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.     

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.     

Microbial transformation of hydrocarbons. II. Growth constants and cell composition of microbial cells derived from n-alkanes

Cultivation of Norcardia sp., Mycobacterium phlei, and Candida lipolytica in inorganic salt solution containing n-alkanes C₁₀–C₂₀ as solo carbon and energy source was investigated. Generation times of 0.5–7.0 hr were typical during the exponential growth phase. The final cell concentrations (dry weight) were usually 9–26 g/l with n-alkane mixtures ranging from n-decane through n-eicosane. A linear dependence was found between the production of cell mass and the consumption of n-alkanes. The rest concentration of n-alkanes in the cell mass is in all experiments smaller than 0.5% (w/w). Cell yields were Y_sub 60–142% and for Y_e 50–97% based on n-alkane utilization. In one case, with the Nocardia NBZ 23, the substrate specifity on hydrocarbons and on a n-alkane mixture C₁₀-C₂₀ was studied. The cell mass recovered from the fermentations contained 47.8–57.7% carbon, 5.6–9.95% nitrogen, 7.2–9.4% hydrogen, 35–62% crude protein, and 6–36% lipid. Cellular protein and lipid synthesized by an organism is influenced by the type of nitrogen source. The amino acid, glucosamine, muramic acid, 2,6-diaminopimelinic acid, and fatty acid distribution in organisms grown on n-alkanes compared with a corresponding fermentation on glucose as sole carbon source were also estimated.     

Comparative lipid content ofCandida lipolytica grown on glucose and onn-hexadecane

Candida lipolytica, grown onn-hexadecane as the sole source of carbon and energy, contained 17.1% lipids in the logarithmic phase of growth, and 7.3% lipids in the stationary phase of growth. When the yeast was grown on glucose, it contained 6.2% lipids in the logarithmic phase of growth, and 3.6% lipids in the stationary phase of growth. Fatty acids, that could be extracted by petroleum ether after saponification, constituted the major part of the fatty acids ofC. lipolytica in its logarithmic phase of growth on glucose. They constituted only a minor amount of the fatty acids in the stationary phase of growth on glucose. The reverse was true when the yeast was grown onn-hexadecane. The broth contained more free, petroleum ether-soluble fatty acids when the cellular lipid content was high than when it was low. Overnight starvation ofC. lipolytica grown onn-hexadecane in a carbon-free nutrient medium, removed the residual cell-bound hydrocarbon, increased the cell population by one half and decreased the cellular lipid content (as % of dry yeast) by one third. Various methods for the determination of lipids, described as appropriate for yeasts were compared. The highest yields were obtained by extraction of the freeze-dried paste, at room temperature, with a 1:1 chloroform-methanol mixture.     

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.     

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.     

Protein-like Activator for n-Alkane Oxidation by Pseudomonas aeruginosa S7B1

During the study of the n-alkane oxidation by Pseudomonas aeruginosa S₇B₁, a nondialysable activator for n-alkane oxidation was discovered in the culture broth of the strain. The activator was purified, as judged by cellulose acetate membrane electrophoresis, by ammonium sulfate precipitation and chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-75 columns.

The purified activator, which was positive in protein color reactions, remarkable stimulated only the oxidation of n-hexadecane, though it was not observed in case of palmitic acid or glucose oxidation.

Co-operative action between the activator and rhamnolipid, which had been isolated as a growth stimulant of P. aeruginosa S₇B₁ on n-hexadecane by the authors, was observed not only in the oxidation of n-hexadecane but also in the growth on n-hexadecane.     

Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats

Cell growth, lipid accumulation and cellular lipid composition of Yarrowia lipolytica growing on mixtures of industrial fats containing stearic, oleic, linoleic and palmitic acid have been studied. During growth, the strain incorporated oleic and linoleic acids more rapidly than the saturated fatty acids. Relatively high lipid accumulation (up to 0.44 g of lipids per g of dry matter) was observed when stearic acid was included in the culture medium. In contrast, substrates rich in oleic acid did not favor cellular lipid accumulation. The accumulated lipids, mainly composed of triacylglycerols (45-55% w/w), demonstrated a different total fatty acid composition compared with that of the substrate; in all cases, the microorganism showed the unusual capacity to increase its cellular stearic acid level, even if this fatty acid was not found in high concentrations in the substrate. This permitted the synthesis of interesting lipid profiles with high percentages of stearic acid and non-negligible percentages of palmitic and oleic acid, with a composition resembling that of cocoa-butter.     

Sucrose Lipids of Arthrobacteria,Corynebacteria and Nocardia Grown on Sucrose

Arthrobacter paraffineus KY 4303, when grown on sucrose as the sole carbon source, produced novel glycolipids, either of which was different from trehalose lipid produced from n-alkane by the same microorganism. Two kinds of glycolipids were isolated by chromatography on silicic acid columns. Major components of these lipids were sucrose and α-branched β-hydroxy fatty acid. One of the lipid (SL–1, having high polarity) was identified as 6-O-monofattyacyl glucosly-β-fructoside. Another (SL–2, having low polarity) was partly characterized as sucrose ester of at least two moles of the fatty acid.

Formation of sucrose lipids was also demonstrated in sucrose-grown cells of several microorganisms of Corynebacteria, Nocardia and Brevibacteria, which were isolated as hydrocarbon-utilizing bacteria and could produce a considerable amount of trehalose lipid from n-alkane.     

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.     

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.     

Production of Dicarboxylic Acids by Candida cloacae Mutant Unable to Assimilate n-Alkane

Strain MR-12 which was derived from Candida cloacae M-l as a mutant unable to assimilate n-alkane showed marked increase in dicarboxylic acid (DC) productivity from n-alkane.

Resting cells of strain MR-12 produced 42.7g/liter of n-tetradecane 1,14-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) after 72 hr’ incubation. DC degradation activities of strain M-1 and MR-12 were found to be markedly reduced and their activities against DC-16 decreased to 40% and 10% of that of the parent strain, respectively.

Strain M-1 and MR-12 produced DC from the various oxidized derivatives of n-alkane such as alcohol, diol, aldehyde, fatty acid and methyl- or ethylester of fatty acid other than n-alkane.

The carbon balance in n-C₁₆ oxidation was determined by using resting cells of strain MR-12 and about 60% of utilized carbon was recovered as DC-16 and about 40% was recovered as CO₂.     

Subcellular localization of long-chain alcohol dehydrogenase and aldehyde dehydrogenase in n-alkane-grown Candida tropicalis

Long-chain alcohol dehydrogenase and longchain aldehyde dehydrogenase were induced in the cells of Candida tropicalis grown on n-alkanes. Subcellular localization of these dehydrogenases, together with that of acyl-CoA synthetase and glycerol-3-phosphate acyltransferase, was studied in terms of the metabolism of fatty acids derived from n-alkane substrates. Both longchain alcohol and aldehyde dehydrogenases distributed in the fractions of microsomes, mitochondria and peroxisomes obtained from the alkane-grown cells of C. tropicalis. Acyl-CoA synthetase was also located in these three fractions. Glycerol-3-phosphate acyltransferase was found in microsomes and mitochondria, in contrast to fatty acid -oxidation system localized exclusively in peroxisomes. Similar results of the enzyme localization were also obtained with C. lipolytica grown on n-alkanes. These results suggest strongly that microsomal and mitochondrial dehydrogenases provide long-chain fatty acids to be utilized for lipid synthesis, whereas those in peroxisomes supply fatty acids to be degraded via -oxidation to yield energy and cell constituents.     

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.     

The inducible microsomal fatty alcohol oxidase of Candida (Torulopsis) apicola

In the transition phase of Candida apicola IMET 43747 from logarithmic to stationary growth a pyridine-nucleotide-independent alcohol oxidase was induced coinciding with the beginning of sophorose lipid production. This enzyme was not repressed by glucose and was measurable in stationary cells grown on glucose or on a mixture of n-hexadecane and glucose. An NAD⁺-dependent aldehyde dehydrogenase behaved in the same way. Both enzymes were localized in the microsomal fraction. The alcohol oxidase accepted long-chain (fatty) aliphatic alcohols (C₈ to at least C₁₆) and diols starting from decanediol. Trace activities were found with -hydroxy fatty acids. Aromatic, secondary and tertiary alcohols were not oxidized. In the stationary growth phase the substrate specificity of the alcohol oxidase tends to be changed to more hydrophobic substrates. The physiological role of both enzymes, the alcohol oxidase and aldehyde dehydrogenase, is discussed including their possible involvement in the synthesis of sophorose lipid. Correspondence to: R. K. Hommel     

The lipid compositions of high-Antarctic notothenioid fish species with different life strategies

Lipid and fatty acid compositions of five notothenioid fishes from the Antarctic Weddell and Lazarev Seas were investigated in detail with regard to their different modes of life. The pelagic Aethotaxis mitopteryx was the lipid-richest species (mean of 61.4% of dry mass, DM) followed by Pleuragramma antarcticum (37.7%DM). The benthopelagic Trematomus lepidorhinus had an intermediate lipid content of 23.2%DM. The benthic Bathydraco marri (20.8%DM) and Dolloidraco longedorsalis (14.5%DM) belonged to the lipid-poorer species. Triacylglycerols were the major lipid class in all species. Important fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:1(n-7), 20:5(n-3) and 22:6(n-3). The enhanced proportions of the long-chain monounsaturated fatty acids, 20:1 and 22:1, in the lipid-rich pelagic fishes clearly reflected the ingestion of the two copepod species, Calanoides acutus and Calanus propinquus, which are the only known Antarctic zooplankters rich in these fatty acids. Although wax esters are the major storage lipid in many prey species, they were absent in all notothenioid fishes studied. Thus, wax esters ingested with prey are probably converted to triacylglycerols via fatty acids or metabolised by the fishes. The enhanced lipid accumulation with increasingly pelagic lifestyle has energetic advantages, especially with regard to improved buoyancy. It is still unknown to what extent these lipids are utilised as energy reserves, since it has been suggested that not only the benthic but also the pelagic Antarctic fishes are rather sluggish, with a low scope for activity and hence low metabolic requirements. Accepted: 14 May 2000     

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the β-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized β-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation. Received: 22 December 1995 / Accepted: 12 March 1996     

Role of polymer complexes in the formation of biofilms by corrosive bacteria on steel surfaces

The composition of exopolymer complexes (EPCs), synthesized by the monocultures Desulfovibrio sp. 10, Bacillus subtilis 36, and Pseudomonas aeruginosa 27 and by microbial associations involved in the corrosion of metal surfaces has been studied. An analysis of the monosaccharide composition of carbohydrate components, as well as the fatty acid composition of the lipid part of EPCs, was carried out by gasliquid chromatography (GLC). It was found that bacteria in biofilms synthesized polymers; this process was dominated by glucose, while the growth of bacteria in a suspension was marked by a high rhamnose content. Hexouronic acids and hexosamine have been revealed as a part of B. subtilis 36 and P. aeruginosa 27 EPCs. Qualitative differences were revealed in the fatty acid composition of exopolymers in biofilms and in a bacterial suspension. It was shown that the transition to a biofilm form of growth led to an increase in the unsaturation degree of fatty acids in the exopolymers of associative cultures. The results can be used to develop methods to control microbial corrosion of metal surfaces.     

Anaerobic n-Alkane Metabolism by a Sulfate-Reducing Bacterium, Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803^T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight