Optimization of production of docosahexaenoic acid (DHA) byThraustochytrium aureum ATCC 34304

By varying culture carbon source, lipid content in mycelium ofThraustochytrium aureum ATCC 34304 varied widely in the range 1–25% of biomass weight. Docosahexaenoic acid (DHA) content of mycelium lipid was higher (65–76%) when biomass lipid content was very low (1–2%) and lower (40–50%) when biomass contained a high lipid content (14–18%). DHA yields from glucose, starch and maltose were 270, 325 and 334 mg/L, respectively. DHA yield and content of biomass was optimal at an initial culture pH of 6.0. During the culture cycle ofT. aureum, DHA content in lipids remained relatively constant with optimal DHA yield being observed after six days. Biomass, lipid content in biomass, DHA content in biomass and DHA yield were all optimal at a cultivation temperature of 28°C. However, the proportion of DHA in lipids declined with increase in temperature. Biomass, lipids in biomass and DHA yields were 13%, 42% and 47% higher, respectively, din light-exposed cultures as compared to dark cultures. A maximum yield of DHA of 511 mg/L was observed in light- exposed cultures containing 2.5% starch, where lipids accounted for over 20% of biomass dry weight. To whom correspondence should be addressed.     

Production of eicosapentaenoic acid bySaprolegnia sp. 28YTF-1

Saprolegnia sp. 28YTF-1, isolated from a freshwater sample, is a potent producer of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). The fungus used various kinds of carbon sources, such as starch, dextrin, sucrose, glucose, and olive oil for growth, and olive oil was the best carbon source for EPA production. The EPA content reached 17 mg/g dry mycelium (0.25 mg/L) when the fungus was grown in a medium that contained 2.5% olive oil and 0.5% yeast extract, at pH 6.0 and 28°C for 6 d with shaking. Accompanying production of arachidonic acid (AA; 3.2 mg/g dry mycelia, EPA/AA = 5.1) and other ω6 polyunsaturated fatty acids was low. Both EPA content and EPA/AA ratio increased in parallel by lowering growth temperature. Triglyceride was the major mycelial lipid (ca. 84%), but EPA comprised only 2.2% of the total fatty acids of this lipid. About 40% of the EPA produced was found in polar lipids, such as phosphatidylethanolamine (EPA content, 28.2%), phosphatidylcholine (13.6%), and phosphatidylserine (21.2%).     

Process for production of arachidonic acid concentrate by a strain of mortierella alpina

Various Mortierella alpina fungi were screened for their capacities to produce arachidonic acid. A strain of M. alpina was found to show the highest productivity. Arachidonic acid content of biomass and overall yield per litre of culture was highest in soya flour supplemented medium which produced dispersed mycelium. When the glucose concentration in the medium was varied from 30 to 100 g/L, biomass, lipid, arachidonic acid content of biomass and arachidonic acid yield increased with increasing glucose concentration. Several natural oils, when added to the growth medium, stimulated arachidonic acid production. After fermentation in a 20-L fermenter under optimal culture conditions, the arachidonic acid yield was 5.3 g/L, representing 34.2% w/w of total fatty acids and 13.7% w/w of biomass. An extract containing 72.5% w/w arachidonic acid was prepared from the recovered mycelium.     

Growth and eicosapentaenoic acid production byPhaeodactylum tricornutum in batch and continuous culture systems

Maximum specific growth rate (μ_max) ofPhaeodactylum tricornutum increased with increasing culture reactor surface-to-volume ratio. Values for μ_max of 0.647, 0.377 and 0.339 day⁻¹ were observed for the 75-mL tube, 5.6-L tank and the 16-L tank, respectively. Higher biomass was achieved in the 75-mL batch culture tube under continuous light as compared with light cycle conditions. Palmitic acid, palmitoleic acid and eicosapentaenoic acid (EPA) accounted for over 60% of total fatty acids in the batch tube culture, with EPA content increasing to a maximum after three days. In chemostat cultures, run at dilution rates of 0.15 day⁻¹ (0.45 of μ_max) and 0.3 day⁻¹ (0.9 of μ_max), cell concentration reached a steady state of 2.18 and 0.7 g/L, respectively, while contents of EPA per liter of culture at steady state were 100.9 and 82.5 mg/L, respectively. At both dilution rates, EPA content of total fatty acids was the same (35.0–35.2%). At a dilution rate of 0.3 day⁻¹, the continuous culture system manifested productivities of 0.51 g/L/d and 25.1 mg/L/d for biomass and EPA, respectively.     

