Changes in Cellular Fatty Acid Composition of Cephalosporium acremonium during Cephalosporin C Production

Cephalosporium acremonium was cultivated in fermentation medium containing sucrose or methyl oleate as a carbon source for cephalosporin C production. The level of antibiotic production was 48 g of cephalosporin C per liter under optimum conditions when methyl oleate was used. The C_18:1 (oleic acid) methyl ester appeared to be utilized faster than the C_18:2 (linoleic acid) methyl ester in fermentation broth. Physiological characteristics of C. acremonium were investigated by determining the fatty acid composition of the total cellular free lipid. Significant changes in cellular fatty acid composition occurred during inoculum cultivation and fermentation. The percentage of C_18:1 increased from 19.1 to 38.5%, but the percentage of C_18:2 decreased from 56.7 to 36.1%, and there was an increase in pH during inoculum cultivation. The cellular fatty acid composition of C. acremonium grown in fermentation medium containing methyl oleate (methyl oleate medium) was significantly different from that in fermentation medium containing sucrose (sucrose medium). The major fatty acids detected were C_16:0 (palmitic acid), C_18:1, and C_18:2. In methyl oleate medium, the ratio of C_18:1 to C_18:2 increased from 0.34 to 1.37, while the cell morphology changed from hyphae to arthrospores and conidia. In contrast, in sucrose medium, the ratio of C_18:1 to C_18:2 decreased from 0.70 to 0.43, and most of the cells remained hyphal at the end of fermentation. We observed that hyphae contained a higher proportion of C_18:2 but arthrospores and conidia contained a higher proportion of C_18:1.     

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.     

Microbial fatty acid specificity

Strains ofRhodotorula sp.,Candida spp. andLangermania sp. cultivated on polyunsaturated oil preferentially incorporated more unsaturated fatty acids. These fatty acids were used mainly for growth needs whereas the saturated ones accumulated in the microbial cell. The cellular oil and the remaining oil in the culture had a lower degree of unsaturation as compared to the initial oil, and a modified fatty acid composition.Candida lipolytica, in a chemostat continuous culture, incorporated C₁₈ fatty acids in the order of C_18:3>C_18:2>C_18:1>C_18:0, and accumulated mostly the saturated ones. The specific productivity of the cellular oil and of the oil remaining in the culture medium was 0.036 and 0.487 gg⁻¹ h⁻¹, respectively, at dilution rateD=0.2/h.     

Optimization of feed conditions in a 2.5-l fed-batch culture using rice oil to improve cephalosporin C production by <Emphasis Type="Italic">Cephalosporium acremonium</Emphasis> M25

Summary Rice oil significantly affected cephalosporin C production in a 2.5-l bioreactor culture of Cephalosporium acremonium M25. To improve cephalosporin C production, the feed conditions of rice oil were optimized. Reducing the feed rate of rice oil improved cephalosporin C production to 1.01 g/l when the consumption rate of rice oil decreased. Overall, under optimal feed conditions in the 2.5-l fed-batch culture, cephalosporin C production increased about four times compared to before optimization.     

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.     

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.     

Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C₁₂–C₁₈ fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the ξ-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.     

Saponified palm kernel oil and its major free fatty acids as carbon substrates for the production of polyhydroxyalkanoates in Pseudomonas putida PGA1

The synthesis of polyhydroxyalkanoates (PHA) by Pseudomonas putida PGA1, using saponified palm kernel oil (SPKO), was investigated. The PHA produced from SPKO was compared with those produced by the major free fatty acids found in the palm kernel oil. Owing to the absence of lipase activity in P.␣putida, palm kernel oil did not support cell growth. However, SPKO could support cell growth and produced relatively high yield of both dry cells and PHA. The polyester produced was similar in properties to those derived from lauric (C_12:0) and myristic (C_14:0) acids, while oleic acid (C_18:1) gave rise to PHA that was sticky and of broader molecular mass distribution. Nuclear magnetic resonance and gas chromatography showed that these PHA were copolymers consisting mainly of n-alkanoate monomers ranging from C₆ to C₁₄, with C₈ as the predominant component. PHA derived from SPKO and oleic acid also contained a small amount of unsaturated monomers. Received: 25 March 1996 / Received last revision: 30 September 1996 / Accepted: 18 October 1996     

Transformation of vegetable oils by an oleaginous yeast:Rhodotorula glutinis IIP-30

Summary A locally isolated oleaginous yeastRhodotorula glutinis IIP-30 was grown on vegetable oils obtained from coconut, ground nut and til. The fatty acid composition of yeast oil was quite similar to that of the substrate oil in case of ground nut and til, while it was different with coconut oil. Utilization of C₁₂ and C₁₄ fatty acids of coconut oil to yield higher proportions of C₁₈₁ and C₁₈₂ fatty acids was observed.     

