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.     

Effects of agingmortierella mycelium on production of arachidonic and eicosapentaenoic acids

Arachidonic acid and eicosapentaenoic acid (EPA) are important intermediates of eicosanoid metabolism and are presently the subject of extensive nutritional and medical research. The effects of mycelial aging on production of these fatty acids were investigated as part of a research program directed toward examining the feasibility of economically producing these products by fungal fermentation. Arachidonic acid content ofM. alpina ATCC 32222 increased from 4.1–8.3% to 13–16% during aging while lipid content of mycelium increased from 14–18% to 33–45%. Maximum lipid content produced in biomass during storage declined as harvesting time was increased from 3 to 6 days while maximum arachidonic acid content in lipid increased. Maximum lipid and arachidonic acid was produced during aging at pH 8, whereas arachidonic acid content of lipids was highest in mycelium aged at pH 6. EPA content ofM. elongata NRRL 5513 biomass increased during aging, reaching a maximum after 22–28 days. When the pH of the culture prior to harvesting was adjusted in the range of pH 4–9, pH values for development of maximum EPA in biomass and in lipids during storage were found to be 6 and 7, respectively. Temperature of aging had little effect on arachidonic acid or EPA content.     

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 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.     

Production of eicosapentaenoic acid by a bacterium isolated from mackerel intestines

Optimization of culture conditions for the growth rate, 5,8,11,14,17-cis-Eicosapentaenoic acid (EPA) content and EPA productivity of a bacterium isolated from Pacific mackerel intestines was investigated by use of a culture medium containing 1.00 wt% peptone and 0.50 wt% yeast extract in an artificial sea water (ASW). Cultivation temperature affected the growth rate and cellular EPA content of the bacterium. The cellular EPA content at 8°C was as great as 16.8 mg/g of dry cells, which was more than two times greater than that at 25°C (7.3 mg/g of dry cells), although the growth rate showed a maximum at 25°C. Both the yield of bacterial cells and the cellular EPA content at 25°C reached maximum values when the pH of the culture medium was nearly 7.0 and when the concentration of ASW was 100% (v/v). Under optimum culture conditions [25°C pH 7.0 and 100% (v/v) ASW], the amount of EPA accumulated in the cellular lipids reached 45.6 mg/L of culture broth after 8 hr.     

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 high yields of docosahexaenoic acid by Schizochytrium sp. strain SR21

The culture conditions for high-yield production of docosahexaenoic acid (DHA) by Schizochytrium sp. strain SR21 were investigated in a fermenter. With increasing carbon (glucose) and nitrogen (corn steep liquor and ammonium sulfate) sources (up to 12% glucose) in the medium, DHA productivity increased without a decrease in growth rate, i.e., 2.0, 2.7, and 3.3 g DHA/L/d with 6, 10, and 12% glucose, respectively. Eventually, 48.1 g dry cells/L and 13.3 g DHA/L were produced in 4 d with 12% glucose. DHA productivity was decreased with 15% glucose, i.e., 3.1 g/L/d. With 12% glucose, the lipid content was 77.5% of dry cells, and DHA content was 35.6% of total fatty acids. The lipid was composed of about 95% neutral lipid and 5% polar lipid. In polar lipids, the contents of phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylinositol were 74, 11, and 5%, respectively. The PC profile was simple, 70% of PC molecules were 1-palmitoyl-2-DHA-PC and 1.2-di-DHA-PC. These results indicate that Schizochytrium sp. strain 21 is an excellent source for microbial DHA production, including not only the acid form of DHA but also 2-DHA-PC.     

Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid

Because of the complexity of marine lipids, polyunsaturated fatty acid (PUFA) derivatives in highly purified form are not easily prepared by any single fractionation technique. The products are usually prepared as the ethyl esters by esterification of the body oil of fat fish species and subsequent physicochemical purification processes, including short-path distillation, urea fractionation, and preparative chromatography. Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils. At room temperature in the presence of Pseudomonas sp. lipase and a stoichiometric amount of ethanol without any solvent, efficient transesterification of fish oil was obtained. At 52% conversion, a concentrate of 46% eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) was obtained in excellent recovery as a mixture of mono-, di-, and triacylglycerols. The latter can be easily separated from the saturated and monounsaturated ethyl esters and converted into ethyl esters either by conventional chemical means or enzymatically by immobilized Candida antarctica lipase. Urea-fractionation of such an intermediary product can give an EPA+DHA content of approximately 85%.     

Production of arachidonic acid by filamentous fungus, Mortierella alliacea strain YN-15

A filamentous fungus producing significant levels of arachidonic acid (AA, C20∶4n−6) was isolated from a freshwater pond sample and assigned to the species Mortierella alliacea. This strain, YN-15, accumulated AA mainly in the form of triglyceride in its mycelia. An optimized culture in 25 L of medium containing 12% glucose and 3% yeast extract yielded 46.1 g/L dry cell weight, 19.5 g/L total fatty acid, and 7.1 g/L AA by 7-d cultivation in a 50-L jar fermenter. Assimilation of soluble starch by YN-15 was notably enhanced by the addition of oleic acid, soybean oil, ammonium sulfate, or potassium phosphate to a starch-based medium. Using starch as a main carbon source in the pre-pilot scale cultivation improved the production of AA by up to 5.0 g/L. Mortierella alliacea strain YN-15 is therefore a promising fungal isolate for industrial production of AA and other polyunsaturated fatty acids.     

