Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid

Crude glycerol is a major byproduct of the biodiesel industry; previous research has proved the feasibility of producing docosahexaenoic acid (DHA, 22:6 n − 3) through fermentation of the algae Schizochytrium limacinum on crude glycerol. The objective of this work is to investigate the cell growth kinetics, substrate utilization efficiency, and DHA production of the algae through a continuous culture. Steady-state biomass yield, biomass productivity, growth yield on glycerol, specific glycerol consumption rate, and fatty acid composition were investigated within the range of dilution rate (D) from 0.2 to 0.6 day⁻¹, and the range of feed crude glycerol concentration (S₀) from 15 to 120 g/L. The maximum specific growth rate was determined as 0.692 day⁻¹. The cells had a true growth yield of 0.283 g/g but with a relatively high maintenance coefficient (0.2216 day⁻¹). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L due to the higher DHA content at S₀ = 90 g/L. The biomass and DHA productivity of the continuous culture was comparable to those of batch culture, while lower than the fed-batch culture, mainly because of the lower DHA content obtained by the continuous culture. Overall, the results show that continuous culture is a powerful tool to investigate the cell growth kinetics and physiological behaviors of the algae growing on biodiesel-derived crude glycerol.     

Effect of growth phase on the fatty acid composition of Thermus spp.

Two yellow and two red pigmented strains of Thermus were monitored for changes in fatty acid content and composition with reference to growth phase at the optimum temperature. Fatty acid content per mg of dry weight increased as the cultures aged. In addition the quantities of iso C 15:0, iso C 17:0 and iso C 16:0 increased in yellow pigmented strains, but in red pigmented strains, an increase was seen in iso C 15:0, but C 16:0 and iso C 16:0 levels decreased. Thus the fatty acid composition of these organisms varies with growth phase, and shows also strain specific variability.     

Lipid content and fatty acid composition in hypogeous organs ofHelleborus species (Ranunculaceae)

Root and rhizome of fourHelleborus species:H. viridis, H. odorus, H. niger, H. foetidus were examined throughout a two-year period in order to determine the variation of lipid content and fatty acid composition during the ontogenetic cycle. In the deciduous geophytesH. viridis andH. odorus subsp.laxus the lipid content reaches its maximum during the quiescent phase, whereas in the evergreen geophyteH. niger the lipid content is highest during the main growth period. In the chamaephyteH. foetidus lipid content is always very low and it does not show variation throughout the year. In all species the fatty acids detected in the neutral fraction are myristic, palmitic, stearic, oleic and linoleic acid. Palmitic and linoleic acid are always present in larger quantities. The pattern of the relative content of saturated/unsaturated fatty acids seems to be linked to the life form of these species.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Characterization of fatty acid composition in healthy and bleached corals from Okinawa, Japan

Under bleaching conditions, corals lose their symbiotic zooxanthellae, and thus, the ability to synthesize fatty acids (FAs) from photosynthetically derived carbon. This study investigated the lipid content and FA composition in healthy and bleached corals from the Odo reef flat in Okinawa, southern Japan, following a bleaching event. It was hypothesized that the FA composition and abundance would change as algae are lost or die, and possibly microbial abundance would increase in corals as a consequence of bleaching. The lipid content and FA composition of three healthy coral species (Pavona frondifera, Acropora pulchra, and Goniastrea aspera) and of partially bleached and completely bleached colonies of P. frondifera were examined. The FA composition did not differ among healthy corals, but differed significantly among healthy, partially bleached, and completely bleached specimens of P. frondifera. Completely bleached corals contained significantly lower lipid and total FA content, as well as lower relative amounts of polyunsaturated FAs and higher relative amounts of saturated FAs, than healthy and partially bleached corals. Furthermore, there was a significantly higher relative concentration of monounsaturated FAs and odd-numbered branched FAs in completely bleached corals, indicating an increase in bacterial colonization in the bleached corals.     

