Effect of temperature and glucose supply on the production of polyunsaturated fatty acids by the fungus Mortierella alpina CBS 343.66 in fermentor cultures

The fungus Mortierella alpina CBS 343.66 was grown in a fermentor at different pH, temperatures and supplies of carbon source (glucose) in order to optimize the culture conditions for rapid biomass and lipid production with a high proportion of polyunsaturated fatty acids, especially arachidonic acid (AA). Good growth and lipid production with 31% AA was obtained at pH 6.5 and 25°C. A temperature decrease to 18°C gave a significantly higher degree of polyunsaturated fatty acids. Eicosapentaenoic acid was not detected at 25°C, but was formed at 18°C at about 10%. The AA concentration but was similar for 18°C about 10%. The AA concentration was similar for 18°C and 25°C (around 30%): 18°C allowed relatively good growth and had a beneficial effect on the fungus morphology, i.e. pellets were formed. Best lipid production and a AA content of up to 33% was achieved at an excess of glucose (carbon source) and a deficit of ammonium chloride (nitrogen source). The percentage of AA of the total fatty acid composition was constant as ong as glucose was present. At glucose exhaustion, the proportion of AA increased to 57%. The increase in AA corresponded to a decrease in palmitic acid, stearic acid and oleic acid. Correspondence to: G. Molin     

Heat Shock Induced Lipid Changes and Solute Leakage in Germinating Seeds of Pigeonpea

Heat shock (HS) reduced total lipid and phospholipid contents and their synthesis in germinating seeds of pigeonpea [Cajanus cajan (L.) Millspaugh]. Lipid peroxidation was also enhanced with increasing temperature and HS duration. HS influenced lipid metabolism to a higher extent at 45°C than at 40°C. This altered lipid metabolism and lipid peroxidation was associated with the loss of various solutes from the germinating seeds, and modification of growth and development. Pretreatment of germinating seeds at 40°C for 1 h or at 45°C for 10 min followed by incubation at 28°C for 3 h prior to 45°C for 2 h ameliorated solute leakage due to reduced lipid peroxidation and improvement in lipid content and membrane function.     

Temperature-dependent lipid content and fatty acid composition of three thermophilic bacteria

The lipid content and fatty acid composition of a strain ofBacillus caldolyticus and of two facultative thermophiles (B. flavothermus and strain NZ-2) were analysed after growth at different temperatures. In all three strains the amount of membrane, as a fraction of total cellular dry mass, was found to increase with temperature, however, in varying degrees. Changes of lipid content and protein/lipid ratio inB. caldolyticus between 60°C and 100°C and in strain NZ-2 between 45°C and 70°C were minor; inB. f avothermus the alterations in the 50°C–70°C range were more pronounced. The same was found for changes observed in the phospholipid/total lipid and phospholipid/membrane ratios, and also in the amounts of individual phospholipids. The alterations of the fatty acid composition were most significant inB. caldolyticus, especially between 80°C and 95°C. In contrast, the main changes inB. flavothermus and NZ-2 were found to occur between 30°C and 50°C, and between 45°C and 60°C, respectively. Based on these data, strain NZ-2 could be characterized as the least andB. flavothermus as the most versatile of the three organisms.     

The effect of temperature on the growth and lipid composition of the extremely halophilic coccus,Sarcina marina

Sarcina marina (NCMB 778) grew over the temperature range 20–45°C but no growth was recorded at 15°C or 50°C. At the optimum growth temperature of 34°C the doubling time was 14.5 h.The major polar lipid components, tentatively identified as the diether analogues of phosphatidyl glycerophosphate (PGP), phosphatidyl glycerol (PG), diglycosyl diglyceride (DGD) and triglycosyl diglyceride (TGD), and the major neutral lipid components, tentatively identified as squalene, dihydrosqualene, tetrahydrosqualene, vitamin MK8, geranyl geraniol and di-O-phytanyl glycerol, are identical to those found in other extremely halophilic rods and cocci.The total lipid content varied with growth conditions from 0.6 – 3.2% of the dry cell weight, polar lipids accounted for between 94.3 and 83.6% of the total lipid, the remainder being neutral lipid.In response to both the transition from exponential to stationary phase and a reduction of 14°C in growth temperature, batch cultures showed: (i) an increase in total lipid content; (ii) a decrease in PG and (iii) an increase in PGP. Specific responses to the temperature decrease were (i) increased total lipid content; (ii) no decrease in neutral lipids in stationary phase; (iii) marked reduction in PG and (iv) raised DGD. (i) and (ii) could be mechanisms for increasing membrane fluidity.In common with all other extreme halophiles investigated the alkyl side chains of S. marina polar lipids were identified as the phytanyl (3R, 7R, 11R, 15-tetramethylhexadecyl) group. Its structure did not appear to vary with temperature so that the normal mechanisms for modifying the structure of lipid alkyl side chains to modulate membrane fluidity in response to temperature changes probably does not occur in this group of microorganisms.     

Lipid formation in the oleaginous mould Entomophthora exitalis grown in continuous culture: effects of growth rate,temperature and dissolved oxygen tension on polyunsaturated fatty acids

Summary The oleaginous fungus Entomophthora exitalis was grown in continuous culture at a constant dilution rate (0.04 h^–1) and over a range of temperatures (20–30° C). As the growth temperature was decreased from 30 to 20° C the percentage of polyunsaturated fatty acids (PUFA) increased proportionally from 18 to 27% (w/w) of the total fatty acids. The increase in unsaturation was as a result of an increased proportion of n-6 PUFA (particularly arachidonic acid) in the phospholipid and sphingo- plus glycolipid fractions. The triacylglycerol fraction of lipids displayed a negligible change. The proportion of phospholipids within the extracted lipid increased between 26 and 20° C without any change in the lipid content of the fungus. Although the changes in lipid unsaturation correlated, at first inspection, to the culture dissolved O₂ tension (DOT), growth of the fungus at a constant dilution rate and temperature (22° C) over a range of DOT values failed to influence lipid unsaturation. Thus temperature is the principal regulation factor of the degree of unsaturation in the lipids of this organism. Offprint requests to: C. Ratledge     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing.In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for ³¹P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 °C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 °C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Alterations in normal fatty acid composition in a temperature-sensitive mutant of a thermophilic bacillus

A temperature-sensitive mutant of a thermophilic bacillus was isolated which was unable to maintain membrane integrity at high temperature. The mutant appeared to lose cytoplasmic contents, as indicated by a decrease in turbidity and cell refractivity, when shifted from a permissive (52° C) to a restrictive (65° C) temperature. Cell number remained fairly constant, however. At the approximate onset of the decline in turbidity, viability decreased and net synthesis of ribonucleic acid, deoxyribonucleic acid, and protein ceased. Both chloramphenicol and sucrose were effective in retarding the decline in turbidity at 65° C. An abnormal fatty acid composition at high temperature suggested that the lesion in the mutant involved lipid synthesis. The proportion of fatty acids with a high melting point (> 55° C) increased in the parent from 42% at 42° C to 69% at 65° C. Similar changes were not made by the mutant. An abnormal phospholipid composition was also observed in the mutant at 42° C and 52° C. However, at 58° C, the maximum growth temperature of the mutant, the proportion of major phospholipids (phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin) was similar to the parent strain. The mutant's apparent loss of membrane stability at high temperature and its inability to regulate fatty acid and phospholipid composition in a normal manner suggested that (i) the temperature-sensitivity of the mutant may be a result of a defect in normal lipid metabolism at high temperature and (ii) the normal changes in fatty acid composition observed at increased growth temperatures may be an essential feature of thermophily.A preliminary report of this work was presented at the 73rd Annual Meeting of the American Society for Microbiology, Miami Beach, Florida, May 6–11, 1973.     

Influence of cultivation conditions on lipid production by Cryptococcus albidus

Summary Cryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.     

Nitrogen and carbon mineralization during incubation of two Bangladesh soils in relation to temperature

Summary At temperatures of 20°, 30°, 40°, 50° and 60°C in a Gangetic alluvial soil (G soil, pH 7.6) N-mineralization and nitrification increased with temperature up to 40°C and mineralized N accumulated entirely as nitrate. At 50° and 60°C mineralized N was relatively low and no nitrification occurred. In the Red soil (R soil, pH 5.2) mineralized N increased with temperature up to 40°C, was somewhat less at 50°C and was at a maximum at 60°C. Nitrification was maximum at 30°C but did not occur at 50° and 60°C. In the G soil C-mineralization increased considerably with temperature, whilst in the R soil there were only small differences due to temperature.     