Medium-Chain-Length Poly(β-Hydroxyalkanoate) Synthesis from Triacylglycerols by Pseudomonas saccharophila

Pseudomonas saccharophila NRRL B-628 is capable of utilizing agricultural lipids for growth. The organism exhibited good growth with triacylglycerol substrates that contained saturated fatty acyl moieties such as coconut oil (CO; C_10–12 fatty acids) and tallow (T; C_16–18 fatty acids). Electron micrographs of the triacylglycerol-grown cells showed the presence of intracellular granules indicative of poly(β-hydroxyalkanoate) (PHA) production. Cells grown in a 250-ml CO-containing medium produced ca. 0.2 g of medium-chain-length (mcl)-PHA. Gas chromatographic analysis showed that β-hydroxyoctanoic acid (30%), β-hydroxydecanoic acid (40%), and β-hydroxydodecanoic acid (16%) were the major monomer repeat-units of the CO-derived polymer. The estimated mean molecular mass of the CO-derived mcl-PHA as determined by gel permeation chromatography was 13.1 × 10⁴ g/mol with a polydispersity of 3.16. Received: 15 August 1998 / Accepted: 25 September 1998     

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.     

Fatty acid composition as an indicator of physiological condition of the cyanobacterium Microcystis aeruginosa

The present study examined the fatty acid composition of Microcystis aeruginosa grown in a batch culture and that of Microcystis-dominated plankton collected in an experimental enclosure in a shallow, eutrophic embayment of Lake Biwa (Akanoi Bay). In pure culture, we detected 16 : 0, 18 : 2ω6, 18 : 3ω3, 18 : 3ω6, and 18 : 4ω3 acids as major fatty acids of M. aeruginosa, with trace amounts of C₂₀ polyunsaturated fatty acids. In both pure culture and the field enclosure, the ratio of total fatty acid weight to dry weight decreased with decreasing availability of dissolved inorganic nitrogen. The ω3/ω6 ratios of C₁₈ polyunsaturated fatty acids [(18 : 3ω3 + 18 : 4ω3)/(18 : 2ω6 + 18 : 3ω6)] varied greatly (range, 2–5) in response to the changes in physical and chemical conditions for Microcystis growth. Most notably, the ω3/ω6 fatty acid ratios were significantly positively correlated with the growth rate of cells in a batch culture. We suggest that the fatty acid composition is a useful indicator of the physiological state of Microcystis in freshwater lakes. Received: March 2, 2001/ Accepted: December 19, 2001     

Endogenous Inhibitors for Pollen Germination and Pollen Tube Elongation in Pinus densiflora SIEB,et Zucc.

Sulfur metabolism in Cephalosporium acremonium was investigated using a mutant, 8650⁺/ OAH^?/SeMe^R, which could not convert cysteine or inorganic sulfur to methionine. The production of cephalosporin by the mutant depended on the amount of S-sulfocysteine in a chemically defined medium supplemented with a low level of methionine sufficient to support optimal growth. S-Sulfocysteine was detected in an extract of cells grown in the presence of sodium thiosulfate and l-serine. Furthermore, an NADPH-linked reduction of S-sulfocysteine to cysteine was demonstrated in a cell-free extract. These facts suggest that S-sulfocysteine is a direct precursor in cysteine biosynthesis in C. acremonium and an alternative pathway involving the compound is one of the most important ones in cephalosporin C production by this fungus.     

Oleyl Oleate and Homologous Wax Esters Synthesized Coordinately from Oleic Acid by Acinetobacter and Coryneform Strains

Newly isolated Acinetobacter (NRRL B-14920, B-14921, B-14923) and coryneform (NRRL B-14922) strains accumulated oleyl oleate and homologous liquid wax esters (C_30:2–C_36:2) in culture broths. Diunsaturated oleyl oleate preponderated in 75 mg liquid wax esters (280 mg lipid extract) recovered from 100-ml cultures of Acinetobacter B-14920 supplemented with 810 mg oleic acid–oleyl alcohol. With soybean oil instead of oleic acid, wax esters (260 mg) were increased to approximately 50% of the lipid extract. Production of wax esters by cultures supplemented with combined fatty (C₈–C₁₈) alcohols and acids suggests a coordinated synthesis whereby the exogenous alcohol remains unaltered, and the fatty acid is partially oxidized with removal of C₂ units before esterification. Consequently, C₈–C₁₈ primary alcohols control chain lengths of the wax esters. Exogenous fatty acids are presumed to enter an intracellular oxidation pool from which is produced a homologous series of liquid wax esters.     