Effect of salinity of medium on cellular fatty acid composition of marine algaPorphyridium cruentum (Rhodophyceae)

Porphyridium cruentum Näg. (clone 161) was found to grow best in medium containing between 0.45 M and 0.8 M NaCl. From studies done on growing cultures, the palmitic acid content of the cells decreased with increasing NaCl concentration of the medium. Conversely, when the culture was transferred from a 0.8 M NaCl medium to 0.2 M NaCl, the amount of palmitic acid in thePorphyridium cells increased with time of incubation and it contributed up to 64.5% of the total fatty acid content. There appears to be a negative correlation between the cellular content of palmitic acid and the growth lag. The oleic acid content varied only marginally with increasing NaCl concentration. The poly-unsaturated acid content (linolenic and arachidonic acids) decreased initially and then increased with NaCl concentration up to and beyond ca. 0.8 M NaCl respectively. At 1.5 M NaCl, the poly-unsaturated fatty acids amounted to 78.2% of the total fatty acids in the cell. For stationary phaseP. cruentum cultures, a similar relationship existed between fatty acids and NaCl concentration. However, palmitic acid was accumulated up to three-fold more when compared to the exponential culture grown in low salinity. In addition stearic acid was also found in significant quantities.     

Influence of culture temperature on the growth,biochemical composition and fatty acid profiles of six Antarctic microalgae

The growth, biochemical composition and fatty acid profiles of six Antarctic microalgae cultured at different temperatures, ranging from 4, 6, 9, 14, 20 to 30 ^∘C, were compared. The algae were isolated from seawater, freshwater, soil and snow samples collected during our recent expeditions to Casey, Antarctica, and are currently deposited in the University of Malaya Algae Culture Collection (UMACC). The algae chosen for the study were Chlamydomonas UMACC 229, Chlorella UMACC 234, Chlorella UMACC 237, Klebsormidium UMACC 227, Navicula UMACC 231 and Stichococcus UMACC 238. All the isolates could grow at temperatures up to 20 ^∘C; three isolates, namely Navicula UMACC 231 and the two Chlorella isolates (UMACC 234 and UMACC 237) grew even at 30 ^∘C. Both Chlorella UMACC 234 and Stichococcus UMACC 238 had broad optimal temperatures for growth, ranging from 6 to 20 ^∘C (μ = 0.19 – 0.22 day^–1) and 4 to 14 ^∘C (μ = 0.13 – 0.16 day^–1), respectively. In contrast, optimal growth temperatures for NaviculaUMACC 231 and Chlamydomonas UMACC 229 were 4 ^∘C (μ = 0.34 day^–1) and 6–9 ^∘C (μ = 0.39 – 0.40 day^–1), respectively. The protein content of the Antarctic algae was markedly affected by culture temperature. All except Navicula UMACC 231 and Stichococcus UMACC 238 contained higher amount of proteins when grown at low temperatures (6–9 ^∘C). The percentage of PUFA, especially 20:5 in Navicula UMACC 231 decreased with increasing culture temperature. However, the percentages of unsaturated fatty acids did not show consistent trend with culture temperature for the other algae studied.     

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart^® (125 mm×4 mm I.D.) Nucleosil 100-5 μm C₁₈ AB cartridge column, using a gradient elution of MeOH–buffer pH 3.9 1:99→15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate–citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H₃PO₄ 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2±2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (Cl_R) and systemic (Cl_S) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney Cl_R/Cl_S calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73±0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87±0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.     

Effect of growth temperature on the fatty acid composition of Mycobacterium phlei

In Mycobacterium phlei, fatty acid unsaturation increased with decreasing temperature. The 10-hexadecenoic acid content increased as the temperature was reduced from 35°C to 26–20°C. At lower temperatures tuberculostearic acid decreased while oleic and linoleic acids increased, the latter being found in M. phlei for the first time. Concomitantly palmitic acid content decreased, and the 6- and 9-hexadecenoic acids increased slightly on reducing the temperature from 35 to 10°C. Thus, down to 26–20°C palmitic acid was mainly replaced by 10-hexadecenoic acid. From this range down to 10°C, palmitic and tuberculostearic acids were replaced by oleic and linoleic acids. Consequently, fatty acid branching decreased and mean chain length increased, as the temperature was reduced. These observations support the view that regulation of membrane fatty acid composition is part of microbial temperature adaptation, and that themechanism behind the responses might be more complex than generally believed.Abbreviations ACP acyl carrier protein - FAS I (Type I) fatty acid synthetase I - FAS II (Type II) fatty acid synthetase II - MGLP methylglucose containing lipopolysaccharide - MMP methylmannose containning polysaccharide     

Effect of ploidy variation on amino acid composition ofDatura innoxia (Solanaceae)

In haploid, diploid, triploid, tetraploid and hexaploid cytotypes of the scopalamine-yieldingDatura innoxia, the amino acid profiles were studied. The results suggest that the amino acid composition is influenced quantitatively by increasing ploidy levels, but in an irregular way.     

