Differential temperature effect on the production of enhanced gamma linolenic acid in <Emphasis Type="Italic">Mucor rouxii</Emphasis> CFR-G15

Mucor rouxii CFR-G15, a locally isolated phycomycetous fungus, on cultivation at room temperature produced more than 30% (w/w) lipid in their dry cell weight, in which 14.2% accounted to be GLA content of the total fatty acids. It was observed that when incubation temperature lowered at 14°C, GLA content of the mycelium increased significantly (P<0.05) from 14.2% to 21.97%. In order to optimize the cultural conditions for high biomass and lipid production with high GLA content, the fungus was grown in association of two different temperatures and supply of additional glucose in culture medium. Maximum lipid and GLA were obtained 23.56 and 19.5% respectively, when the culture was grown at 28°C for four days and followed by addition of glucose (5%), and lowered the incubation temperature to 14°C for another four days. The presence of GLA in the oil obtained from M. rouxii CFR-G15 was confirmed by the gas chromatography-mass spectrometry. Gamma linolenic acid (GLA, n-6) is gaining importance in pharmaceutical and nutraceutical industries because of clinical evidence demonstrated that it has various beneficial effects in human health. In this paper temperature played a major role in enhancing the GLA content which has been described.     

Temperature responses of growth, photosynthesis, fatty acid and nitrate reductase in Antarctic and temperate Stichococcus

Stichococcus, a genus of green algae, distributes in ice-free areas throughout Antarctica. To understand adaptive strategies of Stichococcus to permanently cold environments, the physiological responses to temperature of two psychrotolerants, S. bacillaris NJ-10 and S. minutus NJ-17, isolated from rock surfaces in Antarctica were compared with that of one temperate S. bacillaris FACHB753. Two Antarctic Stichococcus strains grew at temperature from 4 to 25°C, while the temperate strain could grow above 30°C but could not survive at 4°C. The photosynthetic activity of FACHB753 at lower than 10°C was less than that of Antarctic algae. Nitrate reductase in NJ-10 and NJ-17 had its optimal temperature at 20°C, in comparison, the maximal activity of nitrate reductase in FACHB753 was found at 25°C. When cultured at 4–15°C a large portion of unsaturated fatty acids in the two Antarctic species was detected and the regulation of the degree of unsaturation of fatty acids by temperature was observed only above 15°C, though the content of the major unsaturated fatty acid αC18:3 in FACHB753 decreased with the temperatures elevated from 10 to 25°C. Elevated nitrate reductase activity and photosynthetic rates at low temperatures together with the high proportion of unsaturated fatty acids contribute to the ability of the Antarctic Stichococcus to thrive.     

Modulation in fatty acid composition influences salinity stress tolerance in Frankia strains

Frankia strains, isolated from Hippöphae salicifolia D. Don, were utilized to examine the utility of lipid amendments in the strains’ strategic survival against salinity. Frankia strains are known to withstand severe temperature fluctuations (?20 °C to +30 °C), nitrogen deprivation and low soil water content. It was interesting to note that these strains were also able to tolerate a considerable range of salinity. Strains were subjected to 250 mM (500 mM for HsIi10) and 750 mM NaCl treatment, which were the critical and inhibitory NaCl concentrations, respectively, for the experimental strains. Their lipid profiles showed dynamic modifications in saline environment; 16–18 carbon chain fatty acids were of predominant occurrence in the lipid membrane. In the critical NaCl environment, there was an increase in fatty acid unsaturation (measured in terms of MUFA/PUFA ratio), which preserved normal membrane fluidity. Conversely, at the inhibitory salinity level, increased fatty acid saturation made the membrane highly rigid and susceptible to breakage and electrolyte loss. The differential capability of fatty acid desaturation could be a major factor in variation of salt sensitivity/tolerance patterns among these strains. Also, management of the lipid profile in response to salinity was found to be a strain-specific character.     

Fatty acid composition of chlorenchyma membrane fractions from three desert succulents grown at moderate and high temperatures

To help understand the tolerances of desert succulents to extremely high temperatures (above 60°C), the effect of growth temperature on fatty acid composition of various membrane fractions from three species was investigated. When maintained at day/night air temperatures of 30°C/20°C, their chlorenchyma fatty acid compositions were similar to one another and to those of mesophytic leaves, except that desert succulents had appreciably less linolenic acid (18:3) and more oleic acid (18:1) and hence greater fatty acid saturation. The differences were observed in the chloroplast, mitochondrial and microsomal fractions and were more apparent in the nonpolar lipids than the total lipids. For all membrane fractions of Ferocactus acanthodes, a shift to 50°C/40°C resulted in a decrease in 18:3 and an increase in 18:1 and hence an increase in fatty acid saturation level. For Agave deserti and Carnegiea gigantea, however, increasing the day/night air temperatures did not result in increased fatty acid saturation, although their high-temperature tolerances increase about as much as that of F. acanthodes as the air temperature is increased. Thus, acquisition of high-temperature tolerance need not be accompanied by marked changes in fatty acid saturation or composition.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.     