Production of an eicosapentaenoic acid-containing oil by a Δ12 desaturase-defective mutant ofMortierella alpina 1S-4

A Δ12 desaturase-defective mutant of an arachidonic acid (AA)-producing fungus,Mortierella alpina 1S-4, converted α-linolenic acid (18:3ω3) to 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid (EPA). On submerged cultivation at 20°C for 10 d in a 5-L fermentor containing medium comprising 1% glucose, 1% yeast extract and 3% (vol/vol) linseed oil, EPA production amounted toca. 1 g/L culture broth (64 mg/g dry mycelium), which accounted forca. 20% of the total mycelial fatty acids. AA content was 26 mg/g dry mycelium (0.4 g/L), accounting for 7.8% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (4.5%), oleic acid (20.4%), linoleic acid (10.0%), 18:3ω3 (20.3%) and lignoceric acid (4.3%). Most of the EPA produced (ca. 90 mol%) was in triglyceride form.     

Lipids of Haliphthoros philippinensis: An oomycetous marine microbe

Lipids of the marine oomycetous microbe Haliphthoros philippinensis were characterized by chromatographic and spectroscopic techniques. Total lipid content of this organism was relatively low and not very responsive to manipulation of the culture conditions. Neutral lipid comprised 21% of the total lipid and the polar lipids were mainly phosphatidylcholine (44%), phosphatidylethanolamine (15%), and a ceramide-phosphorylethanolamine (19%). Palmitic (16:0) was the primary saturated fatty acid at 25% of the total fatty acids, and arachidonic acid (20:4n-6, ARA) and eicosapentaenoic acid (20:5n-3, EPA) were the major unsaturated fatty acids at 19 and 21%, respectively. Fucosterol was the principal sterol at 59% of the total sterols. The effects of several cultivation variables on growth and EPA production by this species were investigated. Among those tested, glucose and sodium glutamate were the most efficient carbon and nitrogen sources for growth, respectively. When the mycelium was cultivated for 6 d to produce biomass under optimal growth conditions, and then transferred to low temperature for an additional 13 d without glucose, the EPA content reached 31% of the total fatty acids and the yield was 203 mg/L. When the same experiment was performed with glucose supplementation during the low-temperature phase, EPA composed 27% of total fatty acids and yield reached 316 mg/L, or a 285% increase over that from mycelium cultured for 6 d at 24°C, and 56% over that cultured at 16°C for 13 d. ARA production did not respond accordingly.     

Effect of mustard meal on the production of arachidonic acid by Mortierella elongata SC-208

A strain of Mortierella elongata SC-208 that was isolated from soil of a mustardseed extraction plant can produce arachidonic acid in significant amounts, and it was grown in three different media, one of which contained 0.5% deoiled mustard meal. The arachidonic acid content in the lipid part of dry mycelium (23.2 g/L) was as high as 33% w/w from the medium that contained mustard meal, and the overall yield of arachidonic acid was 0.49 g/L. The arachidonic acid contents in the phospholipid fraction and the triglyceride fraction were 39.5 and 30.2%, respectively.     

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.     

Production potential of eicosapentaenoic acid byMonodus subterraneus

Interest in the polyunsaturated fatty acid eicosapentaenoic acid (EPA) as a therapeutic agent is steadily increasing. The microalgaMonodus subterraneus produces EPA, which is concentrated mainly in the galactolipid fraction, as its major fatty acid. Nitrogen starvation increased the fatty acid content but reduced the proportion and content of EPA to 19.5% (of fatty acids) and 1.8% (of dry weight), respectively. Cultivation under low light intensity or high biomass concentration enhanced the proportion of EPA up to 36.7% of fatty acids and the content to 4.4% of dry weight. Maximal EPA productivity of 25.7 mg·L⁻¹·d⁻¹ was obtained at the biomass concentration that resulted in the highest biomass productivity.M. subterraneus is thus one of the most promising candidates for phototrophic production of EPA.     

Temperature shifts in regulation of lipids accumulated byLipomyces starkeyi

The effect of temperature on the accumulating triglycerides ofLipomyces starkeyi was studied in 10-L fermentation experiments. The temperature during the growth, lipid accumulation and postaccumulation phases was altered. The cellular lipid content, the glucose conversion efficiency and the specific lipid production rate were highest when the growth phase temperature was 28°C, instead of 16–18°C. The temperature of the accumulation phase had an influence on the lipid quality at the end of the accumulation. Oleic acid content increased from 52 to over 60% when the accumulation phase temperature was decreased from 28 to 15°C and, concomitantly, palmitic acid decreased from 33 to 26%. The degree of fatty acid unsaturation was the highest (0.75 Δmol⁻¹) when the accumulation phase temperature was the lowest (15°C) andvice versa. The temperature shift after the lipid accumulation phase affected neither the composition nor the amount of the accumulated lipids. In conclusion, the temperature profile for the highest lipid yield with the most desired composition should be: (i) a growth phase temperature that gives the maximum growth rate and (ii) an accumulation phase temperature that produces the desired ratio of palmitic to oleic acid.     

Polar and nonpolar lipids and their fatty acid composition of a fewFusarium species

A few species ofFusarium have been evaluated for their potential to produce lipids. The isolates under investigation exhibited wide variation with respect to the mycelial weight, total lipid content and percentage composition of polar and nonpolar lipids in which triglycerides were the major components (81–90%). Palmitic, stearic, oleic and linoleic acids were the major fatty acids in both the fractions. The polar lipids contained higher levels of linoleic acid, whereas nonpolar lipids contained oleic acid as the predominant acid. Nonpolar lipids were more saturated than polar lipids.     

Muscle Lipids and Fatty Acid Profiles of the Sea Catfish (Arius madagascariensis) in Madagascar Inland Waters

Muscle lipids and fatty acids (FA) of catfish Arius madagascariensis were determined in catfish caught in the Betsiboka River, Madagascar, during a 5-month sampling period. Total lipids from muscle were extracted and quantified. Fatty acids were identified by means of gas chromatography–mass spectrometry of FA methyl esters and FA pyrrolidides, leading to the identification of 42 FA. Lipid content was relatively high in our fish sample and ranged from 4.3 to 6.6% of wet muscle. Three FA dominated the FA composition: palmitic acid (C16:0, 22.9–32.6%), oleic acid (C18:1n-9, 11.3–13.4%) and stearic acid (C18:0, 10.8–12.0%). A number of polyunsaturated FA (PUFA) were present in appreciable amounts, including arachidonic acid (C20:4n-6, 4.7–7.6%), docosahexaenoic acid (C22:4n-6, 3.0–8.1%), eicosapentaenoic acid (C20:5n-3, 0.6–1.0%), n-3 docosapentaenoic acid (C22:5n-3, 1.1–1.6%), n-6 docosatetraenoic acid (C22:4n-6, 0.7–1.2%) and n-6 docosapentaenoic acid (22:5n-6, 0.9–1.8%). The sum of the n-6 PUFA and n-3 PUFA was 11.3–18.8 and 7.5–13.4%, respectively. These results indicate that A. madagascariensis, an abundant freshwater fish in Madagascar rivers, may be good source of dietary PUFA.     

Recovery of eicosapentaenoic acid from fungal mycelia by solvent extraction

Utilization of lipids containing eicosapentaenoic acid (EPA) produced by microorganisms requires processes for their efficient recovery from microbial cells. Recovery of EPA from mycelia of the fungusPythium irregulare by solvent extraction with hexane-isopropanol (HIP) in a pilot-plant colloid mill was investigated. Extraction efficiencies of 96% for lipid and EPA were achieved with a 3∶2 (vol/vol) HIP mixture by milling wet, filtered mycelia for 5 min at a solvent/dry solids ratio of 100 L/kg. The process yielded a crude extract that contained up to 96% lipid and an EPA content as high as 24% (with no selectivity for EPA).     

