Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.     

Effect of different carbon sources on lipase production by Candida rugosa

Different carbon sources affecting growth and lipase production in Candida rugosa were studied by using batch cultures on defined medium. Carbohydrates and acids non-related to fats did not induce lipase production. The highest yields of enzyme were obtained with lipids or fatty acids as carbon sources. Tween 80 stimulated lipase biosynthesis and secretion outside the cell. Combinations of two types of substrates, carbohydrates and fatty acids, did not improve lipase production, and in some cases, their consumption was produced in a sequential pattern. Glucose presented a repressing effect on lipase production. Moreover, glucose was found to be effective in stimulating lipase secretion by cells with a high level of cell-bound lipase activity because of their previous growth in oleic acid.     

Exploring low-cost carbon sources for microbial lipids production by fed-batch cultivation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Volatile fatty acids (VFAs), acetic acid, acetates, and ethanol were used as carbon sources for the production of microbial lipids using Cryptococcus albidus in batch cultures. C. albidus utilized organic acids less than glucose in the production of lipids, resulting in a lipid yield coefficient on VFAs of 0.125 g/g. In a two-stage batch culture, the lipid content increased to 43.8% (w/w) when VFAs were used as the sole carbon source in the second stage, which was two times higher than that of the batch culture. Furthermore, a 192 h, two-stage fed-batch cultivation of C. albidus produced a dry cell weight, lipid concentration, and lipid content of 26.4 g/L, 14.5 g/L, and 55.1% (w/w), respectively. The fed-batch culture model used in this study featured pure VFA solutions, with intermittent feeding, under oxygen-enriched air supply conditions. This study investigated several alternative carbon sources to reduce the cost of microbial lipids production and proved the feasibility of using VFAs as the carbon source for the provision of a high lipid content and productivity.     

An Analysis of the Nutritional Requirements for Fatty Acids of Paramecium aurelia

SYNOPSIS. Axenically cultivated Paramecium aurelia , stock 299, required a fatty acid for growth. This need was satisfied by oleic acid and oleic acid-containing lipids. These included: TEM-4T (tartaric acid esters of tallow monoglycerides), certain phospholipids (crude as well as highly purified preparations), Tween 80, 85, Span 80 and glyceryl monoleate. High concentrations of oleic acid in the medium inhibited growth. This inhibition was partially released or annulled by certain mixtures of "non essential" fatty acids or by increasing the stigmasterol content of the growth medium. Definite but non-stoichiometric levels of oleic acid and sterol were required for optimal growth. Tween 60, a non-ionic emulsifier similar in its surfactant properties to Tween 80, stimulated growth in the presence of suboptimal amounts of oleic acid but failed itself to replace oleic acid as a growth requirement.     

Fatty acid requirement of Treponema denticola and Treponema vincentii

Treponema denticola and Treponema vincentii were cultured in a medium supplemented with either 0.2 or 0.4% (w/v) alpha globulin in place of serum. The active factor(s) in alpha globulin was stable at pH 7.0 to autoclaving and was nondialyzable. Extraction of lipids from alpha globulin showed that both protein and lipid, supplied by the alpha globulin, were required for maximal growth of these two oral treponemes. The lipid component was investigated by adding sodium salts of long-chain fatty acids to the basal medium supplemented with 0.4% delipified alpha globulin. The lipid component of alpha globulin was replaced by either oleic acid (cis-18:1(9)) or by elaidic acid (trans- 18:1 (9)0. No other saturated or unsaturated fatty acid tested could support good growth. Tween 80 (polysorbitan monooleate) was the only Tween compound able to support maximal growth of T. denticola. The cellular lipids of T. denticola, grown with oleate in broth supplemented with 0.4% delipified alpha globulin, were extracted and analyzed by gas chromatography. The principle fatty acids were myristic, pentadecanoic, and palmitic acids. Lesser amounts of oleic acid, eicosadienoic acid, and an unidentified fatty acid (retention time, 88 min) were also detected. Treponema denticola appears to be capable of limited synthesis of cellular fatty acids such as myristic, pentadecanoic, and palmitic acids from oleic acid.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

Stability of Lactic Acid Bacteria to Freezing as Related to Their Fatty Acid Composition

The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-¹⁴C]acetate but had no inhibitory effect on the incorporation of exogenous [1-¹⁴C]oleate (or [1-¹⁴C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-¹⁴C]oleate and cerulenin was associated mainly with cyclopropane Δ19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.     

