Iso-alkane oxidation by aPseudomonas Part I.—Metabolism of 2-methylhexane

Summary Pseudomonas cells adapted to 2-methylhexane oxidize 5-methylhexanoic andiso-valeric acids rapidly. The oxidation rates of 2-methylhexanoic and propionic acids are appreciably lower. It can be concluded that primary attack on the C₆ atom is distinctly favoured over that on C₁. By use of gas-liquid chromatography, 2-methylhexanoic, 5-methylhexanoic andiso-valeric acids were shown to be formed when heptane-grown cells were incubated with 2-methylhexane. When 2-methylhexane-grown cells were used the amount of 5-methylhexanoic acid decreased compared with the amount of the 2-methyl isomer. Moreover,iso-valeric acid could not be detected. The results make it probable that degradation of 2-methylhexanevia C₆, comprising 5-methylhexanoic andiso-valeric acid, is accompanied by a second pathway via 2-methylhexanoic acid. The latter pathway is of minor importance, in particular in 2-methylhexane-adaptedPseudomonas cells.     

The effect of acetic acid concentration on the growth and production of gamma-linolenic acid byMucor circinelloides CBS 203.28 in fed-batch culture

The effect of different initial acetic acid concentrations on the growth of and lipid and gamma-linolenic acid (GLA) production byMucor circinelloides CBS 203.28 was determined in a 14 litre stirred tank reactor operated in a fedbatch, pH-stat mode with acetic acid as carbon source and pH titrant. Increased acetic acid concentrations in the culture resulted in a significant increase in the crude oil content of the biomass. By contrast, all the other parameters such as the biomass concentration, GLA and oil yield on acetic acid, the GLA content of the biomass and oil, the growth rate and volumetric rate of GLA production decreased with an increase in acetic acid concentration. The best results were obtained with acetic acid at 2 g/1, which gave 39.8 mg GLA/g biomass and 15.6% GLA in the neutral lipid fraction, amounting to 340 mg GLA/1 culture. A decrease in the glyco- and phospho-lipid fractions during the cultivation coincided with an increase in the neutral lipid fraction. The GLA content of the biomass remained within rather narrow limits of 3.5% to 4% of the biomass, irrespective of the oil content of the biomass. The fatty acid profile was not greatly affected by the acetic acid concentration. The hyphae of the fungus were characterized by the accumulation of large intracellular oil droplets and some septa delimited the hyphae.     

Mucor—a source of cocoa butter and gamma-linolenic acid

Lipid analyses were performed on 28 strains of various species of the genus Mucor. In shake flasks with glucose as carbon source, the gamma-linolenic acid (GLA) content in the neutral lipid (NL) fraction of some Mucor species was up to 38 mg GLA/g dry biomass. Some Mucor species produced more than 20% (w/w) stearic acid (18:0) and more than 60% of their NL content as symmetrical triacylglycerols (SUS-TAGs) which corresponded to those of cocoa butter. Three Mucor species were evaluated in terms of the production of SUS-TAGs and GLA in pH-stat, fed-batch cultures in an air-lift fermenter with acetic acid as titrant and carbon source. Mucor circinelloides f. circinelloides CBS 108.16 accumulated 27% 18:0 in the NL fraction, which constituted approximately 40% of the dry biomass. In this case, the NL fraction contained more than 70% (w/w) SUS-TAGs.M.P. Roux, J.L.F. Kock, A. Botha, J.C. du Preez and P.J. Botes are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa; G.V. Wells is with Sasol Waxes, P.O. Box 1, Sasolburg, South Africa.     