The filamentous fungusmortierella alpina,high in arachidonic acid

Arachidonic acid is not available readily, although it is widely distributed in animal tissue. We found that in some strains ofMortierella alpina, arachidonic acid accounted for 68.5–78.8% of the total fatty acids. This is more than twice the arachidonic acid content of any organism previously reported. The content of arachidonic acid per dry cell weight was about 25%. Our findings offer a method for the efficient isolation of arachidonic acid in large amounts.     

Variation in fatty acid composition of the different acyl lipids in seed oils from fourSesamum species

Seeds from different collections of cultivatedSesamum indicum Linn. and three related wild species [specifically,S. alatum Thonn.,S. radiatum Schum and Thonn. andS. angustifolium (Oliv.) Engl.] were studied for their oil content and fatty acid composition of the total lipids. The wild seeds contained less oil (ca. 30%) than the cultivated seeds (ca. 50%). Lipids from all four species were comparable in their total fatty acid composition, with palmitic (8.2–12.7%), stearic (5.6–9.1%), oleic (33.4–46.9%) and linoleic acid (33.2–48.4%) as the major acids. The total lipids from selected samples were fractionated by thin-layer chromatography into five fractions: triacylglycerols (TAG; 80.3–88.9%), diacylglycerols (DAG; 6.5–10.4%), free fatty acids (FFA; 1.2–5.1%), polar lipids (PL; 2.3–3.5%) and steryl esters (SE; 0.3–0.6%). Compared to the TAG, the four other fractions (viz, DAG, FFA, PL and SE) were generally characterized by higher percentages of saturated acids, notably palmitic and stearic acids, and lower percentages of linoleic and oleic acids in all species. Slightly higher percentages of long-chain fatty acids (20∶0, 20∶1, 22∶0 and 24∶0) were observed for lipid classes other than TAG in all four species. Based on the fatty acid composition of the total lipids and of the different acyl lipid classes, it seems thatS. radiatum andS. angustifolium are more related to each other than they are to the other two species.     

The production potential of eicosapentaenoic and arachidonic acids by the red algaPorphyridium cruentum

The red microalgaPorphyridium cruentum is a new source for eicosapentanoic acid (EPA) and arachidonic acid (AA) fatty acids of potential pharmaceutical value. The conditions leading to a high content of either fatty acid were investigated. The highest EPA content was obtained under conditions resulting in high growth rate (2.4% of ash free dry weight in Strain 1380-1d). High AA content was obtained under slow growth conditions and was maximal in th stationary phase or under nitrogen starvation (2.9%). Strain 1380-la had the highest content (1.9%) of arachidonic acid under exponential growth conditions. By imposing nitrogen starvation, it was possible to obtain a lipid mixture which may be separated into AA and EPA rich fractions.     

Production of eicosapentaenoic acid byMortierella fungi

Mycelia of arachidonic acid-producing fungi belonging to the genusMortierella were found to be rich sources of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). Production of EPA by these fungi was observed only when they were grown at low temperature (6–16 C). EPA comprised 5–20% of the total extractable mycelial fatty acids in most strains tested. No significant accumulation of EPA was observed on incubation at high temperature (20–28 C), at which the other major mycelial C-20 fatty acid, arachidonic acid, was still efficiently produced. In a study on the optimization of the culture conditions for EPA production by a selected fungiM. alpina 20–17, a medium containing glucose and yeast extract as major carbon and nitrogen sources, respectively, was found to be suitable. Periodic feeding of glucose during growth of the fungus and cultivation at high temperature (20 C) during the early growth phase followed by temperature shift to 12 C were found to be effective at increasing mycelial yield and reducing cultural period, respectively. Under the optimal culture conditions, the EPA production reached 0.49 mg/ml of culture broth (29 mg/g dry mycelia). This value accounted for 13.5% of the total fatty acids in the extracted lipids. Other major fatty acids in the lipids were palmitic acid (6.0%, by weight), stearic acid (5.3), oleic acid (6.2), linoleic acid (3.0), γ-linolenic acid (3.5) and arachidonic acid (60.0). On leave from Suntory Ltd.     