Effect of maternal fat reserves on the fatty acid composition of sardine (Sardina pilchardus) oocytes

We compared the fatty acid (FA) composition of the muscle and gonads of female Iberian sardines with hydrated oocytes collected during the 2002/03 spawning season off southern Portugal (November and February) and off western Portugal (February). Sardine condition and total FA concentration in the muscle decreased between the two sampling dates, while the gonadosomatic index was similar between samples. Total monounsaturated FA concentrations in sardine gonads were different for the three samples while saturated and polyunsaturated FA concentrations were similar. Significant linear relations were found between FA concentrations in female muscle and oocytes, including eicosapentaenoic acid (EPA; 20:5n − 3) and arachidonic acid (AA; 20:4n − 6), both being essential for normal larval development. The concentration of docosahexaenoic acid (DHA; 22:6n − 3) in oocytes was independent on muscle concentration, probably resulting from its selective transfer to the oocytes. The EPA/DHA ratio was highly conserved in sardine tissues, while DHA/AA and EPA/AA ratios varied significantly between samples. These results indicate that the FA content of eggs produced by sardines varies throughout the spawning season, egg FA concentrations decreasing as females lose condition, and FA composition also shows spatial variability. Both types of variability may have a significant impact on egg quality, particularly on the amount of reserves available to larvae affecting their resistance to starvation, and the appropriate FA composition required for normal growth.     

Changes in fatty acid composition of Mytilus galloprovincialis (Lmk) fed on microalgal and wheat germ diets

Dietary fatty acid incorporation and changes in various lipid and phospholipid classes in the mussel Mytilus galloprovincialis subjected to three different dietary regimens were analysed and compared. Group A was unfed; group B received a diet consisting of 100% Thalassiosira weissflogii, exhibiting the typical fatty acid composition of diatoms, and group C received a diet consisting of 100% wheat germ conferring a 18:2:n-6 abundance. Biochemical analyses of diets and mussels were carried out at the beginning and at the end of the 30-day experimental period. Starvation and T. weissflogii based diet poorly affected mussel growth and fatty acid composition which remained unchanged. On the contrary, the wheat germ-based diet increased the condition index and deeply affected the fatty acid profile of all lipid and phospholipid classes. The high dietary 18:2n-6 level drastically reduced tissue content of 20:4n-6, 20:5n-3 and 22:6n-3. The biosynthesis of Non Methylene Interrupted (NMI) dienoic fatty acid appeared to be insensitive to the high input of 16:1n-7 and 18:1n-9 respectively from diet B and C, and to the PUFA shortage of diet C. Nevertheless the two NMI trienoic derivatives, 20:3Δ5,11,14 and 22:3Δ7,13 16, were found higher in C with respect to other groups, presumably due to the high 18:2n-6 content of this diet.     

Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.     

Diet-induced variations in fatty acid content and composition of two on-grown stages of Artemia Salina

Three species of microalgae, the freshwater Euglena gracilis and themarine Dunaliella salina and Tetraselmis suecica, were fed tothe brine shrimp Artemia salina in order to compare their suitabilityin terms of fatty acid enrichment, and their effect on the biometric parametersof the zooplankter. The fatty acid content and composition were analyzed for the post-larval and pre-adult stages of Artemia fed the algae and theresults compared to the initial content of unfed 24-hour post-hatch nauplii.Differences in the total fatty acid content occurred between the three stages,the fatty acid profile being determined by the composition of the diet. A decreasing trend for almost all the individual fatty acids occurred throughdevelopment from post-larva to pre-adult with each of the three algal diets.Biometrical differences between Artemia fed the marine algae and that fed Euglena were not consistent in the post-larval stage, but became considerable in the pre-adult stage. Artemia fed with Euglena achieved twice the weight of animals fed the marine algae and showed thehighest length. The implications for the use of on-grown Artemia as afeed in larviculture of marine and freshwater fish and crustaceans are considered.     

Ubiquinone system,GC contents and cellular fatty acid composition of species of the form-genusMalbranchea andCoccidioides immitis for chemotaxonomic study

Ubiquinone system, GC contents and cellular fatty acid (CFA) composition were analyzed as criteria for chemotaxonomy ofMalbranchea species andCoccidioides immitis, which are suggested to be phylogenetically related. Based on the major ubiquinone,Malbranchea spp. were divided into two groups, of which one group possessed the same major ubiquinone asC. immitis. Similar GC contents and CFA profiles were obtained for the species ofMalbranchea andC. immitis. On the basis of these criteria the relationships between the fungi are discussed.     

