Lipid synthesis by Micrococcus freudenreichii in media containing unsaturated hydrocarbon's]

The growth and synthesis of lipids by thermotolerant bacteria Micrococcus freudenreichii K-219 were investigated in the mineral medium containing a mixture of unsaturated (I-) and saturated hydrocarbons. The bacteria utilized primarily I-alkenes. In lipids the predominant fractions were phospholipids (57%) and free fatty acids (20%). The content of waxes which were in significant quantities in n-alkane containing media (9%) was not higher than 0.3% dry matter upon utilization of I-alkenes. There was a certain correlation between carbon atoms of synthesized fatty acids and unsaturated hydrocarbons used. Bacteria utilizing I-alkenes showed no elevated unsaturation of cell lipids as compared to those assimilating n-alkanes. These data give evidence for different pathways of oxidation of alkenes and alkanes by the above microbial strain.     

Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay

Thirty-six bacteria that degraded long-chain hydrocarbons were isolated from natural environments using long-chain hydrocarbons (waste car engine oil, base oil or the c-alkane fraction of base oil) as the sole carbon and energy source. A phylogenetic tree of the isolates constructed using their 16S rDNA sequences revealed that the isolates were divided into six genera plus one family (Acinetobacter, Rhodococcus, Gordonia, Pseudomonas, Ralstonia, Bacillus and Alcaligenaceae, respectively). Furthermore, most of the isolates (27 of 36) were classified into the genera Acinetobacter, Rhodococcus or Gordonia. The hydrocarbon-degradation similarity in each strain was confirmed by the 2,6-dichlorophenol indophenol (2,6-DCPIP) assay. Isolates belonging to the genus Acinetobacter degraded long-chain normal alkanes (n-alkanes) but did not degrade short-chain n-alkanes or cyclic alkanes (c-alkanes), while isolates belonging to the genera Rhodococcus and Gordonia degraded both long-chain n-alkanes and c-alkanes.     

Novel wax esters and hydrocarbons in the cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus

The cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus, were found to contain minor amounts of novel wax esters, in addition to the major components, hydrocarbons. The wax esters ranged in carbon number from C19 to C31 and consisted of esters of both odd- and even-numbered alcohols and acids. Each wax ester with a given carbon number eluted at several different retention times indicating possible methyl branching in either the fatty acid or alcohol moiety, or in both moieties. Each eluting peak of wax esters consisted of a mixture of wax esters of the same carbon number in which the fatty acid moiety ranged from C8 to C18, and the alcohol moiety ranged from C8 to C17. Some wax esters were largely found on the head indicating they may be of a glandular origin. The hydrocarbons consisted of: n-alkanes, C23 to C33; odd-numbered n-alkenes, C27 to C35; and the major components, methyl-branched alkanes, C26 to over C49. Notable components of the methyl-branched alkanes were 2-methyltriacontane, and the novel trimethylalkanes with a single methylene between the first and second branch points, 13,15,19-trimethylhentriacontane and 13,15,21-trimethyltritriacontane.     

Cuticular lipids of insects. VI. Cuticular lipids of the grasshoppers Melanoplus sanguinipes and Melanoplus packardii

The cuticular lipids of the grasshoppers Melanoplus sanguinipes and Melanoplus packardii contain 60 and 68% alkanes and 28 and 18% secondary alcohol wax esters, respectively, with lesser amounts of normal and sterol wax esters, triglycerides, alcohols, sterols, and free fatty acids. All the hydrocarbons are saturated, and four types of alkanes are present: n-alkanes, 3-methylalkanes, internally branched monomethylalkanes, and internally branched dimethylalkanes. The principal n-alkanes in both insects are C(29) and C(27), with a range from C(21) to C(33). Trace amounts of 3-methylalkanes of 28, 30, and 32 total carbons are present. The principal internally branched monomethylalkanes are C(32) and C(34), whereas the main dimethylalkane contains 35 carbons. The n-alkanes do not correspond in chain length to the secondary alcohols. The primary alcohols range from C(22) to C(32) in both insects, with C(24) and C(26) predominating. The fatty acids in the triglyceride and free fatty acid fractions range from C(12) to C(24) in M. sanguinipes and from C(12) to C(18) in M. packardii.     

