Harnessing Indigenous Plant Seed Oil for the Production of Bio-fuel by an Oleaginous Fungus,Cunninghamella blakesleeana- JSK2, Isolated from Tropical Soil

Cunninghamella blakesleeana- JSK2, a gamma-linolenic acid (GLA) producing tropical fungal isolate, was utilized as a tool to evaluate the influence of various plant seed oils on biomass, oleagenicity and bio-fuel production. The fungus accumulated 26 % total lipid of their dry biomass (2 g/l) and 13 % of GLA in its total fatty acid. Among the various plant seed oils tested as carbon sources for biotransformation studies, watermelon oil had an effect on biomass and total lipid increasing up to 9.24 g/l and 34 % respectively. Sunflower, pumpkin, and onion oil increased GLA content between 15–18 %. Interestingly, an indigenous biodiesel commodity, Pongamia pinnata oil showed tremendous effect on fatty acid profile in C. blakesleeana- JSK2, when used as a sole source of carbon. There was complete inhibition of GLA from 13 to 0 % and increase in oleic acid content, one of the key components of biodiesel to 70 % (from 20 % in control). Our results suggest the potential application of indigenous plant seed oils, particularly P. pinnata oil, for the production of economically valuable bio-fuel in oleaginous fungi in general, and C. blakesleeana- JSK2, in particular.     

Kinetic study of growth,lipid and carotenoid formation in β-carotene producing <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The oleaginous and red yeast Rhodotorula glutinis CCY 20-2-26 is known for its high-storage lipids accumulation in cells as well as for β-carotene biosynthesis. The work is focused on the study of lipogenesis and carotenoid pigments formation by the yeast grown in media with four different C/N ratios (20:1, 50:1, 70:1 and 100:1). Total fatty acids accumulation in cells reached the maximal value of 48% and yielded up to 8.9 g fatty acid/L media under C/N 70:1. On contrary, while the highest pigment accumulation in the yeast (1268 μg/g of cells) was found at the C/N ratio of 20:1, the maximal carotenoid yield (mainly β-carotene) of 12.7 mg/L was obtained under 50:1 ratio. Calculation of kinetic parameters of metabolites production revealed the metabolic correlations among glucose consumption, lipogenesis and carotenoid biosynthesis. It was demonstrated that glucose exhaustion resulted in reduction of growth and lipid accumulation in cells. Then, storage fatty acids, especially palmitic and oleic acids, begun to be degraded by β-oxidations and formed acetyl-CoA which was especially used for biosynthesis of β-carotene. This is the first study providing the new hypothesis about the metabolic connection between fatty acids and β-carotene metabolism in the red yeasts.     

Zeaxanthin Production by Novel Marine Isolates from Coastal sand of India and its Antioxidant Properties

Zeaxanthin carotenoids are class of commercially important natural products and diverse biomolecules produced by plants and many microorganisms. Bacteria often produce a cocktail of polar and nonpolar carotenoids limiting their industrial applications. Marine members of the family Flavobacteriaceae are known to produce potential carotenoids such as astaxanthin and zeaxanthin. A few bacterial species have been reported for the predominant production zeaxanthin. Here, we report the molecular identification of the zeaxanthin as a major carotenoid produced by two novel bacteria (YUAB-SO-11 and YUAB-SO-45) isolated from sandy beaches of South West Coast of India and the effect of carbon sources on the production of zeaxanthin. The strains were identified based on the 16S rRNA gene sequencing as a member of genus Muricauda. The closest relatives of YUAB-SO-11 and YUAB-SO-45 were Muricauda aquimarina (JCM 11811^T) (98.9 %) and Muricauda olearia (JCM 15563^T) (99.2 %), respectively, indicating that both of these strains might represent a novel species. The highest level of zeaxanthin production was achieved (YUAB-SO-11, 1.20?±?0.11 mg g^?1) and (YUAB-SO-45, 1.02?±?0.13 mg g^?1) when cultivated in marine broth supplemented with 2 % NaCl (pH 7) and incubated at 30 °C. Addition of 0.1 M glutamic acid, an intermediate of citric acid cycle, enhanced the zeaxanthin production as 18 and 14 % by the strains YUAB-SO-11 and YUAB-SO-45 respectively. The zeaxanthin showed in vitro nitric oxide scavenging, inhibition of lipid peroxidation, and 2,2-diphenyl-1-picryl hydrazyl scavenging activities higher than the commercial zeaxanthin. The results of this study suggest that two novel strains YUAB-SO-11 and YUAB-SO-45 belonging to genus Muricauda produce zeaxanthin as a predominant carotenoid, and higher production of zeaxanthin was achieved on glutamic acid supplementation. The pigment showed good in vitro antioxidant activity, which can be exploited further for commercial applications.     

Microbial Lipid Production from Enzymatic Hydrolysate of Pecan Nutshell Pretreated by Combined Pretreatment

Biodiesel is a fuel composed of monoalkyl esters of long-chain fatty acids derived from renewable biomass sources. In this study, biomass waste pecan nutshell (PS) was attempted to be converted into microbial oil. For effective utilization of PS, sequential pretreatment with ethylene glycol–H₂SO₄–water (78:2:20, wt:wt:wt) at 130 °C for 30 min and aqueous ammonia (25 wt%) at 50 °C for 24 h was used to enhance its enzymatic saccharification. Significant linear correlation was obtained about delignification-saccharification (R ² = 0.9507). SEM and FTIR results indicated that combination pretreatment could effectively remove lignin and xylan in PS for promoting its enzymatic saccharification. After 72 h, the reducing sugars from the hydrolysis of 50 g/L pretreated PS by combination pretreatment could be obtained at 73.6% yield. Using the recovered PS hydrolysates containing 20 g/L glucose as carbon source, microbial lipids produced from the PS hydrolysates by Rhodococcus opacus ACCC41043. Four fatty acids including palmitic acid (C16:0; 23.1%), palmitoleic acid (C16:1; 22.4%), stearic acid (C18:0; 15.3%), and oleic acid (C18:1; 23.9%) were distributed in total fatty acids. In conclusion, this strategy has potential application in the future.     

Fatty Acid Content and Profile of the Aerial Microalga Coccomyxa sp. Isolated from Dry Environments

Aerial algae are considered to be highly tolerant of and adaptable to severe conditions including radiation, desiccation, high temperatures, and nutrient deficiency, compared with those from aquatic habitats. There are considerable variations in the fatty acid (FA) composition of aerial microalgae from dry environments. A new species with a high lipid level was found on concrete surfaces and was identified as Coccomyxa sp. KGU-D001 (Trebouxiophyceae). This study characterized its FA content and profile in a bath culture. The alga showed a constant specific growth rate (0.26 day^?1) ranging in light intensity from 20 to 80 μmol photons m^?2 s^?1. The algal cells started to form oil bodies in the early stationary phase of growth, and oil bodies occupied most of the cells during the late stationary phase when the cells accumulated 27 % total fatty acids (TFA). The process of lipid body formation accumulating large amounts of triacylglycerols (TAG) appeared to be very unusual in response to stress conditions persisting for a relatively long culture time (50 days). This study could indicate that aerial microalgae will be a candidate for biodiesel production when a new cultivation method is developed using extreme stresses such as nutritional deficiency and/or desiccation.     

Correlations Between FAS Elongation Cycle Genes Expression and Fatty Acid Production for Improvement of Long-Chain Fatty Acids in Escherichia coli

Microorganisms have been used for biodiesel (fatty acid methyl ester) production due to their significant environmental and economic benefits. The aim of the present research was to develop new strains of Escherichia coli K-12 MG1655 and to increase the content of long-chain fatty acids by overexpressing essential enzymes that are involved in the fatty acid synthase elongation cycle. In addition, the relationship of β-ketoacyl-acyl carrier protein (ACP) synthase (fabH), β-ketoacyl-ACP reductase (fabG), β-hydroxyacyl-ACP dehydrase (fabZ), and β-enoyl-ACP reductase (fabI) with respect to fatty acid production was investigated. The four enzymes play a unique role in fatty acid biosynthesis and elongation processes. We report the generation of recombinant E. coli strains that produced long-chain fatty acids to amounts twofold over wild type. To verify the results, NAD⁺/NADH ratios and glucose analyses were performed. We also confirmed that FabZ plays an important role in producing unsaturated fatty acids (UFAs) as E. coli SGJS25 (overexpressing the fabZ gene) produced the highest percentage of UFAs (35 % of total long-chain fatty acids), over wild type and other recombinants. Indeed, cis-9-hexadecenoic acid, a major UFA in E. coli SGJS25, was produced at levels 20-fold higher than in wild type after 20 h in culture. The biochemically engineered E. coli presented in this study is expected to be more economical for producing long-chain fatty acids in quality biodiesel production processes.     

Single Cell Oil Production from Hydrolysates of Inulin by a Newly Isolated Yeast <Emphasis Type="Italic">Papiliotrema laurentii</Emphasis> AM113 for Biodiesel Making

Microbial oils are among the most attractive alternative feedstocks for biodiesel production. In this study, a newly isolated yeast strain, AM113 of Papiliotrema laurentii, was identified as a potential lipid producer, which could accumulate a large amount of intracellular lipids from hydrolysates of inulin. P. laurentii AM113 was able to produce 54.6% (w/w) of intracellular oil in its cells and 18.2 g/l of dry cell mass in a fed-batch fermentation. The yields of lipid and biomass were 0.14 and 0.25 g per gram of consumed sugar, respectively. The lipid productivity was 0.092 g of oil per hour. Compositions of the fatty acids produced were C_14:0 (0.9%), C_16:0 (10.8%), C_16:1 (9.7%), C_18:0 (6.5%), C_18:1 (60.3%), and C_18:2 (11.8%). Biodiesel obtained from the extracted lipids could be burnt well. This study not only provides a promising candidate for single cell oil production, but will also probably facilitate more efficient biodiesel production.     

Marine Diatom, Navicula sp. Strain JPCC DA0580 and Marine Green Alga, Chlorella sp. Strain NKG400014 as Potential Sources for Biodiesel Production

Marine diatom, strain JPCC DA0580, and marine green microalga strain NKG400014 were selected as high neutral lipid-producers from marine microalgal culture collection toward biodiesel production. These strains were tentatively identified as Navicula sp. and Chlorella sp., respectively, by 18S rDNA analysis. Growth and lipid accumulation conditions of both strains were analyzed by changing nutrient concentrations in growth media and initial illuminance intensity. The highest productivity of fatty acid methyl ester (FAME) reached to 154 mg/L/week for NKG400014 and 185 mg/L/week for JPCC DA0580. Gas chromatography/mass spectrometry analysis indicates that FAME fraction from NKG400014 mainly contained 9-12-15-octadecatrienoate (C18:3) and that from JPCC DA0580 mainly contained methyl palmitate (C16:0) and methyl palmitoleate (C16:1). Furthermore, calorimetric analysis revealed that the energy content of strain was 4,233?±?55 kcal/kg (i.e., 15.9?±?0.2 MJ/kg) for NKG400014 and 6,423?±?139 kcal/mg (i.e., 26.9?±?0.6 MJ/kg) for JPCC DA0580, respectively. The value from JPCC DA0580 was equivalent to that of coal. The strains NKG400014 and JPCC DA0580 will become a promising resource that can grow as dominant species in the open ocean toward production of both liquid and solid biofuels.     

Enhanced Lipid Production by Co-cultivation and Co-encapsulation of Oleaginous Yeast Trichosporonoides spathulata with Microalgae in Alginate Gel Beads

This study attempted to enhance biomass and lipid productivity of an oleaginous yeast Trichosporonoides spathulata by co-culturing with microalgae Chlorella spp., optimizing culture conditions, and encapsulating them in alginate gel beads. The co-culture of the yeast with microalgae Chlorella vulgaris var. vulgaris TISTR 8261 most enhanced overall biomass and lipid productivity by 1.6-fold of the yeast pure culture at 48 h and by 1.1-fold at 72 h. After optimization and scale-up in a bioreactor, this co-culture produced the highest biomass of 12.2 g/L with a high lipid content of 47 %. The dissolved oxygen monitoring system in the bioreactor showed that the microalgae worked well as an oxygen supplier to the yeast. This study also showed that the co-encapsulated yeast and microalgae could grow and produce lipid as same as their free cells did. Therefore, it is possible to apply this encapsulation technique for lipid production and simplification of downstream harvesting process. This co-culture system also produced the lipid with high content of saturated fatty acids, indicating its potential use as biodiesel feedstock with high oxidative stability.     

Low-Cost Production of Green Microalga Botryococcus braunii Biomass with High Lipid Content Through Mixotrophic and Photoautotrophic Cultivation

Botryococcus braunii is a microalga that is regarded as a potential source of renewable fuel because of its ability to produce large amounts of lipid that can be converted into biodiesel. Agro-industrial by-products and wastes are of great interest as cultivation medium for microorganisms because of their low cost, renewable nature, and abundance. In this study, two strategies for low-cost production of B. braunii biomass with high lipid content were performed: (i) the mixotrophic cultivation using molasses, a cheap by-product from the sugar cane plant as a carbon source, and (ii) the photoautotrophic cultivation using nitrate-rich wastewater supplemented with CO₂ as a carbon source. The mixotrophic cultivation added with 15 g L^?1 molasses produced a high amount of biomass of 3.05 g L^?1 with a high lipid content of 36.9 %. The photoautotrophic cultivation in nitrate-rich wastewater supplemented with 2.0 % CO₂ produced a biomass of 2.26 g L^?1 and a lipid content of 30.3 %. The benefits of this photoautotrophic cultivation are that this cultivation would help to reduce accumulation of atmospheric carbon dioxide and more than 90 % of the nitrate could be removed from the wastewater. When this cultivation was scaled up in a stirred tank photobioreactor and run with semi-continuous cultivation regime, the highest microalgal biomass of 5.16 g L^?1 with a comparable lipid content of 32.2 % was achieved. These two strategies could be promising ways for producing cheap lipid-rich microalgal biomass that can be used as biofuel feedstocks and animal feeds.     

Rôle du Tween-80 utilisé dans la culture des corynébactéries cutanées (groupe JK) sur la composition en acides gras cellulaires

This study of 8 strains of cutaneous corynebacteria, including 5 strains of «JK corynebacteriaå, concerns the fatty acid composition of cells grown in the presence or not of Tween 80, which enhances their growth rate. The fatty acid methyl esters were analysed by gas-liquid chromatography. The fatty acid profile of the 8 strains agreed with the general pattern of the genus Corynebacterium, with major amounts of palmitic acid (C16:0) and oleic acid (C18:0) and with only trace amounts of 10-methyl octodecanoic acid (tuberculostearic acid). Iso and anteiso branched acids were not present. The 5 strains had high levels of stearic acid (C18:0). When cells were grown in the presence of Tween-80, the fatty acid composition was modified for all strains: their profiles showed high levels of oleic acid (C18:1) and smaller quantities of palmitoleic acid (C16:1).These results confirm the advantage of fatty acid composition for identification of Corynebacteria, and particularly for cutaneous strains, and emphasize the influence of the culture medium, especially that of Tween-80.     

<em>In Vitro</em> Anti-diabetic Activities and Chemical Analysis of Polypeptide-k and Oil Isolated from Seeds of <em>Momordica charantia</em> (Bitter Gourd)

The amino acid and fatty acid composition of polypeptide k and oil isolated from the seeds of Momordica charantia was analysed. The analysis revealed polypeptide k contained 9 out of 11 essential amino acids, among a total of 18 types of amino acids. Glutamic acid, aspartic acid, arginine and glycine were the most abundant (17.08%, 9.71%, 9.50% and 8.90% of total amino acids, respectively). Fatty acid analysis showed unusually high amounts of C18-0 (stearic acid, 62.31% of total fatty acid). C18-1 (oleic acid) and C18-2 (linoleic acid) were the other major fatty acid detected (12.53% and 10.40%, respectively). The oil was devoid of the short fatty acids (C4-0 to C8-0). Polypeptide k and oil were also subjected to in vitro α-glucosidase and α-amylase inhibition assays. Both polypeptide k and seed oil showed potent inhibition of α-glucosidase enzyme (79.18% and 53.55% inhibition, respectively). α-Amylase was inhibited by 35.58% and 38.02%, respectively. Collectively, the in vitro assay strongly suggests that both polypeptide k and seed oil from Momordica charantia are potent potential hypoglycemic agents.     

Ionic Liquid-Based Extraction of Fatty Acids from Blue-Green Algal Cells Enhanced by Direct Transesterification and Determination Using GC × GC-TOFMS

Blue-green algae commonly referred to as cyanobacteria are known to grow in freshwater bodies when they are provided with suitable growth conditions such as nutrients, temperature and light. Algae biomass is known to contain a large amount of lipids, such as saturated and unsaturated fatty acids. In this study, fatty acids from algal cells were extracted using a newly developed extraction protocol using ionic liquid enhanced by direct transesterification at an elevated temperature. The identification and quantification of fatty acids was performed using gas chromatography coupled to a time-of-flight mass spectrometer (GC × GC-TOFMS). The extracted fatty acids were dominated by those with carbon chain of C16 and C18; [i.e. 7-hexadecenoic acid (C16:1) and hexadecanoic acid (C16:0) for C16, whereas C18 includes γ-linolenic acid (γ-C18:3); linoleic acid (C18:2); linolenic acid (C18:3); 6,9,12,15-octadecatetraenoic acid (C18:4); oleic acid (C18:1) and octadecanoic acid (C18:0)]. The obtained fatty acid composition was then compared with that obtained by organic solvent extraction using a mixture of chloroform and methanol. Statistical evaluation was performed using one-way ANOVA and found that there was no statistically significant difference (P = 0.908) between the two extraction methods, a finding which indicates the usefulness of ionic liquid as a solvent to replace volatile organic solvent to minimize environmental pollution.     