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentrationon the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) wascultured in media with different nitrogen sources and concentrations.During the pre-logarithmic phase, the alga grew faster with ammoniumas N source than with nitrate, but the reverse applied during thepost-logarithmic phase. The alga grew poorly in N-free mediumor medium with urea as the sole N source. In the same growth phase,ammonium medium resulted in higher yield of total lipid, but the EPA yielddid not differ significantly different from that using nitrate medium. Themaximum growth rate occurred in medium containing 1.28 mmolL^-1 sodium nitrate, while maximum EPA and total lipid contents werereached at 1.92 mmol L^-1, when EPA accounted for 27.9% totalfatty acids. The growth rate kept stable when NH₄Cl ranged from0.64 to 2.56 mmol L^-1, and the maximum content of total lipidand EPA occurred in the medium with 2.56 mmol L^-1NH₄Cl. The EPA content was higher in the pre- thanpost-logarithmic phase, though the total lipid content was lower. Thehighest EPA content expressed as percent total fatty acid was 27.9% innitrate medium and and 39.0% in ammonium medium.     

Phospholipid composition and fatty acid desaturation in the roots of rye during acclimatization of low temperature

When the roots of rye plants grown at 20°C were cooled to 8°C the concentration of phospholipid in them more than doubled over a 7 d period in comparison with that in roots remaining at 20°C. The relative abundance of lecithin (PC) declined while that of phosphatidyl ethanolamine (PE) increased; this change was completed after 2 d cooling. Labelling with ³²P suggested that turnover of phospholipids may be inhibited by low temperature. Acyl lipids contained an increased proportion of linolenic acid (18:3) and reduced proportion of linoleic acid (18:2) when roots were cooled at 8°C for 7 d. The ratio of these acids is a relatively more sensitive indicator of desaturation than is the double bond index. Cooling brought about no change in the abundance of the principal saturated acid, palmitic (16:0). In the first 3 days of cooling PC and PE desaturated markedly while there was no change in galactosyl and neutral lipids. Desaturation did not appear to be greatly sensitive to the concentration of dissolved O₂ and was only partly inhibited in 8°C solutions where the oxygen concentration was lowered to 0.5–2.0%. Positional analysis of acyl chains in PC and PE showed that more than 90% of all 16:0 is associated with position I while 65% of the 18:2+18:3 is associated with position II. When roots are cooled the abundance of 18:3 increases in both chains but the relative distribution of saturated and unsaturated fatty acids remains constant in positions I and II. At both 20°C and 8°C there is a high probability that a saturated chain in position I will be paired with the polyunsaturated one in position II.Abbreviations PC Lecithin - PE phosphatidyl ethanolamine - TLC thin layerchromatography - BHT butylatedhydroxytoluene     

Seed oil fatty acid patterns of theAconitum-Delphinium-Helleborus complex (Ranunculaceae)

Many members ofRanunculaceae contain unusual fatty acids in their seed oils. This leads to rather typical genus-specific fatty acid patterns or fingerprints in these seed oils. The members of theDelphinioideae and/orHelleboroideae, however, do not contain highly unusual fatty acids. Nevertheless, their seed oil fatty acid fingerprints are also fairly typical and genus-specific, and the patterns found are rather consistent throughout several species of one genus. It was found that species ofAconitum do not contain fatty acids with 20 carbon atoms.Delphinium, Consolida, Helleborus, Nigella and others do contain C₂₀ fatty acids. In allHelleborus species, for example, there was a consistent C₂₀ fatty acid pattern of 20:020:120:2>20:3. Species ofNigella andGaridella contain high levels,Helleborus low levels, of 20:2n-6 in their seed oils.Delphinium andAconitum both contain low levels of 18:3n-3, whereasHelleborus spp. consistently show high levels of this fatty acid. The genus-specific fatty acid patterns found are discussed, and a correlation with the subfamily and tribe affiliation of the genera investigated here is attempted.     

Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids

In the presence of sublethal concentrations of phenol, 4-chlorophenol, and p-cresol in the growth medium, cells of Escherichia coli modified the fatty acid composition of their lipids. The result of these changes was an increase in the degree of saturation of lipids probably in order to compensate an increase of fluidity of the membrane induced by the phenols. Supplementation of the growth medium with saturated fatty acids could also enhance the degree of lipid saturation due to the incorporation of the acyl chains in the phospholipids. At the same time the growth of cells was less inhibited than in unsupplemented cells. The increase of tolerance of cells by manipulating the lipid composition indicates that the membrane structure plays a crucial role in the mode of action of phenols.