Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary The yeast Candida maltosa precultivated on liquid n-alkanes utilized different solid n-alkanes (especially C₂₀–C₂₅) in the presence of pristane as an organic phase with rates comparable to, or somewhat larger than, those of liquid n-alkanes. Analysis of cellular fatty acids indicated an assimilation of solid n-alkanes via monoterminal oxidation. The resulting fatty acids with substrate chain length were chain-shortened by C₂ units down to an optimal range of chain length from C₁₆ to C₁₈ and incorporated into cellular, lipids directly or after desaturation. The intermediates of chain-shortening with numbers of carbon atoms higher than C₁₈, as well as the unusually long-chain fatty acids of substrate chain length, were detected in trace amounts only. Even-carbon-numbered and odd-numbered fatty acids predominated in experiments with evenchain and odd-chain n-alkanes, respectively. Studies with cerulenin indicated that de novo synthesis of fatty acids was negligible. Oxidation of solid n-alkanes by the yeast C. maltosa yielded fatty acid patterns similar to those of cells grown on liquid n-alkanes.     

Effect of cell lipid composition on the formation of nonspecific antibiotic resistance in alkanotrophic rhodococci

The antibiotic resistance and lipid composition of rhodococci grown in rich organic media with gaseous or liquidn-alkanes were studied. Hydrocarbon-grown rhodococci exhibited an increased resistance to a wide range of antibiotics (aminoglycosides, linkosamides, macrolides, β-lactams, and aromatic compounds). The enhanced antibiotic resistance of rhodococci grown onn-alkanes correlated with an increased content of total cell lipids (up to 14–28%) and saturated straight-chain fatty acids (C_16:0, C_18:0, C_21:0) and was accompanied by the appearance of cardiolipin and phosphatidylglycerol in cells. These lipid compounds are supposed to promote the formation of nonspecific antibiotic resistance in rhodococci by decreasing the permeability of their cell envelope to antibiotics.     

Candida lipolytica mutants defective in an acyl-CoA synthetase

Summary Mutant strains of Candida lipolytica NRRL Y-6795, which are defective in fatty acyl-CoA synthetase I linking to the system incorporating the fatty acyl moiety into cellular lipids (Kamiryo, et al., 1977), were cultivated on various carbon sources including odd-chain n-alkanes (C₁₁ to C₁₇) and their fatty acid compositions were examined.In the case of the wild-type strain grown on odd-chain n-alkanes (from C₁₃ to C₁₇), the proportions of odd-chain cellular fatty acids to total cellular fatty acids were markedly high, reaching 98–99% in the n-pentadecane- and n-heptadecane-grown cells. Those of the mutant strains, however, were drastically low, being at most 12–13% even in the n-heptadecane-grown cells. The total fatty acid contents in the mutant cells were 4–5% in dry weight, being slightly lower than those of the wild strain (4–7% in dry weight).The growth rates of the mutants on glucose, n-undecane and n-tridecane were comparable to those of the wild strain. When n-pentadecane, n-heptadecane, or oleic acid was used as carbon source, the mutants had lower, but still practicable, growth rates.The results obtained indicate that these mutant strains of Candida lipolytica will be useful as sources of biomass with low content of nonnatural odd-chain fatty acids.     

Heavy oils,principally long-chain n-alkanes secreted by Aureobasidium pullulans var. melanogenum strain P5 isolated from mangrove system

In this study, the yeast strain P5 isolated from a mangrove system was identified to be a strain of Aureobasidium pullulans var. melanogenum and was found to be able to secrete a large amount of heavy oil into medium. After optimization of the medium for heavy oil production and cell growth by the yeast strain P5, it was found that 120.0 g/l of glucose and 0.1 % corn steep liquor were the most suitable for heavy oil production. During 10-l fermentation, the yeast strain P5 produced 32.5 g/l of heavy oil and cell mass was 23.0 g/l within 168 h. The secreted heavy oils contained 66.15 % of the long-chain n-alkanes and 26.4 % of the fatty acids, whereas the compositions of the fatty acids in the yeast cells were only C_16:0 (21.2 %), C_16:1(2.8 %), C_18:0 (2.9 %), C_18:1 (39.8 %), and C_18:2 (33.3 %). We think that the secreted heavy oils may be used as a new source of petroleum in marine environments. This is the first report of yeast cells which can secrete the long-chain n-alkanes.     