Stability of diether C(25,25) liposomes from the hyperthermophilic archaeon Aeropyrum pernix K1

Temperature and pH effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the Aeropyrum pernix. We determined the permeability of C_25,25 liposomes using fluorescence intensity of released calcein. At pH 7.0 and 9.0, and from 85 °C to 98 °C, only 10% of entrapped calcein was released. After 10 h at 90 °C, calcein release reached 27%, independent of pH. Fluorescence anisotropy measurements of hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene revealed gradual changes up to 60 °C. At higher temperatures, the anisotropy did not change significantly. Fluorescence alone did not provide detailed and direct structural information about these C_25,25 liposomes, so we used electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC). From EPR spectra, mean membrane fluidity determined according to maximal hyperfine splitting and empirical correlation times showed continuous increases with temperature. Computer simulation of EPR spectra showed heterogeneous membranes of these C_25,25 liposomes: at low temperatures, they showed three types of membrane regions characterized by different motional modes. Above 65 °C, the membrane becomes homogeneous with only one fluid-like region. DSC thermograms of C_25,25 liposomes reveal a very broad and endothermic transition in the temperature range from 0 °C to 40 °C.     

Enriching marine macroalgae with eicosatetraenoic (arachidonic) and eicosapentaenoic acids by chilling

Summary Twelve macroalgae belonging to the Chlorophyta, Phaeophyta and Rhodophyta were collected from the Arabian Gulf. Field samples and samples that were first incubated at 5° C and 24° C in the light for 1 week were analysed for lipids and fatty acids. The lipid contents varied according to the macroalga and, within the Chlorophyta and Phaeophyta, some representatives accumulated more lipids at 5° C and others at 24° C. All samples of algae had similar lipid composition with only quantitative differences. The temperature did not have a common effect on the lipid composition of representative algae, although changes in the relative concentration of specific classes were recorded. The Phaeophyta and Rhodophyta were as a rule richer than the Chlorophyta in eicosatetranoic (20:4) and eicosapentaenoic (20:5) but poorer in linolenic (18:3) acids. In most of the algae, incubation at 5° C was associated with lowering the proportion of palmitic acid (16:0) in the total lipids, and, but only in the Phaeophyta and Rhodophyta, increasing the concentration of 20:4 and 20:5. These polyunsaturated fatty acids occurred in high levels in monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) of the Phaeophyta and Rhodophyta but not the Chlorophyta, the MGDG and DGDG of which were rich in 18:3 and hexadecatrienoic acid (16:3). Offprint requests to: R. H. Al-Hasan     

Differential effects induced by α- and β-endosulfan in lipid bilayer organization are reflected in proton permeability

The effects of two insecticides isomers, α- and β-endosulfan, on the passive proton permeability of large unilamellar vesicles (LUV) reconstituted with dipalmitoylphosphatidylcholine (DPPC) or mitochondrial lipids were reported. In DPPC (LUV) gel phase, at 30 °C, the global kinetic constant (K) of proton permeability (proportional to the proton permeability) initially increased slightly with the increase of α-endosulfan/lipid molar ratio up to 0.143. In the range from 0.143 to 0.286, a discontinuity in the increment occurred and, above this range, the proton permeability increased substantially. In DPPC fluid phase, at 48 °C, the proton permeability showed a behavior identical to that observed in gel DPPC, with a sharp increase for α-endosulfan/lipid molar ratios ranging from 0.143 to 0.286. At these and higher concentrations, α-endosulfan induced phase separation in the plane of DPPC membranes, as revealed by differential scanning calorimetry (DSC). Conversely to α-endosulfan, β-endosulfan induced only a slight increase in the proton permeability, either in the fluid or the gel phase of DPPC, for all β-endosulfan/lipid molar ratios tested. Additionally, the effects of the endosulfan isomers on the proton permeability of mitochondrial fluid lipid dispersions, at 37 °C, are similar to those described for DPPC. The β-isomer induced a very small effect, and α-endosulfan, at low concentrations, increased slightly the proton permeability, but for insecticide/lipid molar ratios above 0.143 the permeability increased substantially. Consequently, the membrane physical state of synthetic and native lipid dispersions, as affected by the structural features of α- and β-endosulfan, influenced the proton permeability. The effects here observed in vitro suggest that the formation of lateral membrane domains may underlay the biological activity of α-endosulfan in vivo, contributing to its higher degree of toxicity as compared with β-endosulfan.     