Observation on the composition and biosynthesis of egg wax lipids in the cattle tick,Boophilus microplus

The biosynthesis of wax lipids by Gené's organ, the egg waxing organ in ticks, was investigated. Gené's organ, a complex dermal gland system, applies a superficial wax layer to the eggs during oviposition which prevents desiccation and is essential for egg viability. The detailed anatomy and histology of the three gland cell types are unambiguously described. Serial sectioning of ticks showed that all three gland cell types are capable of contributing to the egg wax. The wax synthetic ability of these three gland types was characterized. The composition of wax lipids extracted from the surface egg wax, and from the three types of wax gland dissected from ovipositing ticks, was analysed using thin-layer and gas-liquid chromatography. Injection of ovipositing ticks with radiolabelled acetate resulted in the incorporation of the label into wax lipids by gland cells of Gené's organ. The egg wax was a complex mixture of long-chain alkanes and fatty acid esters. The gland cells contained a greater proportion of shorter chain alkanes than were present in the egg surface wax. Some unsaturated long-chain fatty acids were present, and these were more abundant in the gland cells than in the surface wax of oviposited eggs, suggesting that oxidation occurs after oviposition. The results confirm that the tubular glands, acinar accessory glands and lobular glands of Gené's organ all contribute to the egg waxes, although the lipid components differed in relative abundance. The results are also consistent with alkane synthesis from fatty acids in Gené's organ by a chain-elongation-decarboxcylation pathway.     

植物角质层蜡质的化学组成研究综述

角质层是植物与外界的第一接触面,而角质层蜡质则是由位于角质层外的外层蜡质和深嵌在角质层中的内层蜡质两部分构成。植物角质层蜡质成分极其复杂,具有重要的生理功能。综述了有关植物角质层蜡质的化学组成信息,探讨了目前植物角质层蜡质化学成分研究中存在的一些问题,展望了角质层蜡质成分的研究前景。     

Untersuchungen zur chemischen Zusammensetzung der Lipidfraktion von Acinetobacter calcoaceticus

Lipids were extracted from the cells of Acinetobacter calcoaceticus EB 10 C IMET B 395 grown on gas oil (Bp. 240–360 °C) with benzine/alcohol (80 : 20). The lipid-hydrocarbon-extract obtained by this extraction method was 18.4%. The extract was composed of hydrocarbons, waxes, phospholipids, fatty acids, glycerides, and ubiquinones. The main components among the lipids were waxes. The compositions of phospholipid, fatty acid, wax, and ubiquinone fractions were analysed.     

Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed

The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.     

Cuticular waxes from potato (Solanum tuberosum) leaves

The qualitative and quantitative compositions of leaf cuticular waxes from potato (Solanum tuberosum) varieties were studied. The principal components of the waxes were very long chain n-alkanes, 2-methylalkanes and 3-methylalkanes (3.1-4.6 microg cm(-2)), primary alcohols (0.3-0.7 microg cm(-2)), fatty acids (0.3-0.6 microg cm(-2)), and wax esters (0.1-0.4 microg cm(-2)). Methyl ketones, sterols, beta-amyrin, benzoic acid esters and fatty acid methyl, ethyl, isopropyl and phenylethyl esters were found for the first time in potato waxes. The qualitative composition of the waxes was quite similar but there were quantitative differences between the varieties studied. A new group of cuticular wax constituents consisting of free 2-alkanols with odd and even numbers of carbon atoms ranging from C25 to C30 was identified.     

Diversity of cuticular wax among Salix species and Populus species hybrids

The leaf cuticular waxes of three Salix species and two Populus species hybrids, selected for their ability to produce high amounts of biomass, were characterized. Samples were extracted in CH(2)Cl(2) three times over the growing season. Low kV SEM was utilized to observe differences in the ultrastructure of leaf surfaces from each clone. Homologous series of wax components were classified into organic groups, and the variation in wax components due to clone, sample time, and their interaction was identified. All Salix species and Populus species hybrids showed differences in total wax load at each sampling period, whereas the pattern of wax deposition over time differed only between the Salix species. A strong positive relationship was identified between the entire homologous series of alcohols and total wax load in all clones. Similarly strong relationships were observed between fatty acids and total wax load as well as fatty acids and alcohols in two Salix species and one Populus species hybrid. One Salix species, S. dasyclados, also displayed a strong positive relationship between alcohols and alkanes. These data indicate that species grown under the same environmental conditions produce measurably different cuticular waxes and that regulation of wax production appears to be different in each species. The important roles cuticular waxes play in drought tolerance, pest, and pathogen resistance, as well as the ease of wax extraction and analysis, strongly suggest that the characteristics of the cuticular wax may prove to be useful selectable traits in a breeding program.     

