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.     

Fatty Acid Compositions of Corynebacterium simplex Grown on 1-Alkenes

The utilization of 1-alkenes by Corynebacterium simplex ATCC 6946 was studied with respect to the characteristic fatty acid profiles resulting from the growth at the expense of these substrates.

It was indicated that the synthetic pathways of the cellular fatty acids in Corynebact. simplex grown on various n-alkanes or 1-alkenes changed markedly according to the chain lengths of the substrates. From shorter chain hydrocarbons (C₁₂, C₁₄) the fatty acids were found to be synthesized mainly via de novo synthesis pathway in a similar manner to those from glucose, while chain elongation and intact incorporation occurred to a very small extent. On the other hand, an intact incorporation mechanism was preferential in the cells grown on longer ones (C₁₆, C₁₈). When n-pentadecane or 1-pentadecene was used as the substrate, these three mechanisms seemed to operate simultaneously.     

Microbial Incorporation of Fatty Acids Derived From n-Alkanes Into Glycerides and Waxes

When n-alkanes with 13 to 20 carbon atoms were fed to a Nocardia closely related to N. salmonicolor, the produced cellular triglycerides and aliphatic waxes invariably contained fatty acids with an even or an odd number of carbon atoms subject to this feature of the n-alkane substrate. Beta-oxidation and C₂ addition are both operative, as evidenced by the spectra of fatty acids incorporated into the cellular lipid components. There is no distinction in the rate of microbial incorporation of the odd-or even-numbered carbon chains. The fatty acids are apparently directly derived from the long chain n-alkanes, rather than synthesized via the classic C₂-condensation route. The alcohol component of waxes produced by the Nocardia is invariably of the same chain length as the n-alkane substrate.     

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 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.     

Fatty Acid Composition of Hydrocarbon-Assimilating Yeast

As a part of extensive program on microbial utilization of hydrocarbons, lipid components of Candida petrophillum SD-14 grown on n-alkanes and glucose as carbon sources were studied. In any carbon source, cellular fatty acids of the yeast contained palmitic, palmitoleic, stearic, oleic and linoleic acids as major components.

When n-tridecane was fed to the yeast, fatty acids with odd- and even-number of carbon atoms were produced in almost identical quantity. Another yeast, Torulopsis petrophillum SD-77, also gave a very similar fatty acid pattern by n-tridecane substrate. These phenomena indicate the existence of C₂ addition and β-oxidation of the fatty acid formed in the yeasts.

In the cases of n-tridecane, n-hexadecane and glucose as substrate, about a half of SD-14’s lipid was phospholipid, which consisted of phosphatidyl ethanolamine and phosphatidyl choline principally. Free alcohol and wax were not detected in any case.     

Assimilation of hydrocarbons

When cultivating different yeast genera on the mixture of C₁₁−C₂₃ n-alkanes with predominant C₁₆ and C₁₇ content C₁₆ and C₁₇ fatty acids predominated in the synthetized cell fat. Besides these C_11∶0, C_15∶0 and C_18∶1 fatty acids were also present in higher quantities in all strains tested. This conclusion is in agreement with results of the residualn-alkanes estimation where during the fermentation most of the C₁₆ and C₁₇ hydrocarbons decreased from the original hydrocarbon mixture. From these experiments it can be provisionally concluded that the qualitative composition of the cell fat is not dependent on the yeast strain investigated, the amount of fat on the dry weight basis depends on the strain.     

Degradation of Hydrocarbons by Members of the Genus Candida II. Oxidation of n-Alkanes and 1-Alkenes by Candida lipolytica

Candida lipolytica ATCC 8661 was grown in a mineral-salts hydrocarbon medium. n-Alkanes and 1-alkenes with 14 through 18 carbon atoms were used as substrates. Ether extracts of culture fluids and cells obtained from cultures grown on the various substrates were analyzed by thin-layer and gas-liquid chromatography. Analyses of fluids from cultures grown on n-alkanes indicated a predominance of fatty acids and alcohols of the same chain length as the substrate. In addition, numerous other fatty acids and alcohols were present. Analyses of saponifiable and nonsaponifiable material obtained from the cells revealed essentially the same products. The presence of primary and secondary alcohols, as well as fatty acids, of the same chain length as the n-alkane substrate suggested that attack on both the methyl and α-methylene group was occurring. The significance of these two mechanisms in the degradation of n-alkanes by this organism was not evident from the data presented. Analyses of fluids from cultures grown on 1-alkenes indicated the presence of 1,2-diols, as well as ω-unsaturated fatty acids, of the same chain length as the substrate. Alcohols present were all unsaturated. Saponifiable and nonsaponifiable material obtained from cells contained essentially the same products. The presence of 1,2-diols and ω-unsaturated fatty acids of the same chain length as the substrate from cultures grown on 1-alkenes indicated that both the terminal methyl group and the terminal double bond were being attacked.     

Growth of Candida albicans in the presence of hydrocarbons: a correlation between sterol concentration and hydrocarbon uptake

Summary Candida albicans KTCC 89062 grown on n-alkanes showed higher levels of sterol content as compared to glucose-grown cells. Certain sterols, such as lanosterol, were significantly reduced in cells grown on n-alkanes, while others, such as ergosterol, increased in these cells. Sterol fractions declined as the chain length of the n-alkanes increased. Ergosterol supplementation of the chemically defined medium showed an increase in the uptake of dodecane (C₁₂) by cells grown on such medium. Increase in the concentration of ergosterol supplementation resulted in an increase in C₁₂ uptake. The uptake of C₁₂ was not stimulated by ergosterol supplementation in the case of non-viable yeast cells.     

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.     

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     

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.     

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.     

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.     

Substrate specificity in hydrocarbon utilizing microorganisms

Three bacteria (designated stram JOB5, 7E4, and 7E1C) isolated from soil by elective culture techniques and capable of growth on a wide variety of hydrocarbons were tested for substrate specificity. Non-proliferating cells of strain JOB5 grown on each of the C₁ to C_N series of normal ahphatic hydrocarbons were assayed for the capacity to oxidize all the alkanes, alcohols, fatty acids, and methyl-ketones in the homologous series of straight chain compounds. Cells of strain 7E4 grown on the gaseous alkanes (C₁ C₄) and strain 7E1C grown on propane were tested for the ability to oxidize the C₁ to C₄ n-alkanes, alcohols, and fatty acids.Contribution from Microbiology, North Carolina Agricultural Experiment Station, Raleigh, North Carolina Published with the approval of the Director of Research as paper No 2395 of the Journal Series.Part of this work was done while the author was associated with the late Dr. Jackson W. Foster, at the University of Texas, Austin.     

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.     

Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases

A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C₁₀H₂₂) to tetracontane (C₄₀H₈₂) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.     

<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.     

ζ-Potential of n-Alkane Emulsion Droplets and Its Role in Substrate Transport into Yeast Cells

The -potentials of n-alkane droplets, formed by fatty acids, were studied in model systems of the culture liquid of yeasts (Candida maltosa) capable of utilizing n-alkanes. The value of the -potential was found to depend on the droplet size. The negative -potential of submicron droplets was so high that it prevented the droplets from coagulating with cells also possessing a high negative -potential. The dominant role of submicron n-alkane droplets in the kinetics of yeast growth could be accounted for by the existence of a mechanism regulating the contact interactions of individual cells with the droplets followed by the uptake of the substrate.