raw water, water purification processes, and distribution systems. The viable microbial biomass, estimated on the basis of PLFAs, increased with increasing water residence time in both distribution systems. The quantitative amount of LPS 3-OH-FAs increased with increasing development time of biofilms of distribution system B. In distribution system A, LPS 3-OH-FAs were below the detection limit.     

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.     

The microbial community structure of drinking water biofilms can be affected by phosphorus availability.

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 microg of phosphorus liter(-1) and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1 omega 7c and 18:1 omega 7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of 13C-labelled fatty acids

A method for the detection of physiologically active autotrophic bacteria in complex microbial communities was developed based on labelling with the stable isotope 13C. Labelling of autotrophic nitrifying, sulphur-oxidizing and iron-oxidizing populations was performed in situ by incubation with NaH[13C]O3. Incorporated label into fatty acid methyl esters (FAMEs) was detected and quantified using gas chromatography-mass spectrometry in single ion monitoring mode. Before the analyses of different environmental samples, the protocol was evaluated in pure culture experiments. In different environmental samples a selective labelling of fatty acids demonstrated which microbial taxa were responsible for the respective chemolithoautotrophic activity. The most strongly labelled fatty acids of a sample from a sulphide treating biofilter from an animal rendering plant were cis-7-hexadecenoic acid (16:1 cis7) and 11-methyl hexadecanoic acid (16:0 11methyl), which are as-yet not known for any sulphide-oxidizing autotroph. The fatty acid labelling pattern of an experimental biotrickling filter sample supplied with dimethyl disulphide clearly indicated the presence and activity of sulphide-oxidizing bacteria of the genus Thiobacillus. For a third environmental sample from an acid mining lake sediment, the assignment of autotrophic activity to bacteria of the genus Leptospirillum but not to Acidithiobacillus could be made by this method, as the fatty acid patterns of these bacteria show clear differences.     

The Microbial Community Structure of Drinking Water Biofilms Can Be Affected by Phosphorus Availability

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 μg of phosphorus liter⁻¹ and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1ω7c and 18:1ω7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.     

Mechanisms for ion formation during the electron impact-mass spectrometry of picolinyl ester and 4,4-dimethyloxazoline derivatives of fatty acids

Mass spectral studies have been conducted with isotopically stable labelled and fluorinated picolinyl esters and 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids in order to establish mechanisms of ion formation. Reciprocal hydrogen transfer is shown to be involved in the formation of the ion at m/z 126 with dimethyloxazoline derivatives and for the ion at m/z 164 with picolinyl esters. Inclusion of a fluorine atom alpha to the carboxyl of a fatty acid has been demonstrated to enhance rearrangements for expulsion of internal chain fragments with both methyl ester and dimethyloxazoline derivatives. When two fluorine atoms are inserted into the alpha position a similar rearrangement has been shown to occur with picolinyl esters, although not nearly to the same extent as that observed with either of the other derivatives. Mechanisms for such rearrangements are proposed and discussed. With fatty acid dimethyloxazoline derivatives the M-15 ion arises solely from the loss of a methyl radical from the ring and the M-43 ion has at least three different mechanisms of formation. Such rearrangements make it difficult to establish the identity of the terminal moiety of the alkyl chain. In mass spectrometry terms the picolinyl ester would seem to be the superior derivative for structural characterisation of fatty acids.     

氮肥添加对高寒藏嵩草(Kobresia tibetica)沼泽化草甸和土壤微生物群落的影响

以藏嵩草沼泽化草甸为研究对象,利用磷脂脂肪酸(PLFA)技术,研究连续6年N素添加对地上植被群落数量特征、土壤微生物群落结构的影响。结果表明:①藏嵩草沼泽化草甸群落生物量、枯枝落叶对施肥处理无明显响应,且莎草科植物对土壤氮素的吸收和利用率较低。②施肥增加了0-10 cm土壤微生物类群PLFAs丰富度尤其细菌和革兰氏阳性菌PLFAs,降低了10-20 cm PLFAs丰富度;③磷脂脂肪酸饱和脂肪酸/单烯不饱和脂肪酸、细菌PLFAs/真菌PLFAs的比值随土壤层次增加而增加;④0-10 cm土层革兰氏阳性菌、真菌PLFAs含量与pH、土壤速效磷、速效氮、土壤有机质显著正相关(P0.05或P0.01);10-20 cm土层,细菌、革兰氏阳性菌、真菌和总PLFAs含量与土壤有机质含量显著正相关(P0.05或P0.01)。表明藏嵩草沼泽化草甸微生物PLFAs含量和丰富度对施肥的响应存在明显的土层梯度效应,土壤微生物PLFAs含量和丰富度主要受表层土壤初始养分含量的影响。     

Identification of hydroxy fatty acids by liquid chromatography-atmospheric pressure chemical ionization mass spectroscopy in Euglena gracilis

Hydroxy fatty acids from Euglena gracilis were identified by reverse-phase high performance liquid chromatography coupled to a mass spectrometer run in atmospheric pressure chemical ionization positive ion mode. These metabolites were converted to methyl esters to improve stability and chromatographic properties. A detection limit of 20 pg/microl per injection was determined for 5-HETE methyl ester based on the signal to noise ratio of the m/z 317 ion which corresponds to the loss of a hydroxyl group (M-17) and the major fragment in all HETE methyl esters studied. This is the first report for these metabolites in E. gracilis.     

Community Structure Comparison Using FAME Analysis of Desert Varnish and Soil,Mojave Desert,California

While a number of studies have shown that a close association exists between microorganisms and varnished rocks, there is little hard evidence to support the existence of either specific desert varnish communities, or any role these microbes might play in the genesis of the varnish layers. To this end, we analyzed fatty acid methyl esters (FAMEs) of samples collected from the Mojave desert of southern California to compare the microbial community structure of desert varnish with the adjacent desert soil. These analyses indicated prokaryotic and fungal communities in both desert varnish and soil samples. FAMEs specific to gram-positive bacteria were found more often, and in greater abundance in varnish samples than in adjacent soils. This may represent a higher preservation potential of gram-positive bacteria fatty acids in varnish, a source area of varnish microorganisms dominated by gram-positive bacteria, or a varnish community dominated by gram-positive microorganisms. Heterogeneity in fatty acids was documented between varnished rocks and soils from different localities, as well as between samples collected from the same locality. This heterogeneity suggests that there are significant differences in the community structure of the microbial fauna found in varnish samples compared to the adjacent soil, and that desert varnish in the Mojave desert is not characterized by a unique and ubiquitous microbial community. These results suggest that the varnish is not a homogeneous and unique environment for biota, and provide no support for the hypothesis that the varnish layers are biogenic in origin.     

Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure

Analysis of fatty acid methyl ester (FAME) profiles extracted from soils is a rapid and inexpensive procedure that holds great promise in describing soil microbial community structure without traditional reliance on selective culturing, which seems to severely underestimate community diversity. Interpretation of FAME profiles from environmental samples can be difficult because many fatty acids are common to different microorganisms and many fatty acids are extracted from each soil sample. We used principal components (PCA) and cluster analyses to identify similarities and differences among soil microbial communities described using FAME profiles. We also used PCA to identify particular FAMEs that characterized soil sample clusters. Fatty acids that are found only or primarily in particular microbial taxa-marker fatty acids-were used in conjunction with these analyses. We found that the majority of 162 soil samples taken from a conventionally-tilled corn field had similar FAME profiles but that about 20% of samples seemed to have relatively low, and that about 10% had relatively high, bacterial:fungal ratios. Using semivariance analysis we identified 21:0 iso as a new marker fatty acid. Concurrent use of geostatistical and FAME analyses may be a powerful means of revealing other potential marker FAMEs. When microbial communities from the same samples were cultured on R2A agar and their FAME profiles analyzed, there were many differences between FAME profiles of soil and plated communities, indicating that profiles of FAMEs extracted from soil reveal portions of the microbial community not culturable on R2A. When subjected to PCA, however, a small number of plated communities were found to be distinct due to some of the same profile characteristics (high in 12:0 iso, 15:0 and 17:1 ante A) that identified soil community FAME profiles as distinct. Semivariance analysis indicated that spatial distributions of soil microbial populations are maintained in a portion of the microbial community that is selected on laboratory media. These similarities between whole soil and plated community FAME profiles suggest that plated communities are not solely the result of selection by the growth medium, but reflect the distribution, in situ, of the dominant, culturable soil microbial populations.     

Simultaneous quantitation of indole 3-acetic Acid and abscisic Acid in small samples of plant tissue by gas chromatography/mass spectrometry/selected ion monitoring

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [²H₄]IAA (m/z 194, 134) or [²H₅]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [²H₃]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue.     

Substrate selectivity of lipases in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the transesterification of cis/trans-isomers of linoleic acid methyl ester

The substrate selectivity of several microbial lipases has been examined in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the alcoholysis of isomeric linoleic acid methyl esters with n-butanol. Lipases from Candida cylindracea and Mucor miehei preferred fatty acids and methyl esters with a (first) cis double bond in 9-position, while Chirazyme L-5, a Candida antarctica lipase A, had a preference for trans-9 unsaturated substrates.     