Branched chain amino acids as source of specific branched chain volatile fatty acids during the fermentation process of fish sauce

Summary.  The source of the formation of branched chain volatile fatty acids (VFA) in fish sauce was investigated. Certain branched VFA were derived from the degradation of specific amino acids as iso-butyric acid from valine and iso-valeric acid from leucine. Short and long straight chain VFA were significantly higher in the linoleic acid added sample than in the control but did not significantly bring changes to the branched chain VFA. It is suggested that straight chain VFA developed from fish fats. Alanine and isoleucine did not have a clear influence on the production of volatile fatty acids. Received November 23, 2001 Accepted June 20, 2002 Published online December 18, 2002 RID="*" ID="*" Part of this paper was presented in the 7th International Congress on Amino Acids and Proteins in Vienna, Austria from August 6–10, 2001. Authors' address: Norlita G. Sanceda, Ph.D., Institute of Environmental Science for Human Life, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan, Fax: + 81-3-5978-5805, E-mail: lita@cc.ocha.ac.jp     

Biodegradation and conversion of alkanes and crude oil by a marine Rhodococcus

A hydrocarbon degrader isolated from a chronically oil-polluted marine site was identified as Rhodococcus sp. on the basis of morphology, fatty acid methyl ester pattern, cell wall analysis, biochemical tests and G + C content of DNA. It degraded upto 50% of the aliphatic fraction of Assam crude oil, in seawater supplemented with 35 mM nitrogen as urea and 0.1 mM phosphorus as dipotassium hydrogen orthophosphate, after 72 h at 30 ° and 150 revolutions per minute. The relative percentage of intracellular fatty acid was higher in hydrocarbon-grown cells compared to fructose-grown cells. The fatty acids C₁₆ , C16_{16 :1} C₁₈ and C_{18 : 1} were constitutively present regardless of the growth substrate. In addition to these constitutive acids, other intracellular fatty acids varied in correlation to the hydrocarbon chain length supplied as a substrate. When grown on odd carbon number alkanes, the isolate released only monocarboxylic acids into the growth medium. On even carbon number alkanes only dicarboxylic acids were produced.     

Effects of Dietary Non-Digestible Oligosaccharides on Microbial Characteristics of Ileal Chyme and Faeces in Weaner Pigs

Fructooligosaccharides (FOS) and transgalactooligosaccharides (TOS), which are non-digestible oligosaccharides (NDO), were included at 10 and 40g/kg in an NDO-free control diet at the expense of purified cellulose. Each of the 5 diets was fed to 4 weaner pigs and microbial characteristics of their ileal chyme and faeces were assessed. The NDO-pigs had lower ileal pH than the control pigs. Dietary NDO did not affect the ileal volatile fatty acid concentration, though FOS-pigs had a higher concentration of lactic acid and relatively more iso-valeric acid and less acetic acid than TOS-pigs. The NDO-pigs had lower ileal aerobic bacterial counts than the control pigs, whilst the FOS-pigs had a larger ileal anaerobic bacterial counts than the TOS-pigs. The NDO-pigs had an higher faecal pH and their faecal volatile fatty acid pool contained relatively more iso-butyric acid and iso-valeric acid than the control pigs. The TOS-pigs tended to have higher faecal anaerobic bacterial counts and had a smaller concentration of faecal volatile fatty acid than the FOS-pigs. We concluded that whilst effects at the faecal level may have been partly due to a reduced intake of cellulose, dietary NDO can exert precaecal prebiotic effects in weaner pigs.     

Pathways of hydrocarbon dissimilation by aPseudomonas as revealed by chloramphenicol