Increase of Docosahexaenoic Acid Production by Schizochytrium sp. Through Mutagenesis and Enzyme Assay

The present study focused on improving docosahexaenoic acid (DHA) production by Schizochytrium sp. through N-methyl-N-nitro-N-nitrisiguanidine treatment coupled with ultraviolet radiation based on the metabolic pathway analysis. The activity of glucose-6-phosphate dehydrogenase of the mutant was higher than the parent strain, which indicated that the hexose monophosphate pathway of the mutant was strengthened, and more NADPH was thus produced. Also, the activities of malic enzyme and ATP–citrate lyase in the cell extract of the mutant were higher than the parent strain, which indicated that the screening method increased NADPH and acetyl–CoA supply in vivo effectively. Finally, in the batch culturing of the mutant, 34.84% higher lipid was accumulated with the cell dry weight at the same level compared with the parent strain. Moreover, the DHA percentage of the total fatty acids up to 56.22% was achieved using the mutant, which was 38.88% higher than the parent strain. When the cultures were maintained under appropriate conditions, the final DHA yield was 0.20 and 0.11 g/g dry biomass, for the mutant and parent, respectively.     

Determination of Fatty Acids in Saliva of Smokers and Nonsmokers by HPLC with Fluorescence Detection Using a Hydrazine-Based Difluoro-boraindacene Reagent

1,3,5,7-Tetramethyl-8-daminozide-difluoroboradiaza-s-indacence (TMBODIPY-H), a pre-column fluorescent derivatization reagent, was applied to the analysis of fatty acid by high-performance liquid chromatography with fluorescence detection. Using this reagent, 12 fatty acids from propionic acid (C3) to stearic acid (C18) can be derivatized at room temperature in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide within 2 h. The baseline separation of these derivatives can be achieved within 46 min by gradient elution. The detection limits of these fatty acids are in the range of 0.1–1 nM and the linear ranges of most of them are 0.5–100 nM. This was the first application of hydrazine-based difluoro-boraindacene reagent for the analysis of fatty acids, and the proposed method has been successfully used for the determination of saliva samples of smokers and nonsmokers with recoveries of 88–110 %.     

Quantitative Determination of Isostearic Acid Isomers in Skin Creams by GC-MS-SIM

“Isostearic acid” is frequently listed as an ingredient of skin creams and other cosmetics. In the four skin creams analyzed, “isostearic acid” was esterified with isopropanol, as well as sorbitan or polyglycerols. “Isopropyl isostearate” was isolated by HCl treatment and saponification whereas emulsifiers (sorbitan or polyglycerol isostearates) were enriched by means of a C₁₈-cartridge. Fatty acids in the resulting lipid fraction were transferred into methyl esters. 25:0 and 19:0 methyl esters were used as internal standards. GC-EI-MS was used to determine that “isostearic acid” was a mixture of many methyl-branched isomers of stearic acid (18:0) in all four skin creams. Thus, it may be better termed “isostearic acids”. The branched-chain nature of isostearates was verified by formation and analysis of picolinyl esters of skin cream fatty acids by GC-EI-MS. Twenty-five 18:0 isomers were detected and the main products had one methyl branch on carbons C₁₀–C₁₄. Two late eluting isostearic acid isomers were identified as 16-methyl heptadecanoic acid (i18:0) and 15-methyl heptadecanoic acid (a18:0). GC-EI-MS in the selected ion monitoring (SIM) mode with m/z 87 as quantification ion was used for the determination of i18:0 methyl ester. The quantities of i18:0 in the samples amounted to 10–20 mg g^?1 skin cream. The contribution of i18:0 to the sum of all 18:0 isomers in the four skin cream samples was 8.5 ± 1.1%. Instead of determining all individual isostearates in a product, we suggest the quantitative determination of i18:0 followed by multiplication with factor 11.75 (~reciprocal value of 8.5%, see above) as a simple method for the quantification of isostearates in cosmetics.     