Light,temperature and nitrogen starvation effects on the total lipid and fatty acid content and composition ofSpirulina platensis UTEX 1928

The total lipid and fatty acid content ofSpirulina platensis UTEX 1928 was 7.2 and 2.2% respectively of cellular dry weight under controlled conditions supporting high growth rates. With increases in irradiance from 170 to 870 μmol photon m^?2 s^?1, growth rate increased, total lipid decreased, and fatty acid composition was unaffected. At 1411 μmol photon m^?2 s^?1, total lipid increased slightly and percent composition of the fatty acid gamma linolenic acid increased. Growth and total lipid content ofS. platensis were affected by changes in growth temperature from 25 to 38 °C. With increased growth rate, total lipid content increased. This suggests that the storage of carbon increases at temperatures supporting high growth rates. The degree of saturation increased with temperature. Although the percent composition of gamma linolenic acid was higher at lower growth temperature, production was still primarily a function of growth rate. The effect of temperature on fatty acid content and degree of saturation was of secondary importance. Nitrogen starvation increased total lipid content but decreased fatty acid content as a percentage of dry weight; composition of the fatty acids was unaffected. N-starvation appeared to suspend synthesis of long chain fatty acids inS. platensis, suggesting that some other compound stores fixed carbon when nitrogen is limiting. It was concluded that fatty acid production inS. platensis is maximized by optimizing culture conditions for growth.     

Production and partial purification of γ-linolenic acid and some pigments fromSpirulina platensis

The polyunsaturated fatty acid -linolenic acid (GLA, 18:36) is of potential pharmaceutical value. The cyanobacteriumSpirulina platensis could become an excellent source for this fatty acid, provided that GLA content could be increased and a GLA concentrate could be obtained at a low cost. Increasing the cell concentration inSpirulina platensis enhanced the fatty acid content and thus the GLA content. This effect was used to further enhance the GLA content of GLA-overproducing strains. Separation of the galactolipids and their purification via urea complexes formation, resulted in a GLA concentrate of over 90% purity.     

Optimization of cultivation conditions for fatty acid composition and EPA production in the eustigmatophycean microalga Trachydiscus minutus

We determined the effects of cultivation conditions (nitrogen source, salinity, light intensity, temperature) on the composition of polyunsaturated fatty acids (PUFAs) and the production of eicosapentaenoic acid (EPA) in the laboratory cultured eustigmatophycean microalga, Trachydiscus minutus. T. minutus was capable of utilizing all nitrogen compounds tested (potassium nitrate, urea, ammonium nitrate, ammonium carbonate) with no differences in growth and only minor differences in fatty acid (FA) compositions. Ammonium carbonate was the least appropriate for lipid content and EPA production, while urea was as suitable as nitrates. Salinity (0.2 % NaCl) slightly stimulated EPA content and inhibited growth. Increasing salinity had a marked inhibitory effect on growth and PUFA composition; salinity at or above 0.8 % NaCl was lethal. Both light intensity and temperature had a distinct effect on growth and FA composition. The microalga grew best at light intensities of 470–1,070 μmol photons m^?2 s^?1 compared to 100 μmol photons m^?2 s^?1, and at 28 °C; sub-optimal temperatures (20, 33 °C) strongly inhibited growth. Saturated fatty acids increased with light intensity and temperature, whereas the reverse trend was found for PUFAs. Although the highest level of EPA (as a proportion of total FAs) was achieved at a light intensity of 100 μmol photons m^?2 s^?1 (51.1?± 2.8 %) and a temperature of 20 °C (50.9?±?0.8 %), the highest EPA productivity of about 30 mg L^?1?day^?1 was found in microalgae grown at higher light intensities, at 28 °C. Overall, for overproduction of EPA in microalgae, we propose that outdoor cultivation be used under conditions of a temperate climatic zone in summer, using urea as a nitrogen source.     

Enzymatic and Non-Enzymatic Esterification of long Polyunsaturated Fatty Acids and Lysophosphatidylcholine in Isooctane

Phosphatidylcholine containing large amounts of long polyunsaturated fatty acid, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), was synthesized in isooctane. Immobilized phospholipase A₂ was used as a catalyst. A parallel non-enzymatic esterification reaction was investigated in separate experiments.