Pseudomonas aeruginosa cells pre-grown on a particular hydrocarbon will oxidize other hydrocarbons as well. Degradation of these hydrocarbons proceeds to a point — depending on their structure — where new enzymes are needed for further degradation. Lack of these enzymes causes accumulation of products. However, secondary adaptation processes tend to decrease yields of intermediates, in particular when adaptation rates are high.By inhibiting these secondary adaptation processes with chloramphenicol (CAM), the amounts of various intermediates could be increased.Propionic acid was accumulated from heptane by hexane-grown cells in yields up to 60% (molar) calculated on heptane converted. The effect of CAM suggests that propionic acid is not subject to -oxidation (acrylate pathway) but is degraded via methylmalonate by adaptive enzymes.2-Methylhexane was converted for 30–40% (molar) intoiso-valeric acid by heptane-grown cells. A known pathway ofiso-valeric acid oxidation incorporates a carbon dioxide fixation step, and lack of this enzyme in heptane-grown cells probably causesiso-valeric acid accumulation.Heptene-1 incubation with heptane-grown cells resulted in a 30–40% conversion into 4-pentenoic plus 2, 4-pentadienoic acids. 6-Heptenoic acid was detected occasionally. A predominant attack at C₇ of the heptene-1 molecule is indicated at least for heptane-grown cells. Attack on the saturated end of the molecule seems well in line with the assumption that alkane oxidation by these bacteria is effected by oxygen transferring enzymes operating on a methyl group, as opposed to the action of a dehydrogenase and formation of an -olefin as the intermediate.     

Specificity effects in the biosynthesis of fatty acids in Bacillus acidocaldarius

Addition to Bacillus acidocaldarius of acids which can act as primers for fatty acid synthesis promote the synthesis of corresponding fatty acids competitively. The effective acids are n?C₅ to -?₇ (not C₄ or C₈), iso- and anteiso-C, and ?C, (not C4), and a range of cyclic acids from cyclobutylacetic and cyclopentanecarboxylic to cycloheptylacetic. New non-natural ω-cyclobutyl-, ω-cyclopentyl-, and ω-cycloheptyl-fatty acids are obtainable. The range of acceptable primers and the range of fatty acids produced therefrom indicate, respectively, the substrate specificities of the transacylase which introduces acyl species into fatty acids synthesis and the one which removes them. The specificity of the primer transacylase may be similar to that in some rumen anaerobes.     

Branched-chain amino acid metabolism controls membrane phospholipid structure in Staphylococcus aureus

Branched-chain amino acids (primarily isoleucine) are important regulators of virulence and are converted to precursor molecules used to initiate fatty acid synthesis in Staphylococcus aureus. Defining how bacteria control their membrane phospholipid composition is key to understanding their adaptation to different environments. Here, we used mass tracing experiments to show that extracellular isoleucine is preferentially metabolized by the branched-chain ketoacid dehydrogenase complex, in contrast to valine, which is not efficiently converted to isobutyryl-CoA. This selectivity creates a ratio of anteiso:iso C₅-CoAs that matches the anteiso:iso ratio in membrane phospholipids, indicating indiscriminate utilization of these precursors by the initiation condensing enzyme FabH. Lipidomics analysis showed that removal of isoleucine and leucine from the medium led to the replacement of phospholipid molecular species containing anteiso/iso 17- and 19-carbon fatty acids with 18- and 20-carbon straight-chain fatty acids. This compositional change is driven by an increase in the acetyl-CoA:C₅-CoA ratio, enhancing the utilization of acetyl-CoA by FabH. The acyl carrier protein (ACP) pool normally consists of odd carbon acyl-ACP intermediates, but when branched-chain amino acids are absent from the environment, there was a large increase in even carbon acyl-ACP pathway intermediates. The high substrate selectivity of PlsC ensures that, in the presence or the absence of extracellular Ile/Leu, the 2-position is occupied by a branched-chain 15-carbon fatty acid. These metabolomic measurements show how the metabolism of isoleucine and leucine, rather than the selectivity of FabH, control the structure of membrane phospholipids.     