Protocol for the analysis of n-alkanes and other plant-wax compounds and for their use as markers for quantifying the nutrient supply of large mammalian herbivores

Plant-wax markers can be used for estimating forage intake, diet composition and supplement intake in grazing livestock, wild ruminants and other mammals. We describe protocols for using the saturated hydrocarbons (alkanes) of plant wax as markers for estimating fecal output, intake and digestibility. Procedures for investigating digestion kinetics are also discussed. Alkanes can also be used to estimate diet composition and the procedures required to do this are also described, including the special case where supplementary feed is treated as a component of the diet composition estimate. The long-chain alcohols (LCOHs) and very long-chain fatty acids (VLCFAs) of plant wax show particular promise for discriminating a greater number of species in the diet. The use of all these plant-wax markers in nutrition studies depends on having quantitative, repeatable and mutually compatible assay procedures for alkanes, LCOHs and VLCFAs; we present protocols for these assays in detail. Analysis of a single sample of feces or plant material for all these plant-wax markers can be completed within 2 days; however, it is possible to process up to 50 samples (analyzed in duplicate) per week.     

Major constituents of the foliar epicuticular waxes of species from the Caatinga and Cerrado

The epicuticular waxes of leaves of four species (Aspidosperma pyrifolium, Capparis yco, Maytenus rigida and Ziziphus joazeiro) from the Caatinga, (a semi-arid ecosystem of Northeast Brazil) and four species (Aristolochia esperanzae, Didymopanax vinosum, Strychnos pseudoquina and Tocoyena formosa) from the Cerrado, (a savanna ecosystem covering one third of the Brazilian territory), were analyzed. Six species contained a high content (above 60 microg x cm(-2)) of wax, four of them from the Caatinga. Triterpenoids and n-alkanes were the most frequent and abundant constituents found in the species from both habitats. The distribution of n-alkanes predominated by homologues with 27, 29, 31 and 33 carbon atoms, displayed no consistent differences between species from the two habitats. Lupeol, beta-amyrin, epifriedelinol and ursolic acid were the triterpenoids found. Triterpenoids clearly predominate over alkanes in the waxes from the Cerrado species. The waxes of two evergreen species from the Caatinga yielded n-alkanes as predominant constituents. A comparison of foliar epicuticular waxes of native plants from ecosystems with different hydric constraints is discussed.     

Composition of sugarcane waxes in rum factory wastes

Wastes produced during fermentation and distillation of crude sugarcane juice in rum factories were evaluated as a new source of waxes. The chemical composition of the crude wax extracted from adsorbat of the wastes on fuller's earth was studied by GC-mass spectrometry. Series of linear alkanes (C19-C33), and wax esters constitute the main components. In addition, phytosterols, triterpene methyl ethers, ethyl and methyl esters of fatty acids, and free fatty acids were found as minor components. Acid (predominance of C16 and C18) and alcohol portions (C26-C32) of the wax esters were analysed after saponification.     

Composition of suberin-associated waxes from the subterranean storage organs of seven plants

The waxes associated with the suberin in the periderm of the underground storage organs of parsnip (Pastinaca sativa L.), carrot (Daucus carota L.), rutabaga (Brassica napobrassica Mill.), turnip (Brassica rapa L.), red beet (Beta vulgaris L.), sweet potato (Ipomoea batatas L.) and potato (Solanum tuberosum L.) were isolated, fractionated into hydrocarbon, wax ester, free fatty alcohol and free fatty acid fractions, and analyzed by combined gas chromatography and mass spectrometry. The amount of wax extracted from the periderm of the storage organs ranged from 2 to 32 μg/cm². The hydrocarbons from the suberin layer have a broader chain-length distribution, a predominance of shorter carbon chains, and a higher proportion of even-numbered carbon chains than the leaf alkanes from the same plants. The major components of the free and esterified fatty alcohols and fatty acids have an even number of carbon atoms, and are similar in chain-length distribution to their counterparts found covalently attached to the suberin polymers; however, these suberin components are shorter in chain length than their cuticular analogues from the leaves. Also extracted from the storage organs were polar components which included fatty alcohols and fatty acids in a conjugated form, and ω-hydroxy acids and dicarboxylic acids. Evidence is presented that removal of the wax from the periderm of whole storage organs results in a decrease in diffusion resistance to moisture. Scientific Paper No. 5516, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164, USA     

Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 microg leaf(-1) of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.     