Production of tylosin byStreptomyces fradiae in palm oil medium

Streptomyces fradiae (NRRL 2702) produced tylosin when cultured on a synthetic defined medium M3. Palm oil, palm kernel oil and their fractions, as well as fatty acids and glycerol were investigated to serve as the major carbon source in shake flask culture. The lipids, glycerol and fatty acids, particularly palmitic acid but not oleic or lauric acid, were suitable for growth and tylosin production. For palmitic acid, at 168 h, the dry cell yield and tylosin production were 8.9 mg/ml and 0.84 mg/g cell mass respectively.     

Fatty acid metabolism in Paramecium. Oleic acid metabolism and inhibition of polyunsaturated fatty acid synthesis by triparanol

Paramecium requires oleic acid for growth and can grow in media containing no other fatty acids. In the present study, we have shown that this ciliate utilized oleate mainly as a carbon and energy source, even though this fatty acid was the only substrate available for synthesis of polyunsaturated fatty acids. Culture growth was inhibited by the addition of the drug triparanol. Triparanol decreased the formation of polyunsaturated fatty acids from oleate by preventing desaturation to form the dienoic acid, linoleate. Triparanol inhibition resulted in an altered phospholipid fatty acyl composition, an increased fragility and an altered behavioral response of the cells to a depolarizing stimulation solution. Therefore, although most of the dietary oleate was not used by the cells for polyunsaturated fatty acid synthesis, the desaturation of oleic acid was critical for normal culture growth, cell integrity and swimming behavior, all of which are expected to be dependent on normal membrane lipid composition.     

Effects of the detergent Tween 80 on Thermomonospora curvata

Tween 80 (0.1%, v/v) added to Thermomonospora curvata growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54°C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium.

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

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.     

Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80. The organism had a mu max of 0.31 h-1 and a Ks for glucose of 0.55 mM. At a dilution rate of 0.10 h-1, a maximal yield of 0.10 g biomass (g glucose)-1 was observed. The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 0.10 h-1, the yield of the S. cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition. The major difference between the two yeast strains was that S. cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid. The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase. The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed. This resulted in a reduction in yield and an increase in specific ethanol production. Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth. Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.     

Fatty acid and carotenoid production by Sporobolomyces ruberrimus when using technical glycerol and ammonium sulfate

The production of carotenoids, lipid content, and fatty acid composition were all studied in a strain of Sporobolomyces ruberrimus when using different concentrations of technical glycerol as the carbon source and ammonium sulfate as the nitrogen source. The total lipids represented an average of 13% of the dry weight, and the maximum lipids were obtained when using 65.5 g/l technical glycerol (133.63 mg/ g). The optimal conditions for fatty acid production were at 27 degrees C using 20 g of ammonium sulfate and a pH range from 6 to 7, which produced a fatty acid yield of 32.5+/-1 mg/g, including 1.27+/- 0.15 mg of linolenic acid (LNA), 7.50+/-0.45 mg of linoleic acid (LLA), 5.50+/-0.35 mg of palmitic acid (PA), 0.60+/-0.03 mg of palmitoleic acid (PAL), 1.28+/-0.11 mg of stearic acid (SA), 9.09+/-0.22 mg of oleic acid, 2.50+/-0.10 mg of erucic acid (EA), and 4.25+/-0.20 mg of lignoceric acid (LCA), where the palmitic, oleic, and linoleic acids combined formed about 37% of the total fatty acids. The concentration of total carotenoids was 2.80 mg/g when using 20 g of ammonium sulfate, and consisted of torularhodin (2.70 mg/g) and beta-carotene (0.10 mg/ g), at 23 degrees C and pH 6. However, the highest amount with the maximum specific growth rate was obtained (micromax=0.096 h(-1)) with an ammonium sulfate concentration of 30 g/l.     

Fats and fatty acids as growth factors for Lactobacillus delbrueckii

The effects of various fats and fatty acids on the growth of Lactobacillus delbrueckii strains have been studied using modified MRS broth without Tween 80 as a basic growth medium. Among the six L. delbrueckii strains studied all except one strain required Tween 80 or Tween 20 as a fatty acid supplement for the growth. Tween 40 and Tween 60, which contain solely medium and long chain saturated fatty acids, inhibited the growth of all L. delbrueckii strains when present as a sole fat supplement in MRS broth. Free oleic acid but not free lauric acid could substitute Tween 80 or Tween 20 supplement suggesting that unsaturated fatty acids are essential growth factors for most L. delbrueckii strains. Among the natural food oils tested, the oils containing the lowest amounts of saturated long chain fatty acids promoted the growth of L. delbrueckii most effectively. Especially cellular C18:1 and C19 cyclopropane fatty acid contents of L. delbrueckii were strongly affected by exogenous fatty acid composition and by strain suggesting genetic diversity and polymorphism among the genes encoding and/or regulating cyclopropane synthase. In addition obviously most if not all L. delbrueckii strains lack particular synthase, desaturase and/or dehydrase activities required for de novo synthesis of long chain unsaturated fatty acids. These biochemical features could be used as informative chemotaxonomic characteristics for L. delbrueckii starter strain identification and selection.     