Growth, natural relationships, cellular fatty acids and metabolic adaptation of sulfate-reducing bacteria that utilize long-chain alkanes under anoxic conditions

Natural relationships, improvement of anaerobic growth on hydrocarbons, and properties that may provide clues to an understanding of oxygen-independent alkane metabolism were studied with two mesophilic sulfate-reducing bacteria, strains Hxd3 and Pnd3. Strain Hxd3 had been formerly isolated from an oil tank; strain Pnd3 was isolated from marine sediment. Strains Hxd3 and Pnd3 grew under strictly anoxic conditions on n-alkanes in the range of C₁₂–C₂₀ and C₁₄–C₁₇, respectively, reducing sulfate to sulfide. Both strains shared 90% 16 S rRNA sequence similarity and clustered with classified species of completely oxidizing, sulfate-reducing bacteria within the δ-subclass of Proteobacteria. Anaerobic growth on alkanes was stimulated by α-cyclodextrin, which served as a non-degradable carrier for the hydrophobic substrate. Cells of strain Hxd3 grown on hydrocarbons and α-cyclodextrin were used to study the composition of cellular fatty acids and in vivo activities. When strain Hxd3 was grown on hexadecane (C₁₆H₃₄), cellular fatty acids with C-odd chains were dominant. Vice versa, cultures grown on heptadecane (C₁₇H₃₆) contained mainly fatty acids with C-even chains. In contrast, during growth on 1-alkenes or fatty acids, a C-even substrate yielded C-even fatty acids, and a C-odd substrate yielded C-odd fatty acids. These results suggest that anaerobic degradation of alkanes by strain Hxd3 does not occur via a desaturation to the corresponding 1-alkenes, a hypothetical reaction formerly discussed in the literature. Rather an alteration of the carbon chain by a C-odd carbon unit is likely to occur during activation; one hypothetical reaction is a terminal addition of a C₁ unit. In contrast, fatty acid analyses of strain Pnd3 after growth on alkanes did not indicate an alteration of the carbon chain by a C-odd carbon unit, suggesting that the initial reaction differed from that in strain Hxd3. When hexadecane-grown cells of strain Hxd3 were resuspended in medium with 1-hexadecene, an adaptation period of 2 days was observed. Also this result is not in favor of an anaerobic alkane degradation via the corresponding 1-alkene. Received: 25 June 1998 / Accepted: 29 July 1998     

Fatty Acid Compositions of Triglyceride and Phospholipid from Candida tropicalis Grown on n-Alkanes

The content of total cellular lipid of Candida tropicalis grown on a mixture of n-alkanes (C₁₀–C₁₈) was about 20% of the dry cell weight at the exponential growth phase and 14% at the early stationary phase. Phospholipid corresponded to approximately 70 % of the total lipid independent of the growth phases. The composition of cellular lipid classes did not change significantly during the growth. On the other hand, a drastic time-course change in fatty acid composition was observed. The proportion of odd-chain fatty acids, one of the most specific cellular components of the yeast grown on the n-alkane mixture, increased in both phospholipid and triglyceride along with the yeast growth. In the meantime, the proportion of polyunsaturated fatty acids varied markedly during the course of cultivation, showing a peak at the early growth phase. The high content of polyunsaturated fatty acids at the early stages of growth correlated to the contents of these acids in phospholipid rather than in triglyceride.     

Production of fatty acids and lipid by a Candida sp. growing on a fraction of n-alkanes predominating in tridecane

A Candida sp. was grown on a fraction of n-alkanes (dodecane 22%, tridecane 48%, tetradecane 28%) as sole carbon source. The growth rate was increased most markedly by using high concentrations of n-alkanes (16.7% v/v). When grown in a 5 liter fermentor, the yeast reached its highest yield (60 g. of cell dry wt/l) with a concomitant high yield of fatty acids (21 g of fatty acids/l), by using a nitrogen-deficient medium. To achieve good growth, it was essential to use an inoculum (1 part into 10) of rapidly growing cells and beneficial to increase the agitation rate gradually once growth had begun. After 108 hr maximum conversions of substrate to product were: 71.5% (w/w) for alkanes into cells and 24.8% (w/w) for alkanes into fatty acids. Of the, total fatty acids at the end of the fat-accumulating phase of growth 54% were shorter in chain length than palmitic acid (C₁₆H₃₂O₂). When grown on glucose, as sole carbon source, less than 2% of the total fatty acids were shorter than palmitic acid. When n-alkanes were added to cells growing on glucose, short-chain fatty acids (C₁₀ to C₁₄) were synthesized immediately, indicating a derepressed enzyme system for hydrocarbon assimilation and the absence of diauxie. The production of these acids was at the apparent sacrifice of linoleic acid synthesis. In spite of the high conversion ratios, it is concluded that it would be uneconomical to produce fatty acids, even expensive ones such as lauric acid, by microbial transformation of n-alkanes.     

Fatty acids compositions of polar and non-polar lipid fractions of wheat leaves inoculated with three brown rust races during progressive pathogenesis

The fatty acids composition of the polar and non-polar lipid fractions of wheat leaves was affected due to progressive brown rust infection during early stages of pathogenesis,i.e. 0, 12, 24, 36, 48 and 72 h after inoculation. The three races ofPuccinia recondita differentially affected the composition of saturated and unsaturated fatty acids and their relative occurrence in wheat leaves. The infection of wheat by race 77 resulted in a relative decrease in fatty acid chain length as measured through C₁₆∶C₁₈ fatty acid ratio. An increase in the relative degree of unsaturation (18∶2/18∶3 acids ratio) was recorded in both lipid fractions. Such changes may be taken as one of the earliest characteristics of disease development.     

Extractive compounds of birch buds (<Emphasis Type="Italic">Betula pendula</Emphasis> Roth.): II. Carbonyl compounds and oxides. Esters

This is the first report devoted to study of the hydrocarbon composition of the extract of buds of European birch Betula pendula (family Betulacea). We have identified saturated (C₁₆ to C₂₈, even number of carbon atoms) and unsaturated (linoleic and linolenic) fatty acids, β-caryophyllene, α-humulene, and the components of epicuticular waxes of cover scales, such as n-alkanes (C₂₁ to C₂₆), esters of fatty acids (C₁₆ to C₂₈, even number of carbon atoms), and fatty alcohols (C₁₈ to C₃₀, even number of carbon atoms). The gas chromatographic retention indices of all identified compounds have been determined.     

Oleispira lenta sp. nov., a novel marine bacterium isolated from Yellow sea coastal seawater in Qingdao, China