The potential use of n-alkanes, long-chain alcohols and long-chain fatty acids as diet composition markers: indoor validation with sheep and herbage species from the rangeland of Inner Mongolia of China

To investigate the potential use of n-alkanes (alkanes), long-chain alcohols (alcohols) and long-chain fatty acids (acids) for estimating the diet composition of sheep, in a feeding trial. A total of 18 sheep were assigned randomly to three different diets (diet A, diet B and diet C) containing up to eight herbage species (Leymus chinensis, Leymus dasystachys, Elymus sibiricum, Chenopodium album, Puccinellia chinampoensis, Medicago sativa, Saussurea sinuata and Bromus inermis). Faecal recoveries of alkanes, alcohols and acids were determined, and diet compositions were estimated using different combinations of alkanes, alcohols and acids. The faecal concentrations of individual alkanes, alcohols and acids were corrected using the mean recovery of the dietary treatment that the respective animal belonged to (diet recovery), or the mean recovery across all dietary treatments (general recovery). In general, diets did not affect the faecal recovery values for alkanes, alcohols and acids, and no difference in accuracy was found between diet composition estimates based on dietary recovery and general recovery. The accuracy of diet composition estimates declined as the number of dietary components increased from four to eight herbage species (P < 0.001). Better (P < 0.05) estimates of diet composition were obtained with the combinations of two or three marker types instead of alkanes alone. Moreover, results showed that excluding minor diet components from the calculations decreased (P < 0.05) the accuracy of diet composition estimates, whereas including extra non-grazed herbage species did not reduce (P > 0.05) the quality of diet composition estimates. These results confirmed the usefulness of alkanes, alcohols and acids as markers for determining complex diet composition of sheep. However, a negative impact on the accuracy of diet composition estimates, caused by missing minor diet components from the calculation of diet composition, could happen when plant wax markers are used to estimate the diet composition of free-ranging animals.     

The effects of stress on plant cuticular waxes

Plants are subject to a wide range of abiotic stresses, and their cuticular wax layer provides a protective barrier, which consists predominantly of long-chain hydrocarbon compounds, including alkanes, primary alcohols, aldehydes, secondary alcohols, ketones, esters and other derived compounds. This article discusses current knowledge relating to the effects of stress on cuticular waxes and the ways in which the wax provides protection against the deleterious effects of light, temperature, osmotic stress, physical damage, altitude and pollution. Topics covered here include biosynthesis, morphology, composition and function of cuticular waxes in relation to the effects of stress, and some recent findings concerning the effects of stress on regulation of wax biosynthesis are described.     

n-alkane profile of Argemone mexicana leaves

An n-hexane extract of fresh, mature leaves of Argemone mexicana (Papaveraceae), containing thin-layer epicuticular waxes, has been analysed for the first time by TLC, IR and GLC using standard hydrocarbons. Seventeen long-chain alkanes (n-C18 to n-C34) were identified and quantified. Nonacosane (n-C29) was established as the n-alkane with the highest amount, whilst octadecane (n-C19) was the least abundant component of the extracted wax fraction. The carbon preference index (CPI) calculated for the hydrocarbon sample with the chain lengths between C18 and C34 was 1.2469, showing an odd to even carbon number predominance.     

Cuticular wax composition of Salix varieties in relation to biomass productivity

The leaf cuticular waxes of six Salix clones (one Salix miyabeana, one Salix dasyclados, one Salix eriocephala, two Salix purpurea, and one interspecific hybrid of Salix eriocephala x interior) with different biomass productivities were characterized by gas chromatography-mass spectrometry. Total wax content ranged from 6.3 to 16.8 microg cm(-2), and two distinct patterns of wax were measured. The wax from leaves of S. dasyclados 'SV1' differed from all other clones and was dominated by fatty acids (42%), high concentrations of n-alkanes (25%) and n-alcohols (28%), with low n-aldehyde content (4%). All other clones produced cuticular wax dominated by n-alcohols (32-51%), particularly 1-hexacosanol, with fatty acids (14-37%) and n-aldehydes (19-26%) present in lower abundances. Clones of Salix grown under identical environmental conditions produce noticeably different amounts of cuticular wax. In contrast to previous studies of Salix, total wax content was independent of biomass productivity, measured as basal area, suggesting that wax production is not directly linked with woody biomass production by shrub willows under these site conditions.     

Fatty Aldehydes in Cyanobacteria Are a Metabolically Flexible Precursor for a Diversity of Biofuel Products

We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis.