Analysis of interspecific variation in relative fatty acid composition: use of flow cytometry to estimate unsaturation index and relative polyunsaturated fatty acid content in microalgae

Relative polyunsaturated fatty acid content and unsaturation index are very important composition variables in the use of microalgae both for animal and human nutrition and biofuel production. A readily available technique to rapidly and inexpensively estimate relative fatty acid composition is very important for mass screening of new strains for the production of different types of oil. This study demonstrates the validity of Nile Red staining and flow cytometry for quick estimation of unsaturation index and relative fatty acid content in microalgae. Nile Red staining allows polar and neutral lipid contents to be estimated, and in this study a significant correlation was observed between polar/neutral ratio and fatty acid content in the species studied, corresponding to higher polyunsaturated fatty acid content in the polar lipid fraction of microalgae. This technique enables quick estimation of relative polyunsaturated fatty acid content and interspecific variation, as well as variations caused by culture conditions. In the species studied, most variability in fatty acid composition was due to variation in monounsaturated and polyunsaturated fatty acids, with less variation observed in saturated fatty acid content.     

Neutral lipid production in <Emphasis Type="Italic">Dunaliella salina</Emphasis> during osmotic stress and adaptation

The salt-tolerant green microalga Dunaliella salina can survive both hyper- and hypo-osmotic shock. Upon osmotic shock, the cells transiently and rapidly decreased or increased in size within minutes and slowly over hours acquired their original cell size and volume. Cell size distribution differs significantly in the cultures grown in the salinity range from 1.5 to 15 % NaCl. By using Nile Red fluorescence to detect neutral lipids, it became clear that only hyper-osmotic shock on cells induced transient neutral lipid appearance in D. salina, while those transferred from 9 to 15 % NaCl stimulated the most neutral lipid accumulation. These cells grew well in 9 % NaCl, but they cannot recover a shift to 15 % NaCl and cell division is accordingly slowed down. The transient appearance of neutral lipid could be dependent on the inhibition of cell division experiencing the NaCl shift. Moreover, the effect of nutrient limitation slows down cell division and photosynthesis as a secondary result, which triggers the cells to accumulate neutral storage lipids when they entered the stationary phase, which is seen in all the batch cultures of D. salina grown in the salinity range of 3–15 %. The changes in salt concentration did not significantly influence the overall fatty acid composition in D. salina cells. Although there shows both increased amounts of total lipids and neutral lipids in the cells grown in salinity higher than 9 % NaCl, lipid productivity is however compromised by the slower cell growth rate and lower cell density under this condition.     

Intraspecific Variation of Unusual Phospholipids from Corynebacterium spp. Containing a Novel Fatty Acid

The novel fatty acid trans-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as Arthrobacter globiformis) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of Corynebacterium variabilis DSM 20132^T and DSM 20536. All three strains possessed the novel fatty acid, identifying trans-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of Corynebacterium sp. strain LMG 3820 were similar to those of the type strain but different from those of Corynebacterium variabilis DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132^T and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.     

A comparative study of the fatty acid profile of Artemia franciscana and A. persimilis cultured at mesocosm scale

The goal of this study was to examine the fatty acid (FA) profile of two Artemia species, A. persimilis (Argentina) and A. franciscana (Great Salt Lake,Utah; USA) in coexistence at mesocosm scale. The experiment was carried out to 1) evaluate putative differences in the fatty acid composition of both species while they share resources and 2) to investigate the causes of such differences. Although the coexistence of these species in nature has not yet been observed, it remains possible that this situation may arise in the future mainly due to the invasive ability of A. franciscana. FA analyses were performed on individuals as well as on pooled biomasses of each species, and integrated in multivariate principal components analysis (PCA). Comparison of the relative abundance of FA between the two species revealed that interspecific differences in FA composition are greater than intraspecific variability. Higher percentages of unsaturation were found in the fatty acids of A. persimilis compared to A. franciscana, demonstrating that aside from a high phenotypic effect of diet on the FA composition of the animals, a species-specific genotypic effect should not be discarded.     

Cellular Fatty Acid Composition of Selected Pseudomonas Species

The cellular fatty acid composition of 10 reference strains representing eight species of Pseudomonas was determined by gas-liquid chromatography. A variety of acids were detected in these organisms, including branched and straight-chained acids, cyclopropane, and hydroxy acids. Comparison of the presence and relative amounts of these acids among strains was useful for distinguishing various Pseudomonas species.     

Peculiarities of fatty acid composition of the genusCaulobacter

The fatty acid composition of 35 strains of stalked bacteria belonging to 17 of the hitherto described 19 species and 10 unidentified strains of the genusCaulobacter was studied. ll-Methyl-cis-octadec-11-enoic acid presumably synthesized fromcis-vaccenic acid was detected in all the strains in amounts of 0.4 – 34.7 % and was considered as a chemotaxonomic marker of the genus. During growth on a peptone-yeast medium, the caulobacters synthesized, along with the fatty acids which are typical of gram-negative bacteria, some normal and branched fatty acids with 15 and 17 carbon atoms (1–49 %). The synthesis of these acids was inhibited by glucose. The cell shape of stalked bacteria (fusiform, vibrioid or bacteroid) is not obviously associated with the contents of individual fatty acids.     

Characterization and Differentiation of Filamentous Fungi Based on Fatty Acid Composition

Cellular fatty acid composition of 100 different filamentous fungi, including oomycetes, zygomycetes, ascomycetes, basidiomycetes, and sterile mycelia, was analyzed to determine if they can be differentiated from one another on this basis and how minor variations in culture temperature and age affect this characteristic. Many fungi were found to possess the same fatty acids but produced different relative concentrations of each. Some fungi differed in both the fatty acids produced and in the relative concentrations of others. Multivariate discriminant analysis demonstrated that all of the species included in this study had significantly different (P < 0.001) fatty acid profiles. Each of the three phyla from which representative species were analyzed and the sterile forms had distinctive fatty acid profiles. Significant differences in fatty acid composition were also found at the intraspecific level. Both culture temperature and age affected fatty acid composition in the fungi examined, but when these factors were held constant, variance in fatty acid composition was not a problem and fungal fatty acid profiles could be differentiated statistically.     

Fluorescent measurement of lipid content in the model organism Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is one of the most studied microalgae, which has the potential to be used as a model system to study lipid metabolism. Establishment of a method in this organism for rapid and simple measurement of neutral lipids is desirable. Fluorescent measurement of neural lipids by Nile Red staining has been widely used in various cell types including microalgae. However, a systematic study of Nile Red staining to measure neutral lipids in Chlamydomonas has not been reported. Here, we show that Nile Red staining is suitable for relative and absolute quantification of neutral lipids as well as for possible large-scale screening for mutants defective in lipid accumulation. We have compared and optimized the factors involved Nile Red staining including solvents, cell concentration, staining time, and Nile Red concentration. We determined that 5 % DMSO with 1 μg mL^?1 Nile Red and 5–15-min time window after staining was optimal for measuring lipid content of cells within the range of 1 to 8?×?10⁶ cells mL^?1. The absolute quantification of neutral lipids could be achieved by standard addition method. In addition, we developed a protocol that could be potentially used for large-scale screening for cells with different lipid content. Thus, the work reported here provides timely needed techniques to facilitate Chlamydomonas to be used as a model organism for studying lipid metabolism for biodiesel production.     

Fatty Acids Associated with the Cell Wall in Film Strains of Saccharomyces

Fatty acid contents were estimated in the cell wall of Saccharomyces. The fatty acids responsible for cell wall hydrophobicity were classified by ease of extraction to ‘readily extractable’ and ‘bound’ acids. The readily extractable fatty acids were easily extracted with pentane and chloroform-methanol. The fatty acids extracted with chloroform-methanol were quite effective for cell wall hydrophobicity, but the fatty acids extracted with pentane were not. The bound fatty acids comprised in the phospholipids phosphatidylethanolamine and phosphatidylserine, which were rigidly associated with the cell wall. These phospholipids were not extractable until they were released from the cell wall by pronase. Chloroform-methanol extraction caused a reduction in cell wall phospholipid content, particularly after treatment with pronase. The fatty acid content of the resultant cell wall was lowered to below 7% of initial content. Phospholipids contained more saturated fatty acid than readily extractable lipids. Phospholipids greatly contributed to cell wall hydrophobicity of various film strains of Saccharomyces.     

Distribution of seed fatty acids and the taxonomy of Vochysiaceae

The distribution of seed fatty acids of 16 species of Vochysiaceae native to Brazil, corresponding to the genera Callisthene, Qualea, Salvertia and Vochysia, and amounting to 57 samples, were studied. The methyl esters of the fatty acids were analyzed by GC–EIMS. For most species, fatty acid patterns are seemingly useful for species characterization. Intraspecific variability of samples of Salvertia grandiflora seems to have geographic significance. With one exception, palmitic and oleic acids predominate in the fatty acids distribution of Qualea and Callisthene. Q. grandiflora possesses exceptional amounts of lauric acid. Relatively high amounts of either C_20–C₂₂ (saturated or monounsaturated) or stearic acids characterize Salvertia and Vochysia. UPGMA analysis of fatty acids distribution based on Euclidean distances resulted in a cluster combining Callisthene and all samples of Qualea, except Q. grandiflora. Samples of Vochysia are combined into several clusters. The dendrogram is suggestive of taxonomic relationships, allowing the distinction of taxa down to the sub-sectional level. V. pyramidalis, from section Ciliantha, subsection Ferrugineae, has closer chemical affinities with species of section Vochysiella, subsection Decorticantes. Comparing with monomers of polysaccharide seed cell walls of Vochysiaceae, fatty acid patterns revealed lower intraspecific variability and higher taxonomic resolution.     

Intra-and interspecific variability of fatty acid composition of soft corals

The intra-and interspecific variability of fatty acid (FA) composition of soft corals was examined in the tropical alcyonarian Sarcophyton sp., tropical gorgonian Euplexaura erecta, and boreal alcyonarian Gersemia rubiformis. Characteristic significant differences in the FA composition were found between these species belonging to different taxonomic groups and habitats. We assume that the FA groups 14: 0 + 16: 0 + 18: 3n-6, 16: 2 + 20: 4n-6 + 24: 5n-6, and 18: 1n-7 + 20: 1n-7 + 20: 5n-3 + 24: 6n-3 are characteristic of Sarcophyton sp., E. erecta, and G. rubiformis respectively. There were no significant differences (p > 0.05) between the three soft coral species in the content of oleic, linoleic, and docosahexaenoic acids. The relative content of more than 45% of individual FA did not significantly differ (p > 0.05) between the pairs of species, i.e., intraspecific variations exceeded interspecific ones. The reasons for intraspecific variability of soft coral FA composition are discussed. Control of this variability is needed when using individual FA as chemotaxonomic and food markers.     

Cellular fatty acid profiles for the differentiation of Penicillium species

Cellular fatty acid composition of eighteen species of Penicillium was studied to investigate its taxonomic usefulness. Many fungi included in this study displayed the same fatty acid composition but differed in relative concentration. Several test species presented the same fatty acid composition but differed in relative concentration. The principal fatty acids were palmitic (16:0), oleic (18:1) and linoleic (18:2). The amount of unsatured fatty acids varied between 68.5% and 78.5%. Multivariate analyses of data showed that it is possible to differentiate some species that belonged to different Penicillium groups. The level of agreement of long chain fatty acids with morphological taxonomy was acceptable.     

The Influence of Bacterial Diet on Fat Storage in C. elegans

Background

The nematode Caenorhabditis elegans has emerged as an important model for studies of the regulation of fat storage. C. elegans feed on bacteria, and various strains of E. coli are commonly used in research settings. However, it is not known whether particular bacterial diets affect fat storage and metabolism.