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities

Phospholipid fatty acid (PLFA) as biomarkers, is widely used to profile microbial communities in environmental samples. However, PLFA extraction and derivatization protocols are not standardized and have widely varied among published studies. Specifically investigators have used either HCl/MeOH or KOH/MeOH or both for the methylation step of PLFA analysis, without justification or research to support either one. It seems likely that each method could have very different outcomes and conclusions for PLFA based studies. Therefore, the objective of this study was to determine the effect of catalyst type for methylation on detecting PLFAs and implications for interpreting microbial profiling in soil. Fatty acid samples extracted from soils obtained from a wetland, an intermittently flooded site, and an adjacent upland site were subjected to HCl/MeOH or KOH/MeOH catalyzed methylation procedures during PLFA analyses. The methylation method using HCl/MeOH resulted in significantly higher concentrations of most PLFAs than the KOH/MeOH method. Another important outcome was that fatty acids with a methyl group (18:1ω,7c 11Me, TBSA 10Me 18:0, 10Me 18:0, 17:0 10Me and 16:0 10Me being an actinomycetes biomarker) could not be detected by HCl/MeOH catalyzed methylation but were found in appreciable concentrations with KOH/MeOH method. From our results, because the HCl/MeOH method did not detect the fatty acids containing methyl groups that could strongly influence the microbial community profile, we recommend that the KOH/MeOH catalyzed transesterification method should become the standard procedure for PLFA profiling of soil microbial communities.     

Distribution of C22-, C24- and C26-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄-α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.     

Fatty acid methyl ester (FAME) profiles as a tool to investigate community structure of two agricultural soils

Soil microbiological parameters may be the earliest predictors of soil quality changes. Recently, molecular techniques such as fatty acid methyl ester (FAME) profiles have been used to characterize soil microbial communities. Fatty acid methyl ester (FAME) from whole soil may be derived from live cells, dead cells, humic materials, as well as plant and root exudates. Our objective was to verify differences in FAME profiles from two agricultural soils with different plants. Soil samples were collected from Ritzville and Palouse silt loams for fatty acid analysis. Soil samples from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativum L.), jointed goatgrass ( Aegilops cylindrica L.) and downy brome (Bromus tectorum L.) rhizospheres were also collected for fatty acid analysis. Principal component analysis (PCA) of the two soils explained 42% of the variance on PC1, which accounted for Palouse soil. Ritzville soil accounted for 19% of the variance on PC2. Factor analysis showed that rhizosphere microbial communities from various plant species may differ depending on the plant species. Presence of Gram-positive bacteria as identified by a15:0, i15:0, a17:0 and i17:0 peaks were similar between rhizosphere and nonrhizosphere soils. Gram-negative bacteria characterized by short chain hydroxy acids (10:03OH and 12:03OH) as well as cyclopropane acids (cy17:0) were higher in rhizosphere soil than nonrhizosphere. This indicates a possible shift in the bacterial community to more Gram-negative bacteria and fewer Gram-positive bacteria in the rhizospheres of the plants species studied.     

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.     

Characterization of lyso-galactolipids, C-2 and C-3 O-acyl trigalactosylglycerol isomers, obtained from the lichenized fungus Dictyonema glabratum

A mixture of two lyso isomers of a galactolipid was obtained from Dictyonema glabratum. Aqueous hydrolysis gave rise to galactose and glycerol in a 3:1 molar ratio. ESI-MS spectroscopy gave, in the positive-ion mode, a pseudomolecular ion at m/z 839 and daughter ions with m/z 677, 600, 515 and 353, suggesting three galactosyl units linked to a glycerol moiety, substituted by one O-acyl group. 1D and 2D NMR experiments were used to characterize the glycolipid, and HMQC examination showed three anomeric signals, corresponding to two alpha-Galp and one beta-Galp residue liked to glycerol. The glycolipid structure was shown to be O-alpha-D-Galp-(1-->6)-O-alpha-D-Galp-(1-->6)-O-beta-D-Galp-(1<-->1)-2- and -3-monoacyl-D-glycerol, the latter structures not having been previously found in nature. The fatty acid composition was determined by GC-MS of derived methyl esters: that of palmitic acid C(16:0) was the most abundant, although the presence of C(12:0), C(14:0), C(16:1) and C(18:0) esters was observed.     

Two naturally occurring acyclic diterpene and norditerpene aldehydes from Tetragonia tetragonoides

In addition to neophytadiene, phytol, methyl (2E)- and (2Z)-3-(4-hydroxy-3-methoxyphenyl) propenates, fatty acid methyl esters and fatty acids,     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.