Changes in free fatty acids of awamori during aging

The fatty acid components of awamori during aging were as follows. The total amount of volatile acids calculated as acetic acid ranged from 20 to 140 mg/l, the main acid was acetic acid, and the proportion of acetic acid to total acids ranged from 35 to 80 per cent. The main acids other than acetic acid were propionic acid and i-butyic acid. Differences were observed in fatty acid constituents between awamori and other alcoholic beverages.Certain components tended to increase during maturation in kame (porous earth-enware pots): acetic acid, i-butyric acid, i-valeric acid, valeric acid, capric acid, lauric acid, myristic acid and total fatty acids. Others, however, showed no distinct changes: propionic acid, butyric acid, caproic acid, caprylic acid, palmitic acid, stearic acid, oleic acid and linoleic acid.During maturation in non-porous containers (stainless-steel or glass-linked tanks), on the other hand, caprylic acid, capric acid, lauric acid and myristic acid components tended to increase, while no distinct changes however were shown by acetic acid, propionic acid, i-butyric, butyric acid, i-valeric acid, valeric acid, caproic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and total fatty acids.     

Production of gamma-linolenic acid by Mucor circinelloides and Mucor rouxii with acetic acid as carbon substrate

Summary The production of gamma-linolenic acid (GLA) by Mucor circinelloides CBS 203.28 and M. rouxii CBS 416.77 in fed-batch cultures operated in pH-stat mode with acetic acid as carbon substrate and titrant compared favourably with the performance of M. circinelloides in batch culture on glucose. On acetic acid M. circinelloides accumulated up to 39.8 mg GLA/g biomass, with a crude oil content of 28% containing 91% neutral lipids. The GLA content of the neutral lipid fraction was 15.6%.     

Cheese whey as a renewable substrate for microbial lipid and biomass production by Zygomycetes

Three Zygomycetes, Mortierella isabellina, Thamnidium elegans and Mucor sp., were tested for their ability of producing biomass and lipid‐containing γ‐linolenic acid (GLA) during their cultivation on cheese whey. M. isabellina consumed all of the available lactose and a significant amount of the available protein. On the contrary, the two other fungi seemed incapable of consuming lactose after protein exhaustion. In the second series of experiments, for M. isabellina a supplementary quantity of lactose was added into the medium in order to increase the C/N ratio and hence to increase the production of fat. In the case of T. elegans and Mucor sp., a supplementary quantity of ammonium sulfate was added in order to favor the consumption of lactose and the production of biomass. Indeed, enhancement of lipid production was observed for M. isabellina and biomass production for T. elegans and Mucor sp.. Fatty acid analysis of the microbial lipid showed a composition that presented non‐negligible changes in relation with the age of the culture and the C/N molar ratio of the medium. Further analysis of the fat showed that the quantity of neutral lipids was the more abundant. The fatty acid composition of neutral lipids resembled to that of total lipids. Phospholipids were the more unsaturated fraction for Mucor sp. and M. isabellina. GLA was synthesized in all trials but its concentration presented differences related with the utilized strains and the fermentation time. Growth of M. isabellina on lactose‐supplemented whey resulted in a maximum GLA production of 301 mg/L.     

FATTY ACIDS AND HYDROXY FATTY ACIDS IN THREE SPECIES OF FRESHWATER EUSTIGMATOPHYTES

The monocarboxylic fatty acids and hydroxy fatty acids of three species of freshwater microalgae—Vischeria punctata Vischer, Vischeria helvetica (Vischer et Pascher) Taylor, and Eustigmatos vischeri (Hulbert) Taylor, all from the class Eustigmatophyceae— were examined. Each species displayed a very similar distribution of fatty acids, the most abundant of which were 20:5n-3, 16:0, and 16:1n-7; C₁₈ polyunsaturated fatty acids were minor components. These fatty acid distributions closely resemble those found in marine eustigmatophytes but are quite distinct from those found in most other algal classes. These microalgae also contain long-chain saturated and unsaturated monohydroxy fatty acids. Two distinct types of hydroxy fatty acids were found: a series of saturated α-hydroxy acids ranging from C₂₄ to C₃₀ with a shorter series of monounsaturated α-hydroxy acids ranging from C₂₆ to C₃₀ together with a series of saturated β-hydroxy acids ranging from C₂₆ to C₃₀. The latter have not previously been reported in either marine or freshwater microalgae, although C₃₀ to C₃₄ midchain (ω-18)-hydroxy fatty acids have been identified in hydrolyzed extracts from marine eustigmatophytes of the genus Nannochloropsis, and C₂₂ to C₂₆ saturated and monounsaturated α-hydroxy fatty acids have been found in three marine chlorophytes. These findings have provided a more complete picture of the lipid distributions within this little studied group of microalgae as well as a range of unusual compounds that might prove useful chemotaxonomic markers. The functions of the hydroxy fatty acids are not known, but a link to the formation of the lipid precursors of highly aliphatic biopolymers is suggested.     