The concentrations of lyso-phosphatidylcholine, polyunsaturated fatty acids, water and the enzyme were varied over wide ranges as were the temperature and the reaction time. The type of enzyme, carrier and immobilization procedure were held constant.

The yield of phosphatidylcholine was relatively high (about 21%) when the concentration of polyunsaturated fatty acids was high (300 mg/g of reaction mixture) and the water content was low (below 30% of the dry immobilized enzyme). The highest yield of phosphatidylcholine was found at 80 hours and 75°C. However, at this temperature an extensive non-enzymatic reaction between polyunsaturated fatty acids and lyso-phosphatidylcholine occurred. At 80°C the polyunsaturated fatty acids were partly oxidized. Therefore, a temperature of 45°C to 65°C is probably the optimum temperature for the reaction.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

Lipidomic analysis of psychrophilic yeasts cultivated at different temperatures

Analysis of polar lipids from eight psychrophilic yeasts (Cryptococcus victoriae, Cystofilobasidium capitatum, Holtermaniella wattica, Mrakiella aquatica, M. cryoconiti, Rhodotorula lignophila, Kondoa malvinella and Trichosporon aggtelekiense) grown at 4–28 °C by hydrophilic interaction liquid chromatography/high resolution electrospray ionization tandem mass spectrometry determined 17 classes of lipids and identified dozens of molecular species of phospholipids including their regioisomers. Most of the yeasts were able to grow over the whole temperature range, reaching the highest biomass at 4 or 10 °C. On temperature drop to 4 °C, all eight strains showed a significant decrease of MUFA and a simultaneous increase of PUFA such as α-linolenic acid, the content of which in the biomass reached up to 20%. We also found alterations in the proportions of individual phospholipids (PI, PE and PC), the PC/PE-ratio decreasing with decreasing temperature. With increasing temperature the content of PoO-PC rose while that of LL-PC decreased, the drop in the content of LL-PC being nearly 100-fold while the content of PoO-PC increased more than twice.A change in temperature brought about changes in molecular species of PC (molecular species PO-PC versus OP-PC) as well as PE, i.e. PO-PE and OP-PE. The phase transition temperature of PO-PC differs from OP-PC by 7 °C and the difference between PO-PE and OP-PE is some 10 °C; we thus assume that the cell compensates for the adverse temperature effect by changing the fatty acids in the sn-1 and sn-2 positions.     

Photosynthesis,lipids and proteins in the cyanobacteriumSynechocystis PCC 6803 as affected by temperature

The cyanobacteriumSynechocystis PCC 6803 was grown photoautotrophically in an inorganic medium at constant growth temperatures of 20, 38 (control) or 43°C for 9 h. The up and down-shift of cultivation temperature decreased the growth as measured by culture absorbance and chlorophylla content. However, high temperature slightly increased the oxygen evolution while temperature lower than control inhibited oxygen evolution during the whole incubation period. The protein synthesis studied by¹⁴C-labeled protein declined under low temperature by about 50%. The fatty acid pattern is characterized as lacking in C₂₀/C₂₂ acids but containing large amounts of C₁₆ and C₁₈ polyunsaturated fatty acids, 16:2 and 18:3 in particular. The lower temperature increased the percentage of monounsaturated fatty acids while higher temperature increased the saturated fatty acid content in total lipids and lipid classes studied.     

Increasing γ-linolenic acid content in Spirulina platensis using fatty acid supplement and light–dark illumination

Spirulina platensis, a filamentous cyanobacterium, produces γ-linolenic acid (GLA, 18:3), which is an important anti-inflammatory for pharmaceutical use. Thus, to increase the GLA content in S. platensis, this study investigated the combined effect of a light–dark (LD) two-stage culture and mixotrophic culture including a precursor of GLA. When compared with a photoautotrophic culture, the supplement of a GLA precursor, such as a long- or short-chain carbon source, enhanced the total fatty acid and GLA contents in the cells in the two-stage culture. The highest GLA content of 2% (w/w) and productivity of 27.6?±?4.7?mg?L^?1 were obtained in S. platensis when using 0.01?mM palmitic acid as a supplement in the two-stage culture. This study also suggests that a mixotrophic and LD two-stage culture may represent a method for increasing the total lipid production, which can then be converted to biofuels.     