Carotenoid and fatty acid profiles of bilberries and cultivated blueberries from Romania

The worldwide consumption of blueberries has increased due to their potential health effects. They contain micronutrients, various phytochemicals and anti-oxidant pigments, represented especially by anthocyanins. In addition to these, carotenoids and fatty acids are also present. The aim of this study was the identification and quantification of carotenoids (by HPLC) and fatty acids (by GC with flame ionisation detection) in two bilberry genotypes (Wild 1 and 2) and three cultivars (BlueCrop, Elliot, and Duke). The main carotenoids identified were lutein, ??-cryptoxanthin, and ??-carotene, with the average total carotenoid content of 266 ??g per 100 g of fruit. The ratio of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs) was 0.098 for Wild 1 and 0.11 for Wild 2 genotypes and ranged from 0.14 to 0.28 for cultivars. The main fatty acids identified were linoleic (18:2) and linolenic (18:3) acids in all the samples analysed. In Vaccinium myrtillus the amount of carotenoids and fatty acids was higher than in Vaccinium corymbosum species.     

Isolation of Novel Microalgae from Acid Mine Drainage and Its Potential Application for Biodiesel Production

Microalgae were selected and isolated from acid mine drainage in order to find microalgae species which could be cultivated in low pH condition. In the present investigation, 30 microalgae were isolated from ten locations of acid mine drainage in South Korea. Four microalgae were selected based on their growth rate, morphology, and identified as strains of KGE1, KGE3, KGE4, and KGE7. The dry biomass of microalgae species ranged between 1 and 2 g L^?1 after 21 days of cultivation. The growth kinetics of microalgae was well described by logistic growth model. Among these, KGE7 has the highest biomass production (2.05?±?0.35 g L^?1), lipid productivity (0.82?±?0.14 g L^?1), and C16–C18 fatty acid contents (97.6 %). These results suggest that Scenedesmus sp. KGE 7 can be utilized for biodiesel production based on its high biomass and lipid productivity.     

Preparation of Triacylglycerols Rich in Omega-3 Fatty Acids from Sardine Oil Using a Rhizomucor miehei Lipase: Focus in the EPA/DHA Ratio

The increasing evidence on the differential biochemical effects of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) raises the need of n-3 highly unsaturated fatty acid concentrates with different amounts of these fatty acids. In the present work, physicochemical and enzymatic techniques were combined to obtain acylglycerols, mainly triacylglycerols (TAG), rich in n-3 fatty acids. Sardine oil was obtained by washing sardine (Sardina pilchardus) mince with a NaHCO₃ solution, hydrolyzed in a KOH–ethanol solution, and concentrated with urea. The esterification reaction was performed in the stoichiometric proportion of substrates for re-esterification to TAG, with 10 % level of Rhizomucor miehei lipase based on the weight of substrates, without any solvent, during 48 h. This procedure led to approximately 88 % of acylglycerols, where more than 66 % were TAG and the concentration of n-3 fatty acids was higher than 60 %, the EPA and DHA ratio (EPA/DHA) was 4:1. The content of DHA in the unesterifed fraction (free fatty acids) increased from 20 to 54 %, while the EPA level in the same fraction decreased from 33 to 12.5 % (EPA/DHA ratio ≈1:4). Computational methods (density functional theory calculations) have been carried out at the B3LYP/6-31G(d,p) level to explain some of the experimental results.     

Supercritical carbon dioxide extraction and analysis of lipids from <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> using gas chromatography

The influence of six different supercritical carbon dioxide extraction conditions on the quantity and quality of the extracted lipids from Chlorella vulgaris (C. vulgaris) was examined using experimental design. The investigated parameters included dynamic extraction times (45 and 90 min), pressures (300 and 350 bars) and a modifier (with and without ethanol). By using a grinding method prior to the extraction, cells were disrupted resulting in an enhanced extraction yield. The results showed that the total lipid yields increased by increasing the pressure, extraction time and the modifier. Applying harsher extraction conditions increased the extraction of longer-chain fatty acids (FAs), changed the FA compositions and enhanced the total lipid yield. Using transesterification, the extracted lipids were converted to biodiesel. The biodiesel physiochemical properties were estimated using empirical equations based on the fatty acid methyl ester contents. Furthermore, analysis of the protein content of remaining biomass showed a decrease in protein content with increasing the lipid extraction yield.