Supercritical CO2 extraction of lipids from grain sorghum dried distillers grains with solubles

Experiments were carried out on a lab supercritical CO(2) extraction system to determine the effects of extraction conditions, including mass ratio of CO(2) consumed to distillers dry grain with solubles (DDGS) extracted, extraction pressure, extraction temperature and time, on yield and composition of extracted lipids. A maximum lipid yield of 150 g/kg DDGS was achieved with a mass ratio approximately 45, an extraction pressure at 27.5 MPa, an extraction temperature at 70 degrees C and an extraction time of 4 h. Under these extraction conditions, the contents of tocols, phytosterols, policosanols and free fatty acids were 0.44, 15.6, 31.2 and 155.3 mg/g in the extract. Experimental results indicated that shorter extraction time and higher flow rate of CO(2) can achieve higher contents of tocols, phytosterols and policosanols but lower content of free fatty acids in the lipid extract. Extraction conditions had no observed effects on the composition of free fatty acids in the extract. Palmitic, oleic and linoleic acids were three main free fatty acids extracted and constituted about 94% of all free fatty acids.     

Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture.

Lipid accumulation of Candida 107, grown at dilution rates from 0.03 to the maximum of 0.21/h, with carbon, nitrogen, phosphate, and magnesium limitations in a chemostat, was maximal at about 40% (wt/wt) with nitrogen-limited medium at a dilution rate of 0.06/h, giving an efficiency of substrate conversion of 22 g of lipid per g of glucose consumed. At higher dilution rates the lipid content decreased. With carbon-limited growth, the highest lipid content (14%, wt/wt) was at the maximum dilution rate. High lipid contents also occurred with phosphate + nitrogen as double limitations of growth, with the lipid content of the yeast (about 35%, wt/wt) continuing to be near maximum at dilution rates also near maximum (0.17/h), thus giving the highest specific rate of lipid formation of any growth conditions (0.59 g of lipid/g of yeast per h). However, the efficiency of substrate utilization was only 5.2 g of lipid formed per 100 g of glucose consumed. The composition of the fatty acyl residues within the lipid remained constant over many weeks if the steady-state conditions remained unchanged. With carbon-limited growth, the degree of unsaturation of the fatty acids markedly decreased as the dilution rate was increased, but with nitrogen limitation the reverse trend was seen. In all cases, linoleic and oleic acids were the principal fatty acyl residues affected, and their relative proportions always varied in opposite directions. When magnesium was a limiting nutrient, there was a considerable increase in the proportion of myristic acid produced within the lipid. Neutral lipids (predominantly triglycerides) varied from 66 to 92% of the total lipid from carbon- and nitrogen-limited growth; phospholipids (varying from 2 to 25%) were highest in nitrogen-limited growth. The fatty acyl residues within each lipid fraction showed the same variations with changing growth rates.     

Effects of growth conditions on the lipid composition of Bifidobacterium bifidum subsp. pennsylvanicum

Lipid-phosphorus and lipid-galactose content and phospholipid and fatty acid composition of Bifidobacterium bifidum subsp. pennsylvanicum were examined under a wide variety of growth conditions. Cells from 29-C cultures contained less lipid-phosphorus than did cells from 37-C cultures, but their lipid-galactose content and phospholipid composition did not differ. At both temperatures, the growth phase influenced the lipid composition similarly.Phosphate, Mg²⁺ and K⁺ concentrations in the medium did neither significantly change the cellular lipid-phosphorus content nor the phospholipid composition. Only Mg²⁺-deficiency markedly reduced growth and lowered the content of cellular lipid-galactose.Omission of Tween 80 from the medium did not affect growth, but lowered the content of lipid-galactose and augmented those of lipid-phosphorus and diphosphatidylglycerol in the cell. Increased osmolarity and substitution of other Tweens for Tween 80 caused the same changes in lipid composition, and besides inhibited growth. Omitting Tween 80 and replacing it by other Tweens dramatically reduced the percentage of unsaturated fatty acids. C₁₂- and C₁₄-fatty acids made up about 50% of total fatty acids in cells from Tween 20 cultures and 12–14% in cells from Tween 40 and Tween 60 cultures. The differences in the decline of unsaturated fatty acids and in the degree of replacement of these acids by C₁₂- and C₁₄-fatty acids may be related to the variations in growth in cultures with various Tweens by way of changes in the physical state of the membrane lipids.The present investigations have been carried out with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Netherlands Foundation for Chemical Research (SON).     

耐乙酸粘红酵母合成油脂

乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。