Metabolic flux analysis on arachidonic acid fermentation

The analysis of flux distributions in metabolic networks has become an important approach for understanding the fermentation characteristics of the process. A model of metabolic flux analysis of arachidonic acid (AA) synthesis in Mortierella alpina ME-1 was established and carbon flux distributions were estimated in different fermentation phases with different concentrations of N-source. During the exponential, decelerating and stationary phase, carbon fluxes to AA were 3.28%, 8.80% and 6.97%, respectively, with sufficient N-source broth based on the flux of glucose uptake, and those were increased to 3.95%, 19.21% and 39.29%, respectively, by regulating the shifts of carbon fluxes via fermentation with limited N-source broth and adding 0.05% NaNO₃ at 96 h. Eventually AA yield was increased from 1.3 to 3.5 g·L⁻¹. These results suggest a way to improve AA fermentation, that is, fermentation with limited N-source broth and adding low concentration N-source during the stationary phase. __________ Translated from Journal of Chemical Engineering of Chinese Universities, 2007, 21(2): 316–321 [译自: 高校化学工程学报]     

γ-Linolenic acid in zygomycetous fungi: Syzygites megalocarpus

The fatty acids of over 150 species and isolates of zygomycetous fungi were analyzed, and it was found that γ-linolenic acid (GLA) composed 35 to 62% of the total fatty acids in several species, i.e., Circenella simplex, Mucor indicus, Syzygites megalocarpus (ATCC 18025), and Zygorhynchus moellierie A (UAMH 1556). Further study of S. megalocarpus showed that the total lipid content of the mycelium could be increased from 9.8% of the dry biomass to 20 to 25% when grown in a medium with a high carbon/nitrogen ratio. Under these conditions, the GLA content of the triacyglycerols increased during culture development, even during the stationary phase, but remained relatively constant in the phospholipid fraction. Nonsaponifiable lipid represented 4% of the total lipid, and the major sterol among 14 others detected was ergosterol at 52% of the total. Phospholipids composed 7% of the total lipid with phosphatidylethanol-amine and phosphatidylcholine representing 53 and 39% of the total, respectively.     

Optimization of fatty acid extraction from Phaeodactylum tricornutum UTEX 640 biomass

Fatty acids in the microalga Phaeodactylum tricornutum were isolated using an optimized three-step method: extraction of crude fatty acid potassium salts made by direct saponification of lipids in the microalgal biomass with KOH/ethanol (96%, vol/vol), separation of unsaponifiable lipids by extraction with hexane, and final purification of fatty acids by acidification of the alcoholic solution of potassium soaps followed by extraction of fatty acid into hexane. Direct saponification was carried out in ethanol (96%, vol/vol) using 2.09 mL ethanol (96%) per gram of wet biomass (10 mL/g of dry biomass) mixed with 0.4 g KOH/g of biomass. Under these conditions the fatty acid yield was 87%. The optimal water content of the alcoholic solution for extraction of the unsapononifiables was established as 40%, w/w. Data on equilibrium carotenoid distribution between the alcoholic (40%, w/w water) and hexane phases were determined. These data allow prediction of the carotenoid yields with different volumes of hexane in several extraction steps. The optimal pH of the alcoholic solution before extracting the purified fatty acid was established as pH 6, and the equilibrium fatty acid distribution between the alcoholic and hexane phases was determined. This optimized method permited a 20% reduction in the production costs of highly purified eicosapentaenoic acid (EPA) in the three-step preparative process (extraction of fatty acid, concentration of polyunsaturated fatty acids by the urea method, and EPA fractionation through preparative high-performance liquid chromatography) previously developed by the authors.