The taxonomic position of strain DFH11^T, which was isolated from coastal seawater off Qingdao, People’s Republic of China in 2007, was determined. Strain DFH11^T comprised Gram-negative, motile, strictly aerobic spirilli that did not produce catalase. Comparative 16S rRNA gene sequence analysis revealed that strain DFH11^T shared ~97.2, 93.3, 91.8, 91.7 and 91.5% sequence similarities with Oleispira antarctica, Spongiispira norvegica, Bermanella marisrubri, Oceaniserpentilla haliotis and Reinekea aestuarii, respectively. DNA–DNA hybridization experiments indicated that the strain was distinct from its closest phylogenetic neighbour, O. antarctica. The strain grew optimally in 2–3% (w/v) NaCl, at pH 5.0–10.0 (optimally at pH 7.0) and between 0 and 30°C (optimum growth temperature 28°C). The strain exhibited a restricted substrate profile, with a preference for aliphatic hydrocarbons, that is consistent with its closest phylogenetic neighbour O. antarctica. Growth of the isolate at different temperatures affected the cellular fatty acid profile. 28°C cultured cells contained C_16:1ω7c and/or iso-C_15:0 2-OH (50.4%) and C_16:0 (19.2%) as the major fatty acids. However, the major fatty acids of the cells cultured at 4°C were C_16:1ω7c and/or C_16:1ω6c (40.2%), C_16:0 (17.2%) and C_17:1ω8c (10.1%). The G+C content of the genomic DNA was 42.7 mol%. Phylogeny based on 16S rRNA gene sequences together with data from DNA–DNA hybridization, phenotypic and chemotaxonomic characterization revealed that DFH11^T should be classified as a novel species of the genus Oleispira, for which the name Oleispira lenta sp. nov. is proposed, with the type strain DFH11^T (=NCIMB 14529^T = LMG 24829^T).     

Effects of Chain-length of Alkane Substrate on Fatty Acid Composition and Biosynthetic Pathway in Some Candida Yeasts

Cellular fatty acid compositions of Candida tropicalis pK 233 and Candida lipolytica NRRL Y -6795 and the time-course changes during yeast growth were studied using individual n-alkanes of various chain lengths (from C₁₁ to C₁₈) and a mixture of n-alkanes (C₁₁ to C₁₈) as a sole carbon source. Observed relationships of the chain-length of n-alkane substrate to time-course changes and final patterns of the fatty acid compositions of these yeasts, especially those of the cells grown on odd-carbon alkanes, indicated that “intact incorporation mechanism,” that is, accumulation of the fatty acid having the same chain-length as that of the alkane substrate used was predominant in the yeasts cultivated on a longer alkane such as n-heptadecane and n-octadecane. On the other hand, “chain elongation pathway” and “de novo synthesis pathway” following β-oxidation of substrate were simultaneously operative in the cells growing on a relatively shorter alkane such as undecane and dodecane.     

Effect of Substrate on the Fatty Acid Composition of Hydrocarbon- and Ketone-utilizing Microorganisms

The fatty acid pattern in hydrocarbon- and ketone-utilizing bacteria after growth on various substrates was examined. The fatty acid composition of one hydrocarbon-utilizing organism (Mycobacterium sp. strain OFS) was investigated in detail after growth on n-alkanes, 1-alkenes, ketones, and n-alcohols. n-Alkanes shorter than C₁₃ or longer than C₁₇ were not incorporated into cellular fatty acids without some degradation. Strain OFS incorporated C₁₄ to C₁₇ 1-alkenes into cellular fatty acids as the ω-monoenoic fatty acid. Methyl ketones were incorporated into strain OFS after removal of one- or two-carbon fragments from the carbonyl end of the molecule. An organism isolated by enrichment on methyl ketones was incapable of n-alkane utilization but could grow on, although not incorporate, ketones or long chain n-alcohols into cellular fatty acids.     

Viability and formation of conjugated dienes in plasma membrane lipids of Saccharomyces cerevisiae,Schizosaccharomyces pombe,Rhodotorula glutinis and Candida albicans exposed to hydrophilic,amphiphilic and hydrophobic pro-oxidants