Methodology/Principal Findings

Fat staining of fixed nematodes, as well as biochemical analysis of lipid classes, revealed considerable differences in fat stores in C. elegans growing on four different E. coli strains. Fatty acid composition and carbohydrate levels differ in the E. coli strains examined in these studies, however these nutrient differences did not appear to have a causative effect on fat storage levels in worms. Analysis of C. elegans strains carrying mutations disrupting neuroendocrine and other fat-regulatory pathways demonstrated that the intensity of Nile Red staining of live worms does not correlate well with biochemical methods of fat quantification. Several neuroendocrine pathway mutants and eating defective mutants show higher or lower fat storage levels than wild type, however, these mutants still show differences in fat stores when grown on different bacterial strains. Of all the mutants tested, only pept-1 mutants, which lack a functional intestinal peptide transporter, fail to show differential fat stores. Furthermore, fatty acid analysis of triacylglycerol stores reveals an inverse correlation between total fat stores and the levels of 15-methylpalmitic acid, derived from leucine catabolism.

Conclusions

These studies demonstrate that nutritional cues perceived in the intestine regulate fat storage levels independently of neuroendocrine cues. The involvement of peptide transport and the accumulation of a fatty acid product derived from an amino acid suggest that specific peptides or amino acids may provide nutritional signals regulating fat metabolism and fat storage levels.     

Inter-and intraspecific differences in fatty acid composition of freshwater crustacean zooplankton

1. The composition of fatty acids (FA) from C14 to C18 was measured for edible seston and for individual Daphnia galeata , Diaphanosoma brachyurum and Eodiaptomus japonicus from Lake Biwa using a pyrolysis–gas chromatography (Py–GC) method. Based on the relative abundance of the FA, inter-and intraspecific differences in composition were examined.
2. Among the FA, C16 : 0 was the most abundant, both in the three zooplankton species and in edible seston smaller than 20 μm, suggesting that the composition of the zooplankton was roughly reflected by their diet. However, the abundance of C18 : 1 relative to C16 : 1 and C18 : 0 was much higher in each zooplankton species than in the diet.
3. Comparison of the relative abundance of FA among the three zooplankton species revealed that intraspecific differences in FA composition are greater than interspecific differences. These results indicate that variability in FA composition is not necessarily species-specific, and can be obscured by variation in composition between individuals.     

The effect of environmental factors on the fatty acid composition of copepods and Artemia in the Sfax solar saltern (Tunisia)

The biochemical composition and abundance variation of zooplankton (copepods and Artemia salina) were determined in four ponds of increasing salinity (A5, A16, C41 and M2) in the Sfax solar saltern (Tunisia). The zooplankton community was dominated by copepods in the ponds A5, A16 and C41. The pond M2 was marked by the presence of only Artemia salina. Our results showed the dominance of total saturated fatty acids (SFA), which made up 57%–95% of total fatty acids (TFA). SFA 16:0 and 18:0 dominate in all ponds. A. salina showed the highest amounts of the total monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA), this indicates that this species could be employed in hatcheries and used as food source for some aquarium species. Fatty acids of herbivory, proportion of all diatom markers to all flagellate markers (D/F), were negatively correlated with the total zooplankton (r = −0.998, p < 0.05). A. salina was negatively correlated with a biomarker for carnivory polyunsaturated fatty acids/saturated fatty acids (PUFA/SFA) (r = −0.959, p < 0.05). The dietary quality of zooplankton seems to be dependent on food availability in the four studied ponds.     

A Study of Fatty Acids as a Taxonomic Tool for Dermatophyte Fungi

The fatty acid composition of 64 strains from 15 species of dermatophyte fungi was studied by gas-liquid chromatography. The percentage amounts of palmitic, palmitoleic, stearic, oleic and linoleic acids were measured. These proved to be very similar for all species, with considerable intraspecific variation. As a group, Epidermophyton floccosum strains could be distinguished but individual strains could not be assigned reliably to any species or genus. No significant changes due to age or formaldehyde treatment were noted.     