Growth and hyoscyamine production of ‘hairy root’ cultures of Datura stramonium in a modified stirred tank reactor

Summary The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg·g^–1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g^–1 wet weight). Growth at 30°C was initially faster than at either 25°C or 35°C and by day 12, the drained weight of roots in the fermentor at 30°C was about fourfold greater than at 25°C and twice that at 35°C. The ultimate hyoscyamine levels attained (approximately 0.5 mg·g^–1 wet weight) were similar at both 25°C and 30°C but some 40% lower at 35°C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25°C and the highest production rate of 8.2 mg hyoscyamine l^–1 per day was obtained for roots grown at 30°C. In continuous fermentation at 25°C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30°C or 35°C. Offprint requests to: M. G. Hilton     

Dietary fats,selected body temperature and tissue fatty acid composition of agamid lizards (Amphibolurus nuchalis)

The composition of tissue and membrane fatty acids in ectothermic vertebrates is influenced by both temperature acclimation and diets. If such change in body lipid composition and thermal physiology were linked, a diet-induced change in body lipid composition should result in a change in thermal physiology. We therefore investigated whether the selected body temperature of the agamid lizardAmphibolurus nuchalis (body mass 20 g) is influenced by the lipid composition of dietary fatty acids and whether diet-induced changes in thermal physiology are correlated with changes in body lipid composition. The selected body temperature in two groups of lizards was indistinguishable before dietary treatments. The selected body temperature in lizards after 3 weeks on a diet rich in saturated fatty acids rose by 2.1 °C (photophase) and 3.3 °C (scotophase), whereas the body temperature of lizards on a diet rich in unsaturated fatty acids fell by 1.5 °C (photophase) and 2.0 °C (scotophase). Significant diet-induced differences were observed in the fatty acid composition of depot fat, liver and muscle. These observations suggest that dietary lipids may influence selection of body temperature in ectotherms via alterations of body lipid composition.Abbreviations bm body mass - FA fatty acid(s) - MUFA monounsaturated fatty acids - PUFA polyunsaturated fatty acids - SFA saturated fatty acids - T _a air temperature - T _b body temperature - UFA unsaturated fatty acids     

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I′ band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 °C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 °C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 °C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.     

Lipid Peroxidation and Status of the Antioxidant System in Tissues of Little Suslik (Citellus pygmaeus) in the Course of Hibernation

The rate of lipid peroxidation and activities of superoxide dismutase, catalase, and hydrophilic antioxidants were studied in the hypothalamus, liver, kidney, myocardium, skeletal muscle, and serum of Citellus pygmaeus upon entering hibernation (18–20°C), in early torpor (7–10°C), and after hibernation for a week or three months (5–10°C). During hibernation, lipid peroxidation proved to either decrease or remain at the level characteristic of waking animals. High antioxidant activity was maintained in most tissues, particularly, in the case of prolonged hibernation.     

Temperature dependence of the fluorescence of pyrene labeled crab nerve membranes

Summary A method, using albumin-pyrene complexes, has been developed for labeling, in a controlled manner, crab leg nerves whose excitability was preserved.The excimer-to-monomer fluorescence intensity ratio of pyrene, embedded in nerve membrane lipids and in their crude lipid extracts, is a fluidity parameter which displayed the following features with temperatures. a—a temperature-dependent increase of fluidity b—three breaks (6°, 19° and 37°C) in the physiological medium c—In Ca ⁺⁺-depleted sea water, the 37° characteristic temperature vanished.These breaks may reflect some lateral phase separations of the lipid components of nerve membranes. The calcium dependent temperature break may involve a segregation of acidic phospholipids while the other two breaks (6° and 19°C) may be due to neutral lipids phase separation. The relationship of these findings to nerve function is discussed.     

Pyrolytic characteristics of heterotrophic Chlorella protothecoides for renewable bio-fuel production

Heterotrophic Chlorella protothecoides cells were pyrolyzed in athermogravimetric analyzer to investigate the pyrolytic characteristics anddetermine the kinetic parameters. Heating rates of 15, 40, 60 and 80 ^°C^-1 up to a final temperature of 800 ^°C wereused. The pyrolysis reactions mainly took place between 160–520^°C with a volatile yield of about 80%. The devolatilization stageconsisted of two main temperature zones (I and II) with a transition at300–320 ^°C. Crude lipid in cells decomposed at Zone II whileother main components at Zone I. The increase of heating rate caused alateral shift to higher temperatures in the thermograms, a decrease ofactivation energies for the devolatilization stage and an increase of both theinstantaneous maximum and average reaction rates. The difference ofactivation energies between two zones implied that more energy input forlipid pyrolysis seems needed in comparison with other main components.These data are useful for the design, operation, and modeling of thepyrolysis systems for microalgae.