Effects of four lipid peroxidation-inducing pro-oxidants-amphiphilictert-butyl hydroperoxide (TBHP), hydrophobic 1,1′-azobis(4-cyclohexanecarbonitrile) (ACHN), hydrophilic Fe¹¹ and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-on cell growth and on generation of peroxidation products in isolated plasma membrane lipids were determined in four yeast species (S. cerevisiae, S. pombe, R. glutinis andC. albicans) differing in their plasma membrane lipid composition. TBHP and ACHN inhibited cell growth most strongly, Fe¹¹ and AAPH exerted inhibitory action for about 2 h, with subsequent cell growth resumption.S. cerevisiae strain SP4 was doped during growth with unsaturated linoleic (18∶2) and linolenic (18∶3) acids to change its resistance to lipid peroxidation. Its plasma membranes then contained some 30% of these acids as compared with some 1.3% of 18∶2 acid found in undopedS. cerevisiae, while the content of (16∶1) and (18∶1) acids was lower than in undopedS. cerevisiae. The presence of linoleic and linolenic acids inS. cerevisiae cells lowered cell survival and increased the sensitivity to pro-oxidants. Peroxidationgenerated conjugated dienes (CD) were measured in pure TBHP- and ACHN-exposed fatty acids used as standards. The CD level depended on the extent of unsaturation and the pro-oxidant used. The TBHP-induced CD production in a mixture of oleic acid and its ester was somewhat lower than in free acid and ester alone. In lipids isolated from the yeast plasma membranes, the CD production was time-dependent and decreased after a 5–15-min pro-oxidant exposure. ACHN was less active than TBHP. The most oxidizable were lipids fromS. cerevisiae plasma membranes doped with linoleic and linolenic acids and fromC. albicans with indigenous linolenic acid.     

<Emphasis Type="Italic">n</Emphasis>-Alkanes variability in the diazotrophic cyanobacterium <Emphasis Type="Italic">Anabaena cylindrica</Emphasis> in response to NaCl stress

n-Alkanes pattern in response to NaCl stress has been studied in the cyanobacterium Anabaena cylindrica. Saturated hydrocarbons were separated and identified by gas chromatography-mass spectrometry (GC-MS) using serially coupled capillary column. Light chain n-alkanes in the range of C₉–C₁₇ (43%) and heavy chain n-alkanes in range of C₁₇–C₂₃ (34%) and C₂₃–C₃₁ (23%) were identified as the major components of total hydrocarbons in the NaCl adapted cells of A. cylindrica. In contrast, NaCl-untreated cells of A. cylindrica had dominance of only long chain n-alkanes in the range of C₂₃–C₃₁ comprising about 94% of its total n-alkanes. The persistence of high level (43%) of short chain n-alkanes (C₉–C₁₇) in NaCl adapted cells of A. cylindrica as compared to its negligible level (0.2%) in NaCl untreated counterpart clearly indicates that NaCl stress causes the A. cylindrica to shift towards the synthesis of short chain n-alkanes.     

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C₁₉ phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine. Received: 20 April 1998 / Received revision: 10 July 1998 / Accepted: 29 July 1998     

Enhancement of growth and antibiotic titre inCephalosporium acremonium induced by sesame oil

The role of sesame oil as part of the carbon source on growth and cephalosporin C production byCephalosporium acremonium was studied in shake-flask fermentation. The growth and antibiotic production were maximum on the fifth and sixth day, respectively, irrespective of the presence of sesame oil. Sesame oil enhanced cephalosporin C production by 54%. Analysis of fatty acid profile indicated that C_18∶1, C_18∶2 and C_18∶3 are the major fatty acids inC. acremonium. The percentage of C_18∶2 was higher in the culture grown with sesame oil.     

Hydrocarbon degradation by <Emphasis Type="Italic">Dietzia</Emphasis> sp. A14101 isolated from an oil reservoir model column

The hydrocarbon-degrading strain Dietzia sp. A14101 was isolated from an oil reservoir model column inoculated with oil-field bacteria. The column was continuously injected with nitrate (0.5 mM) from the start of water flooding, which lead to a gradual development of nitrate reduction in the column. Strain A14101 was able to utilize a range of aliphatic hydrocarbons as sole carbon and energy source during aerobic growth. Whole oil gas chromatography analysis of the crude oil phase from aerobic pure cultures showed that strain A14101 utilized the near complete range of aliphatic components and aromatic components toluene and xylene. Longer n-alkanes ≥C₁₇ were utilized simultaneously with the shorter C₁₀ and C₁₅. After 120 days aerobic incubation, the whole oil gas chromatography profile of the crude oil phase was similar to that of heavily biodegraded oils. Anaerobic degradation of hydrocarbons with nitrate was not observed. Nitrate reduction was, however, observed during anaerobic growth on propionate, which suggests that strain A14101 grows on fatty acids in the column rather than on hydrocarbons.     

Effects of zinc deficiency on the fatty acid composition and metabolism in rats fed a fat-free diet

The effects of dietary zinc deficiency (ZD) on the composition and metabolism of the fatty acyl chains of phospholipids in rat liver were investigated with a fat-free diet. The levels of (n−9) fatty acids such as 18∶1 and 20∶3(n−9) in liver phospholipids (PL) were significantly lower in ZD-rats (19.4% and 5.4%, respectively) than in PF-rats (25.2 and 8.3%). On the other hand, the level of (n−6) acids such as 18∶2 and 20∶4 were higher in ZD-rats (3.3 and 19.1%, respectively) than in PF-rats (2.1 and 14.9%). In order to study the metabolism of fatty acids in vivo,¹⁴C-18∶0 or¹⁴C-18∶2 was intravenously injected, and then the conversion to the respective metabolite was examined. After the injection of¹⁴C-18∶0, the radioactivity was found in 18∶0 (49.3% of the total), 18∶1 (33.2%), and 20∶3 (n−9) (9.1%) in liver PL in PF-rats at 24h. In ZD-rats, the radioactivity was dramatically lower in 18∶1 (23.5%) and 20∶ (n−9) (3.6%), suggesting that the conversion of 18∶0 to 18∶1 and 20∶3 (n−9) was strongly inhibited in ZD-rats. When¹⁴C-18∶2 was injected, the radioactivity was mainly found in 18∶2, 20∶3(n−6), and 20∶4. The radioactivity in 20∶4 in ZD-rats was slightly higher than that in control rats. These results indicate that zinc deficiency affects the fatty acid metabolism in liver, in particular, it causes a reduction in δ9 desaturase activity, when rats are fed a fat-free diet.     

Isolation and Identification of n-Butane-assimilating Bacterium

A bacterial strain capable of assimilating gaseous n-alkanes was newly isolated from activated sludge by enrichment culture technique using n-butane as the sole carbon source. The strain was identified as Pseudomonas butanovora sp. nov. It utilised n-alkanes of C₂~C₉, primary alcohols and carboxylic acids for growth, but did not utilize sugars and C₁ compounds. The cell yields on gaseous n-alkanes, such as ethane, propane and n-butane, were 80% or more. The maximum specific growth rate on n-butane was 0.22 hr^?1 at 30°C, pH 7.0. Dried cells of this new isolate grown on n-butane contained 73% pure protein.     

Alterations in growth temperature range and fatty acid composition of Thermus as a result of plasmid elimination

Elimination of plasmids from Thermus flavus, T. thermophilus and three wild Thermus strains caused alterations in growth temperature range, pigmentation and membrane fatty acids without affecting viability. Following plasmid elimination all Thermus strains lost their ability to grow above 70°C. In addition, the minimum growth temperature was lowered by 5–10°C. Fatty acids were reduced by an average of approximately 35%. In addition, the contribution of iso- and anteisobranched fatty acids were altered in four of the five strains. The iso C_15:0/iso C_17:0 ratio approached 1.0 in all strains, whereas the anteiso C_15:0/anteiso C_17:0 was reduced to 0.2. The iso C_16:0/normal-C_16:0 ratio increased in all strains due to an increase in iso C_16:0 in four strains and a reduction in normal-C_16:0 relative to iso C_16:0 in one strain. However, it was evident that the plasmid-free strains were able to compensate for these alterations in membrane fluidity to a certain extent by reducing the average chain length of isobranched acids. Altered fatty acid metabolism at the level of precursors may have influenced membrane composition and consequently growth temperature range.