Effects of dietary lead,fish oil,and ethoxyquin on hepatic fatty acid composition in chicks

Three experiments were conducted with day-old broiler chicks reared to 18 or 19 d of age. The objective was to examine the effects of dietary oil (cottonseed oil vs fish oil), dietary antioxidant (0 vs 75 ppm ethoxyquin), and dietary lead (0 vs 1000 ppm Pb as lead acetate trihydrate) on hepatic fatty acid composition. A 2×2 factorial arrangement was used in all experiments. In Experiment 1, the factors were oil (4% of each) and Pb. In Experiments 2 and 3, the factors were ethoxyquin and Pb in diets containing 3.5% cottonseed oil (Experiment 2) or 3.5% fish oil (Experiment 3). Hepatic fatty acid profiles were measured by gas-liquid chromatography in 10 chicks/treatment (Experiment 1) or 4 chicks/treatment (Experiments 2 and 3). Dietary oils altered the profiles, with cottonseed oil producing the higher values for linoleic acid (18∶2) and arachidonic acid (20∶4). With fish oil, in addition to the lower levels of 18∶2 and 20∶4, there were significant levels of eicosapentaenoic acid (20∶5) and docosahexaenoic acid (22∶6). Pb enhanced the levels of 20∶4, but the effect was greater with cottonseed oil diets compared with fish oil diets. The enhanced 20∶4 levels resulted in lower ratios of 18∶2/20∶4. Ethoxyquin enhanced the level of 18∶2 with the cottonseed oil diet, and of 20∶5 and 22∶6 with the fish oil diet. Ethoxyquin decreased the level of hepatic 20∶4 when fish oil was fed. The results clearly show that all three factors (oil type, Pb level, and ethoxyquin level) after hepatic fatty acid composition. Both oil source and Pb level appeared to exert an effect on the metabolic conversion of 18∶2 and 20∶4. The primary effect of ethoxyquin was to enhance the levels of polyunsaturated fatty acids in liver. The data do not allow the partitioning of possible ethoxyquin effects to protection of polyunsaturated acids in feed vs protection of polyunsaturated acids in liver tissue. Use of trade names implies neither approval by the North Carolina Agricultural Research Service of products named nor criticism of products not named.     

Fatty acids reduce the tensile strength of fungal hyphae during cephalosporin C production in Acremonium chrysogenum

Fragmentation rate constants, which can be used to estimate the tensile strength of fungal hyphae, were used to elucidate relationships between morphological changes and addition of fatty acids during cephalosporin C production in Acremonium chrysogenum M35. The number of arthrospores increased gradually during fermentation, and, in particular, was higher in the presence of rice oil, oleic acid or linoleic acid than in their absence. Because supplementation of rice oil or fatty acids increased cephalosporin C, we concluded that differentiation to arthrospores is related to cephalosporin C production. To estimate the relative tensile strengths of fungal hyphae, fragmentation rate constants (k _frag) were measured. When rice oil, oleic acid, or linoleic acid were added into medium, fragmentation rate constants were higher than for the control, and hyphal tensile strengths reduced. The relative tensile strength of fungal hyphae, however was not constant presumably due to differences in physiological state.     

Toxigenic fungi and the deterioration of Nigerian poultry feeds

Growth of toxigenic strains of Aspergillus clavatus Des. and Aspergillus flavus Link at 30°C on milled poultry feeds led to a considerable decrease in the protein, oil and crude fibre contents of the feed substrate. A corresponding increase in the free fatty acid fractions of the feeds due to the activities of these microbes was also recorded. Rapid degradation of the feedstuff by both species was recorded at a temperature of 25°C and 30°C and a pH range of 4.8–6.4. When grown on feed infusion broth at 30°C, the highest amounts of mycelial production with sporulation of both fungal species occurred within the 8-day incubation period. A determination of their extra-cellular enzyme profile showed the production of amylases, pectate lyase, cellulases, proteases, lipases, xyalanases, DNase and RNase.All the carbon and nitrogen sources used (except L-sorbose and DL-tryptophan), supported good mycelial growth with sporulation. An optimal CN ratio of 5.04.5 and 7.53.0 was recorded for growth and sporulation of A. clavatus. For A. flavus, a CN ratio of 7.54.5 was found best for growth and 5.03.0 for sporulation.     

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.     

Media Formulation using Complex Organic Nutrients for Improved Activity,Productivity, and Yield of Candida rugosa Lipase and Esterase Enzymes

Abstract

Candida rugosa is an excellent source of multiple lipase and esterase enzymes; therefore, it is of technological importance to formulate the medium that provides high activity for each enzyme. In this work, the cultivation medium comprising complex nutrients that provided the highest activity, productivity, and yield of C. rugosa enzymes individually was formulated. Time courses of the extracellular and intracellular lipase and esterase activities of C. rugosa were represented and the role of protease in the cultivation progress was discussed. Urea, soy-peptone, yeast extract, a mixture of soy-peptone and yeast extract, cheese whey, and wheat mill bran were tested for their lipolytic and esterasic activities. Urea provided considerably higher extracellular lipase activity when compared to other nitrogen sources; however, soy-peptone provided the highest extracellular esterase activity. Hazelnut, olive, sesame, soybean, and flax seed oils affected the enzyme activities to different extents related to their fatty acid compositions. Hazelnut oil and olive oil provided the highest extracellular lipase and esterase activities, respectively, whereas sesame oil produced the highest biomass. High C₁₈ and C₁₆ ester contents of vegetable oils promoted high lipase and esterase productions, respectively. A temperature of 30°C yielded the highest extracellular and intracellular lipase and esterase activities; however, 35°C produced the highest biomass.     

The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum

Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed. Received: 13 October 1998 / Revised revision: 14 January 1999 / Accepted: 22 January 1999