Phospholipid and acid composition of Nocardia and nocardoid bacteria as criteria of classification

Phospholipid and acid composition of 5 strains of ‘true’ Nocardia and 4 strains of nocardoid bacteria have been studied. A great homogeneity was found in all the Nocardia species: phospholipids consist of cardiolipin, phosphatidyl ethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Streptomyces (Nocardia) mediterranei did not contain phosphatidylinositol and Oerskovia (Nocardia) turbata had no phosphatidyl ethanolamine. The fatty acid composition of these phospholipids was determined and was found different in Nocardia and nocardoid species. Nocardia were rich in straight chain fatty acids and tuberculostearic acid while the phospholipids of nocardoid bacteria contained greater amounts of branched fatty acids. The fatty acids from acetone soluble lipids consisted of hydroxy and non-hydroxy compounds. Hydroxy acids were found in Nocardia which contained nocardic acids: high MW β-hydroxy α-branched acids and in S. mediterranei which contained β-hydroxy acids with 15–17 carbon atoms. Non-hydroxy acids were essentially palmitic and tuberculostearic acids in Nocardia species while S. mediterranei and O. turbata contained great amounts of iso acids from C₁₄ to C₁₇. Phospholipid and acid composition are discussed as criteria of taxonomic classification of Nocardia and related Actinomycetes.     

Volatile acid production from threonine,valine, leucine and isoleucine by clostridia

The amounts of the volatile acids produced from thereonine, valine, leucine and isoleucine by growing cultures of clostridia have been measured. The species used were Clostridium sporogenes; C. caloritolerans; C. botulinum proteolytic type A; C. botulinum proteolytic type B; C. botulinum proteolytic type F; C. botulinum proteolytic type G; C. putrificum; C. difficile; C. ghoni; C. bifermentans; C. sordellii; C. mangenoti; C. cadaveris; C. lituseburense; C. propionicum; C. sticklandii; C. scatologenes; C. subterminale; C. putrefaciens; C. histolyticum; C. tetanomorphum; C. limosum; C. lentoputrescens; C. tetani; C. melanomenatum; C. cochlearium; C. sporospheroides. Most of the species tested gave increased yields of propionic acid when grown in the threonine medium; in addition, some species resembled C. propionicum and produced n-butyric acid when grown in this medium. C. histolyticum produced only acetic acid in the basal medium; all seven strains of this species produced more acetic acid when grown in the threonine medium than in the basal medium. Species which oxidize valine to iso-butyric acid also oxidize leucine to 3-methyl butyric acid and isoleucine to 2-methylbutyric acid. The iso-caproic fraction produced by some species is shown to be derived from leucine. The identitity of the branched-chain acids produced by C. sporogenes has been confirmed by gas liquid chromatography/mass spectrometry.Abbreviations GLC gas liquid chromatography - RCM reinforced clostridial medium - VFA volatile fatty acid     

Biosynthesis of acyclic methyl branched polyunsaturated hydrocarbons inPseudomonas maltophilia

Summary The hydrocarbon composition ofPseudomonas maltophilia was determined by gas chromatography-mass spectrometry. Mono-, di- and tri-unsaturated alkenes were identified with a predominance of polyunsaturated components. The carbon chains of the alkenes contained single methyl branches iniso andanteiso position and double methyl branches in theiso-iso andanteiso-anteiso configurations. The composition of the hydrocarbons from cells grown in synthetic media enriched with amino acids or volatile fatty acids demonstrated that the probable precursors incorporated into individual hydrocarbons were branched and normal fatty acid chains in the range from C₃ to C₁₆. The probable fatty acid precursors which were connected together to form the major triunsaturated hydrocarbon chains were two monounsaturated chains, whereas the major liunsaturated chains resulted from condensation of saturated and monounsaturated chains. The probable precursors for the major monounsaturated hydrocarbons were C₁₄ (C₁₅) and C₁₆ (C₁₅) fatty acids. The accumulation of hydrocarbons was not detected until the cells were in the late exponential phase of growth; the maximal levels were reached at the mid-stationary phase of growth.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Biosynthesis of gamma-linolenic acid and beta-carotene by Zygomycetes fungi

Due to increasing demand for natural sources of both polyunsaturated fatty acids (PUFAs) and beta-carotene, 28 Zygomycetes fungal soil isolates were screened for their potential to synthesize these biologically active compounds. Although all fungi produced C₁₈ PUFAs, only nine strains also formed beta-carotene. Although Actinomucor elegans CCF 3218 was the best producer of gamma-linolenic acid (GLA) (251 mg/L), Umbelopsis isabellina CCF 2412 was found to be the most valuable fungus because of the dual production of GLA (217 mg/L) and beta-carotene (40.7 mg/L). The calculated ratio of formed PUFAs provided new insight into activities of individual fatty acid desaturases involved in biosynthetic pathways for various types of PUFAs. The maximal activity of delta-9 desaturase was accompanied by high accumulation of storage lipids in fungal cells. On the other hand, maximal activity of delta-15 desaturase was found in strains synthesizing low amounts of oleic acid due to diminished delta-9 desaturase. Activities of delta-6 desaturase showed competition for fatty acids engaged in n3, n6, and n9 biosynthetic pathways. Such knowledge about fatty acid desaturase activities provides new challenges for the regulation of biotechnological production of PUFAs by Zygomycetes fungi.     

Attractants for the gravid Queensland fruit fly Dacus tryoni

The vapours of certain pure chemicals, typical of ripe fruits, elicited characteristic components of ovipositional behaviour from gravid Dacus tryoni (Froggat) in an olfactometer: the flies walked and flew upwind to the source of the vapour and then probed with their ovipositors. A range of alcohols, acids, ketones and esters having 2–6 carbon atoms were effective (1 and 10% of iso-butyric acid, n-butyric acid, methyl butyrate, ethyl butyrate, 2-butanone, ethyl lactate and ethyl acetate; and 10% concentrations of ethanol and 2-propanone). The most effective were 4–6 carbon acids, esters and ketones. Behavioural threshold for n-butyric acid vapour at 26°C was obtained from a 5×10^–3% dilution in paraffin oil; maximum fly response occurred at about 200 times this concentration. Low concentrations of the 15-carbon sesquiterpene, -farnesene, were also very effective, despite its lower volatility. These results suggest that at least three different types of alfactory sensory neurones are involved in the identification of fruit attractants by gravid D. tryoni.     

Lipids of the Green Alga BotryococcusCultured in a Batch Mode

The lipid fraction of the green alga Botryococcuscultured in a batch mode was found to contain polar lipids (more than 50% of the total lipids), di- and triacylglycerols, sterols and their esters, free fatty acids, and hydrocarbons. In aging culture, the content of polar lipids somewhat decreased and that of triacylglycerols increased by more than four times. The content of hydrocarbons in the algal biomass did not exceed 0.9% and depended little on the culture age. Intracellular lipids contained saturated and unsaturated (mono-, di-, and trienoic) fatty acids. The maximum content of C_{16 : 3}and -C_{18 : 3}fatty acids (up to 35% of the total fatty acids) was detected in the phase of active growth. The extracellular and intracellular lipids of the alga differed in the proportion of particular lipids and in the fatty acid pattern.