Estimation of methanotroph abundance in a freshwater lake sediment

The numbers of methane-oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture-independent methods. Quantitative slot-blot hybridizations were performed with type I and type II methanotroph-specific probes. These data were compared to data from quantitative hybridizations using a pmoA-specific probe and a eubacterial probe. From the combined hybridization data, the methanotroph population in Lake Washington was estimated to be 3.6 x 10(8)-7.4 x 10(8) cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1 omega 8) and type II (18:1 omega 8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 x 10(8)-9.4 x 10(9) cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 x 10(8)-1.2 x 10(9) cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two- to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.     

Estimation of cell numbers of methanotrophic bacteria in boreal peatlands based on analysis of specific phospholipid fatty acids

Abstract: Concentrations of two phospholipid fatty acids (PLFAs) specific for methane-oxidizing bacteria (16:1 ω8 and 18:1 ω8), were used to estimate the biomass and cell numbers of this group of bacteria in two Sphagnum-dominated boreal peatlands. Concentration ranges of 16:1 ω8 and 18:1 ω8 were 0.0–73 and 1.0–486 pmol g⁻¹ of wet peat, respectively. Concentrations in the peat of each fatty acid were positively correlated with the potential methane oxidation activity ( V _max), which was used as an independent estimate of methanotrophic biomass. This correlation suggests that the two PLFAs are good biomarkers for the population of methanotrophic bacteria in peatlands. Concentrations of the two PLFAs were transformed to cell numbers using conversion factors for the cell content of PLFAs, average cell volume and percentage of cellular dry matter. The total cell number of methanotrophic bacteria in peat samples from a range of sites and depths ranged between 0.3 and 51 × 10⁶ cells g⁻¹ of wet peat, with similar proportions of type I and type II methanotrophic bacteria in most samples. Within particular peat profiles, numbers of methanotrophic bacteria were highest around the level of the water table, implying that the supplies of methane and oxygen largely determine the biomass distribution of methanotrophic bacteria in this type of peatlands.     

Selective grazing of methanotrophs by protozoa in a rice field soil

Biological methane oxidation is a key process in the methane cycle of wetland ecosystems. The methanotrophic biomass may be grazed by protozoa, thus linking the methane cycle to the soil microbial food web. In the present study, the edibility of different methanotrophs for soil protozoa was compared. The number of methanotroph-feeding protozoa in a rice field soil was estimated by determining the most-probable number (MPN) using methanotrophs as food bacteria; naked amoebae and flagellates were the dominant protozoa. Among ten methanotrophic strains examined as a food source, seven yielded a number of protozoa comparable with the yield with Escherichia coli [10⁴ MPN (g soil dry weight)⁻¹], and three out of four Methylocystis spp. yielded significantly fewer numbers [10²–10³ MPN (g soil dry weight)⁻¹]. The lower edibility of the Methylocystis spp. was not explained either by their growth phase or by harmful effects on protozoa. Incubation of the soil under methane resulted in a higher number of protozoa actively grazing on methanotrophs, especially on the less-edible group. Protozoa isolated from the soil demonstrated a grazing preference on the different methanotrophs consistent with the results of MPN counts. The results indicate that selective grazing by protozoa may be a biological factor affecting the methanotrophic community in a wetland soil.     

[2-3H]GLYCEROL AS A PRECURSOR OF PHOSPHOLIPIDS IN RAT BRAIN: EVIDENCE FOR LACK OF RECYCLING

Abstract— When [2-³H]glycerol was injected intracranially into young rats, it was presented as a pulse label, leaving the brain rapidly and giving up much of its labelled hydrogen to water. [2-³H]glycerol was efficiently incorporated into brain lipids, especially into choline and ethanolamine phospholipids. Following injection of a mixture of [³H]- and [¹⁴C]-labelled glycerol, the ratio of ³H to ¹⁴C in the phospholipids of both whole brain and the microsomal fraction decreased as a function of time after injection. This finding indicated less recycling of the tritium label. This lack of recycling was further indicated by the finding that 94 per cent of the tritium label of phosphatidyl choline was in the glycerol portion of the molecule rather than in the fatty acids. At 2 weeks following injection with [³H]glycerol, 93 per cent of the total radioactivity in brain appeared in the lipid fraction. In contrast, following injection with [¹⁴C]glycerol, only 57 per cent of the radioactivity appeared in lipid, with about 20 per cent in protein.     

Characterization of Microbial Consortia in Paddy Rice Soil by Phospholipid Analysis

Abstract Microbial biomass and community structure in paddy rice soil during the vegetation period of rice were estimated by analysis of their phospholipid fatty acids (PLFA), hydroxy fatty acids of lipopolysaccharides (LPS-HYFA), and phospholipid ether lipids (PLEL) directly extracted from the soil. A clear change in the composition of the community structure at different sampling periods was observed, indicated by the principal component analysis of the PLFA. A dramatic decline of ester-linked PLFA was observed in the soil samples taken at the second sampling time. In contrast to the ester-linked PLFA, the non-ester-linked PLFA composition did not change. The hydroxy fatty acids of lipopolysaccharides as well as ether lipids decreased consecutively during the observation period. Total microbial abundance was estimated to be (4.1–7.3) × 10⁹ cells g^-1 soil (dry weight). About 44% account for aerobic and 32% for facultative anaerobic bacteria, and 24% for archaea, on average. According to the profile and patterns of PLFA in the soil sample, it may be suggested that the paddy soil at the August sampling period contained more abundant facultative anaerobic bacteria (ca. 36%) and archaea (ca. 37%), but the total microbial biomass was significantly lower than in the remaining sampling periods. As the plant approached maturity, the microbial community structure in the soil changed to contain more abundant Gram-negative bacteria and methanotrophs. Received: 23 September 1999; Accepted: 28 February 2000; Online Publication: 12 May 2000     

Consumption of NO by methanotrophic bacteria in pure culture and in soil

Abstract The methanotrophs Methylomonas angile (type I) and Methylosinus trichosporium (type II) produced nitrite, nitrate and N₂O during growth on methane, apparently by heterotrophic nitrification of ammonium. The methanotrophs were also able to consume NO but did not produce it. After incubation of soil from a drained paddy field in the presence of CH₄ the numbers of methanotrophs increased from 10⁵ to 10⁷ per gram dry weigth. The thus enriched soil showed increased rates of NO consumption while rates of NO production did not change.     

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus / Methylocystis , type I methanotrophs related to Methylobacter / Methylosoma and Methylococcus , and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using ¹³CH₄ were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella , which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium , were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.     

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-¹³C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field ¹³C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C . Brocadia fulgida synthesizes C_16:0 and iso C_16:0 fatty acids according to the known pathway of type II fatty acid biosynthesis. The ¹³C-labelling pattern of the C₈ alkyl chain of the C₂₀ [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.     

Equivalence of microbial biomass measures based on membrane lipid and cell wall components,adenosine triphosphate,and direct counts in subsurface aquifer sediments

An uncontaminated subsurface aquifer sediment contains a sparse microbial community consisting primarily of coccobacillary bacteria of relatively uniform size which can be counted directly with appropriate staining. The morphological simplicity and the relatively decreased cell numbers, when compared with surface soils and sediments, make the subsurface an ideal natural community with which to compare the utility of chemical measures of microbial biomass to direct microscopic counts. The membrane phospholipids (estimated as the polar lipid fatty acids, the lipid phosphate, and phosopholipid glycerol phosphate), lipopolysaccharide lipid A (estimated as the LPS hydroxy fatty acids), cell walls (estimated as the muramic acid), and adenosine triphosphate all give essentially identical estimates of cell numbers and dry weight as the direct counts, using conversion factors determined on subsurface microorganism monocultures. Assays of microbial cell components are thus validated by comparison with the classical direct count in at least one soil/sediment.     

Archaebacterial ether lipid diversity analyzed by supercritical fluid chromatography: integration with a bacterial lipid protocol

A strategy has been developed for archaebacterial lipid analysis which provides three times the information to describe archaebacterial isolates and is compatible with simultaneous eubacterial/eukaryotic lipid analysis of environmental samples. Eubacterial and micro-eukaryotic biomass, community structure, and nutritional status have been routinely defined in environmental samples by lipid analysis. Lipid profiles are also useful in eubacterial identification and taxonomy. Polar lipid or whole cell ester-linked fatty acids are generally analyzed by gas chromatography-mass spectroscopy. Archaebacteria are characterized by their ether-linked membrane lipids. There is, however, less diversity in the side chains of archaebacterial membrane lipids as compared the eubacterial ester-linked membrane lipids. The information content of the archaebacterial lipid profile was increased by separately analyzing the polar lipid, glycolipid, and lipid-extracted residue fractions. Identification and quantification were performed by supercritical fluid chromatography. Results are presented for three species of methanogens and four thermoacidophile isolates, and compared with a literature review.     