Fatty acid composition of muscle and heart tissue of Nile perch,Lates niloticus,and Nile tilapia,Oreochromis niloticus,from various populations in Lakes Victoria and Kioga,Uganda

The fatty acid composition in the heart tissue and muscle tissue of the Nile perch, Lates niloticus, and Nile tilapia, Oreochromis niloticus populations from Lakes Kioga and Victoria was determined by methanolysis and gas chromatography of the resulting fatty acid methyl esters. The analytical data were treated by multivariate principal component analysis. The most abundant individual fatty acids were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1n9), vaccenic acid (18:1n7), arachidonic acid (20:4n6) and docosahexaenoic acid (22:6n3). Due to high levels of both n6 and n3 fatty acids, the ratios of n3 to n6 were between 1 and 2, typical for freshwater fish species. Two Lake Victoria and one Lake Kioga populations of Nile tilapia and Nile perch were distinguished by the fatty acid profiles in their heart and muscle tissue. The heart tissue showed better separation than muscle tissue, due to dominance of polar phospholipids. It is rationalised that genetics are more important than diet in determining the fatty acid composition of the tissues.     

Unsaturated fatty acid composition of wild type and respiratory deficient yeasts after aerobic and anaerobic growth

An analysis is given of the fatty acid composition of 18 yeast species, predominantly of the genus Saccharomyces; respiratory deficient mutant strains are included. The results are discussed from chemotaxonomical and physiological viewpoints, with special attention to unsaturated fatty acids and their relation to the petite mutation. The fatty acid composition of anaerobically grown Saccharomyces cerevisiae remains restricted, as far as unsaturated fatty acids are concerned, to those added to the medium and it may thus differ considerably from the composition after aerobic growth. Depending on the acids added, the cells may contain either palmitoleic or linoleic acids as the sole unsaturated fatty acid after anaerobic growth and as the predominant unsaturated fatty acid after aerobic growth. In contrast to all other known eukaryotes, Schizosaccharomyces japonicus seems to possess an anaerobic pathway for synthesis of unsaturated fatty acids.     

Composition of the floral nectar of different subgenera of Argentinian Passiflora species

The composition of the floral nectar sugars and amino acids of four species of Passiflora (P. foetida, P. caerulea, P. suberosa, and P. misera) included in different infrageneric taxa and with distinct pollination mechanisms has been studied. The effect of weather and floral age on nectar volume, existence, and total and relative amounts of the various compounds was explored. The proportion of sugars was rather constant within a given species whereas the composition, number, and total quantity of amino acids showed great intraspecific and intra-plant variability; these nectar properties were independent of floral stage or meteorological conditions. Species belonging to the same subgenus displayed equivalent sugar ratios and similar total amount of amino acids, so these characteristics might be conservative in the genus. For all species, the amino acid concentration surpassed known values for their respective pollination syndromes, viz. bee and wasp-pollinated flowers. No relationship emerged between pollinators with different glossa length and nectars with distinct sugar ratios. Rather, nectar chemical composition seems to reflect taxonomic relationships.     

Phospholipid and acid composition of Nocardia and nocardoid bacteria as criteria of classification

Phospholipid and acid composition of 5 strains of ‘true’ Nocardia and 4 strains of nocardoid bacteria have been studied. A great homogeneity was found in all the Nocardia species: phospholipids consist of cardiolipin, phosphatidyl ethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Streptomyces (Nocardia) mediterranei did not contain phosphatidylinositol and Oerskovia (Nocardia) turbata had no phosphatidyl ethanolamine. The fatty acid composition of these phospholipids was determined and was found different in Nocardia and nocardoid species. Nocardia were rich in straight chain fatty acids and tuberculostearic acid while the phospholipids of nocardoid bacteria contained greater amounts of branched fatty acids. The fatty acids from acetone soluble lipids consisted of hydroxy and non-hydroxy compounds. Hydroxy acids were found in Nocardia which contained nocardic acids: high MW β-hydroxy α-branched acids and in S. mediterranei which contained β-hydroxy acids with 15–17 carbon atoms. Non-hydroxy acids were essentially palmitic and tuberculostearic acids in Nocardia species while S. mediterranei and O. turbata contained great amounts of iso acids from C₁₄ to C₁₇. Phospholipid and acid composition are discussed as criteria of taxonomic classification of Nocardia and related Actinomycetes.