Effect of temperature on fatty acid composition and development of unfed Siberian sturgeon (A. baerii) larvae

The fatty acid metabolism in fish is influenced by various factors, including fish species, water temperature, water environment and diet supply. The aim of present work is to investigate the fatty acid composition of yolk‐stage Siberian sturgeon larvae reared at three different temperatures. Fertilized Siberian sturgeon eggs were transferred to the Lodi Aquaculture Research Center of the University of Milan, divided in three aquaria, each containing three incubators and incubated at 16°C. After hatching the temperature was switched to 16, 19 and 22°C. Larvae sampling was performed at the end of yolk sac reabsorption. No feed was dispensed during the trial. Eggs and larvae were weighed and fatty acid profile was determined by GC‐FID analysis after lipid extraction by chloroform/methanol mixture and fatty acid transesterification by methanolic hydrogen chloride. The fertilized eggs had a weight of 23.27 mg and a lipid content of 2.67 mg/egg. At hatching, the weight was 12.2 (0.17 SD) mg and lipid content 1.9 (0.6 SD) mg/larva. At the end of the trial, larvae mean weight was 33.6 (3.6 SD), 34.7 (1.8 SD) and 36.9 (1.1 SD) mg, while lipid content was 2.0 (0.3 SD), 2.1 (0.3 SD) and 2.0 (0.2 SD) mg for larvae reared at 16, 19 and 22°C respectively, without statistically significant difference. Larvae subjected to the highest water temperature showed a faster yolk‐sac absorption. No differences were found across temperatures regarding survival rates and regarding ontogenic development. The fatty acid composition of larvae was affected by the temperature. Larvae reared at 16°C had the lowest amount of saturated fatty acids, mainly due to a lower palmitic acid content, that was offset by a higher level of linolenic and linoleic acid, if compared with larvae reared at 19°C and 22°C. The study suggests that at a lower temperature sturgeon spare unsaturated fatty acid consuming preferably saturated fatty acids, increasing our knowledge of the fatty acid metabolism in this species.     

An estimate of the minimum amount of fluid lipid required for the growth of escherichia coli

The lipid phase transition of Escherichia coli was studied by high sensitivity differential scanning calorimetry. A temperature sensitive unsaturated fatty acid auxotroph was used to obtain lipids with subnormal unsaturated fatty acid contents. From these studies it was concluded that E. coli can grow normally with as much as 20% of its membrane lipids in the ordered state but that if more than 55% of the lipids are ordered, growth ceases. Studies with wild-type cells show that the phase transition ends more than 10°C below the growth temperature when the growth temperature when the growth temperature is either 25°C or 37°C.     

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger,Neurospora crassa,Penicillium chrysogenum,andTrichoderma reesei)

The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses. Received: 1 March 1995 / Accepted: 8 May 1995     

Lipid production and fatty acid composition of selected strains belonging to Mucorales

The lipid accumulation, fatty acid composition and γ-linolenic acid (GLA) production by 28 strains belonging to Mucorales were investigated. The lipid content varied from 5 to 30% on dry biomass and the percentage of γ-linolenic acid in total intracellular lipid was in a range from 2.5 to 15.4% (w/w). The best yield of γ-linolenic acid (expressed as mg GLA per 1 g biomass) was found for Mucor mucedo CCF – 1384 (28.4) and Cunninghamella echinulata CCF – 103 (25.1).     

Changes of γ-linolenic acid content inRhizopus arrhizus duringl(+)-lactic acid fermentation

Effects of nitrogen source, temperature and pH onl(+)-lactic acid production and γ-linolenic acid (GLA) accumulation byRhizopus arrhizus were examined. The nitrogen source had a minor effect on lactate synthesis but influenced the total lipid content and the fatty acid composition in fungus. Higher temperature favorably influenced the rate of both lactic acid production and lipid formation in the biomass and caused a decrease in the yields of oligounsaturated fatty acids. At higher temperature and after glucose exhaustion, degradation of lactate increased. A low pH value negatively affected the formation of lipids and lactate synthesis. The highest value of GLA in the lipid (25.5%,W/W) was reached at the end of lactate synthesis, but maximum yields of total lipids were achieved when the cultivation continued in the presence of lactate until polyols were exhausted.     

Effects of sodium chloride concentration on phospholipid fatty acid composition of yeasts differing in osmotolerance

The effect of growth medium NaCl concentration on the fatty acid composition of phospholipids of 3 strains of Saccharomyces cerevisiae and 6 osmotolerant yeast strains was examined. The S. cerevisiae strains were characterized by a high content of palmitoleic (C_16:1) acid and by having no polyunsaturated C₁₈ acids, whereas the osmotolerant strains had a low content of C_16:1 and a high proportion of polyenoic C₁₈ acids. An increase of the NaCl concentration from 0% to 8% resulted in a decrease of the cellular phospholipid content on a dry-weight basis, for all strains but one of the osmotolerant strains. For the S. cerevisiae strains increased salinity produced a slight decrease of the proportion of C₁₆ fatty acids with a concomitant increase of C₁₈ acids, whereas the osmotolerant strains showed an increase of the relative content of oleic acid (C_18:1) at the expense of the proportion of polyenoic C₁₈ acids.