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH₄ into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH₄ flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using ¹³C-labelled CH₄ directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to ¹³CH₄ for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.     

Fatty acid, isoprenoid quinone and polar lipid composition in the classification of Renibacterium salmoninarum

Twenty-one strains of Renibacterium salmoninarum were degraded by acid methanolysis and the non-hydroxylated fatty acid esters released examined by thin-layer and gas chromatography. The fatty acid profiles were composed almost exclusively of methyl-branched fatty acids with 12-methyltetradecanoic ( anteiso -C₁₅), 13-methyltetradecanoic ( iso -C₁₅) and 14-methylhexadecanoic ( anteiso -C₁₇) as major components. Polar lipids of the test strains were examined by two-dimensional thin-layer chromatography. All of the organisms possessed very characteristic polar lipid patterns consisting of diphosphatidylglycerol, two major and six or seven minor glycolipids, and two unidentified minor phospholipids. In all cases the major menaquinone components consisted of unsaturated menaquinones with nine isoprene units. The lipid data support the integrity of the genus Renibacterium and can be used to separate it from Corynebacterium and from coryneform bacteria which also contain lysine in the wall peptidoglycan.     

PELIZAEUS-MERZBACHER DISEASE: BRAIN LIPID AND FATTY ACID COMPOSITION

Abstract— Biochemical analysis of the leukodystrophy brain from a case of Pelizaeus-Merzbacher disease, classical type, was performed. A decrease in the amount of solid material present was found. The lyophilized brain weight was reduced to 76% of normal with a slightly greater decrease in the amount of extractable lipid. Total myelin was diminished to 7% of normal. Among specific lipids plasmalogens were present in slightly lowered amounts. Cerebrosides and sulphatides were drastically reduced to 8% of normal, whereas sphingomyelin was less severely affected. Fatty acids from phospholipids were close to normal, only enols being slightly diminished. Analysis of pure cerebrosides and sulphatides revealed that the a-hydroxylated compounds as well as very long chain fatty acids (over C₁₈, especially C₂₃ to C₂₆) were greatly reduced. For chain lengths over C₁₈, the ratio of leukodystrophy fatty acid to normal fatty acid was close to 10%. The defect in very long chain fatty acids is estimated at 99.2% in total brain.
Thus, we have found a marked decrease in the amount of very long chain fatty acids and a less marked decrease in sphingolipids. The reduced amount of these acids appears to be partially offset by an increase in the amount of medium-chain fatty acids in sphingolipids. We conclude that one aspect of Pelizaeus-Merzbacher disease may be a defect in the synthesis of myelin very long chain fatty acids (as these acids are far much reduced than any other myelin molecule).     

Wax Ester Production by the Marine Cryptomonad Chroomonas salina Grown Photoheterotrophically on Glycerol

SYNOPSIS. An age-autolyzed culture of Chroomonas salina , grown under cool-white light with glycerol, produced waxy lipid constituting about 44% of total matter harvested. This lipid was composed of 87% wax ester, 9% triglyceride, 3% polar lipid and 1% hydrocarbon. The major wax ester species were identified by total carbon number as C₂₆(28%), C₂₈(35%), C₃₀(15%). The main fatty acid components of the wax esters were 12:0 (39%), 14:0 (30%), 16:0 (14%), while the main alcohols were 14:0 (53%) and 16:0 (40%). The hydrocarbon fraction showed saturated paraffins ranging from C₁₇ to C₃₃, with odd-numbered chain components predominating. No polyunsaturated components were detected in the wax ester or hydrocarbon fractions. This is the first record of wax ester production by a cryptomonad or a marine phytoplankter.     

On the Role of Long-Chain Aldehydes in Mammalian Plasmalogen Biosynthesis

Abstract: [1-³H, 1-¹⁴C]Palmitaldehyde(³H:¹⁴C= 15) was injected intracerebrally to 18-day-old rats and incorporation of radioactivity into brain lipids was followed over a 24-h period. The substrate was metabolized primarily by oxidation to palmitic acid with loss of tritium and, to a lesser extent, by reduction to hexadecanol. The alkyl moieties of the ethanolamine phospholipids showed considerably lower ³H:¹⁴C ratios than the substrate, indicating a substantial participation in ether lipid synthesis by tritium-free alcohols derived from ¹⁴C-labeled fatty acids. Virtually no ³H radioactivity was found in alkenyl moieties, indicating stereospecificity of both reduction of aldehyde and dehydrogenation of alkyl to alkenyl glycerolipid. The data are consistent with the general concept that plasmalogen biosynthesis proceeds exclusively through fatty alcohols and alkyl glycerolipids and that fatty aldehydes cannot be utilized directly.     

Microbial characterization of a JP-4 fuel-contaminated site using a combined lipid biomarker/polymerase chain reaction--denaturing gradient gel electrophoresis (PCR-DGGE)-based approach

The impact of pollution on soil microbial communities and subsequent bioremediation can be measured quantitatively in situ using direct, non-culture- dependent techniques. Such techniques have advantages over culture-based methods, which often account for less than 1% of the extant microbial community. In 1988, a JP-4 fuel spill contaminated the glacio-fluvial aquifer at Wurtsmith Air Force Base, Michigan, USA. In this study, lipid biomarker characterization of the bacterial and eukaryotic communities was combined with polymerase chain reaction– denaturing gradient gel electrophoresis (PCR–DGGE) analysis of the eubacterial community to evaluate correlation between contaminant (JP-4 fuel) concentration and community structure shifts. Vadose, capillary fringe and saturated zone samples were taken from cores within and up- and down-gradient from the contaminant plume. Lipid biomarker analysis indicated that samples from within the plume contained increased biomass, with large proportions of typically Gram-negative bacteria. Outside the plume, lipid profiles indicated low-biomass microbial communities compared with those within the initial spill site. 16S rDNA sequences derived from DGGE profiles from within the initial spill site suggested dominance of the eubacterial community by a limited number of phylogenetically diverse organisms. Used in tandem with pollutant quantification, these molecular techniques should facilitate significant improvements over current assessment procedures for the determination of remediation end-points.     

Exploring potential use of Australian thraustochytrids for the bioconversion of glycerol to omega-3 and carotenoids production

Marine microbes have the potential for accumulating large quantities of lipids and are therefore suitable candidate as feedstock in unsaturated fatty acid production. The efficient utilisation of glycerol as an alternative carbon source to glucose was demonstrated in the fermentation of newly isolated thraustochytrid strains from the Queenscliff, Victoria, Australia. The isolates exhibited the presence of omega-3 and omega-6 polyunsaturated fatty acids, with the major fatty acids for all isolates being (as percent total fatty acid), palmitic acid (25.1–40.78%), stearic acid (4.24–13.2%), eicosapentaenoic acid EPA (2.31–8.5%) and docosapentaenoic acid (7.24–10.9%). Glycerol as a carbon source gave promising biomass growth with significant lipid and DHA productivity. An approximate three-fold increase in carotenoid content in all isolates was achieved when glycerol was used as a carbon source in the production medium.     

Polar lipid ester-linked fatty acid composition of Lake Vechten seston: an ecological application of lipid analysis

Abstract In order to relate the benthic lipid composition to possible sources in the water column, the sestonic communities of a monomictic lake were profiled using their saponifiable polar lipid fatty acids, which were identified by capillary gas chromatography-mass spectrometry (GC-MS). The epilimnion, dominated by the dinoflagellate alga Ceratium hirundella , was characterized by C_20:5 and C_22:6 polyunsaturated fatty acids. The photic anoxic metalimnion supported a radically different community, dominated by photosynthetic sulfur-oxidizing bacteria ( Chromatium and Chloronema spp.) and a Synechococcus -like cyanobacterium, and was characterized by high concentrations of C₁₆ and C₁₈ monounsaturated fatty acids. The fatty acid compositions of the hypolimnetic seston and the sediment were qualitatively similar to that of the metalimnion. Methyl-branched acids, commonly found in eubacteria, increased with depth in the water column. The concentrations of several unusual fatty acids found in Desulfovibrio spp. Desulfobacter spp. and Desulfotomaculum spp. were inversely related to sulfate concentration in the metalimnion. After the water column mixed in the winter, steep gradients of respiratory terminal electron acceptors developed in the surface sediment and were reflected in the polar lipid fatty acid compositions. The results show that fatty acids derived from the membranes of epilimnetic phytoplankton were efficiently metabolized in the oxic portion of the water column. The fatty acids synthesized by prokaryotic microorganisms at and below the oxycline dominated the sediment. The polar lipid fatty acid composition of the sediment showed seasonal changes which can be associated with concentrations of terminal electron acceptors of microbial respiration, and thus with physiologically distinct bacterial groups.     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.