Biosynthetic pathway of diepoxy bicyclic FA from linoleic acid by Clavibacter sp. ALA2

The biosynthetic pathway of two bicyclic FA, 12∶17, 13∶17-diepoxy-9(Z)-octadecenoic acid (DEOA) and 7-hydroxy-12∶17, 13∶17-diepoxy-9(Z)-octadecenoic acid (hDEOA), by Clavibacter sp. ALA2 was investigated. When cultivated with linoleic acid as a substrate, the strain produced 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA), DEOA, and hDEOA as well as other FA. To clarify the synthetic route to these bicyclic FA, the strain was cultivated with purified THOA as a starting substrate. THOA was consumed almost completely by the strain with sequential generation of DEOA and hDEOA. Moreover, the strain produced hDEOA when cultivated with purified DEOA. Therefore, it was confirmed that THOA was a precursor of these bicyclic FA and that hDEOA was generated from DEOA. Based on our previously reported result that linoleic acid is first converted to 12,13-dihydroxy-9(Z)-octadecenoic acid (DHOA) and the present results, the overall biosynthetic pathway for the diepoxy bicyclic FA from linoleic acid was postulated as: linoleic acid→DHOA→THOA→DEOA→hDEOA.     

Concentration of eicosapentaenoic acid by selective esterification using lipases

The aim of this work was to increase the content of EPA in FFA extracts from a commercial oil (43.1% EPA) and from Phaeodactylum tricornutum oil, a single-cell oil, by selective enzymatic esterification. Initially, the FFA extract was esterified with lauryl alcohol using nine lipases. All the lipases concentrated EPA in the unesterified FFA fraction. The criterion used to choose the best lipase was maximization of the dimensionless effectiveness factor (F_AE). This factor grouped the concentration factor (ratio between the EPA concentrations in the FFA fractions before and after esterification) with EPA recovery in the final FFA fraction. Experiments were carried out to correlate F_AE and the degree of esterification (ED, percentage of initial FA converted to lauryl esters). Lipase AK from Pseudomonas fluorescens was the most effective for concentrating EPA. Studies, of the optimal temperature, substrate molar ratio, solvent/substrate ratio, and treatment intensity (product of the lipase mass and the reaction time) were also carried out using the lipase. The maximum F_AE was obtained when the ED was 60%: EPA concentration was 72%, and recovery was 73%. Finally, this lipase was used to concentrate EPA from a FFA extract from P. tricornutum (23% EPA). The content of EPA in the unesterified FFA fraction increased to 71% at 78% ED (recovery of EPA, 75.5%). Comparison of the results of obtained with the two FFA extracts seemed to indicate that the selectivity of Lipase AK for EPA depended on the content of EPA, with higher contents of EPA in the initial FFA mixture reducing the selectivity for EPA.     

Biodegradation ofVernonia galamensis seed Oil byAcinetobacter andPseudomonas sp.

The biodegradability ofVernonia galamensis seed oil (VO) has been demonstrated with two environmental bacterial strains,Acinetobacter Iwoffi (HU 3955), andPseudomonas sp. (HU 4020). A time-dependent increase in the degradative activities of both bacteria species was apparent. There wasca. 60% decrease in the amount of VO over an eightday incubation period. Additionally, lipolytic activity was evident from the amount of free fatty acid (FFA) that was generated. The percent FFA of the residual oil were 82% for thePseudomonas strain, and 62% for theAcinetobacter strain. The weight per epoxy value of the VO in the fermentation medium remained relatively constant over the incubation period, suggesting the lack of preference for either the epoxidized or nonepoxidized acids present in VO.     

Enhancement of arachidonic acid production by Mortierella alpina 1S-4

The effect of mineral addition on arachidonic acid (AA) production by Mortierella alpina 1S-4 was evaluated. At first, the addition of minerals such as sodium, potassium, calcium, and magnesium was examined in flask cultures, and then the addition of phosphorus with the optimal amounts of the minerals was investigated in a 10-L jar-fermenter. As a result, 1.5% soy flour medium with the addition of 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O, and 0.05% MgCl₂·6H₂O was found to enhance the AA yield 1.7-fold over that without mineral addition. When 1% yeast extract with the above mineral mixture was used, the AA yield was enhanced 1.35-fold over that without minerals. We also verified that an increase in the polar lipid content occurred in the case of only KH₂PO₄ addition, and that the above-mentioned increase in the AA yield was due to the minerals themselves, not a pH buffer effect.     

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).     

富里酸诱导对三角褐指藻EPA合成积累的影响

探究不同富里酸作用浓度对三角褐指藻二十碳五烯酸（EPA）合成积累的影响。结果表明,在20 mg/L富里酸诱导下,三角褐指藻EPA含量和产量均达到最大值,为19.81 g/100 g和738.91 mg/L,较无富里酸诱导EPA含量和产量分别提高2.01和2.54倍。外源富里酸作用提高了三角褐指藻胞内过氧化物酶（POD）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）酶活,使得色素和多酚抗氧化组分比例增加,活性氧（ROS）和丙二醛（MDA）水平降低,整体抗氧化能力获得显著提升,有效阻抑EPA的氧化分解;富里酸作用同时还上调了EPA合成通路脱饱和酶（Δ5-FADS、Δ6-FADS、Δ12-FADS、Δ15-FADS、Δ17-FADS）和延长酶（Δ6-ELOVL）等系列关键酶,使其上调表达量较无富里酸作用分别增加1.27、1.81、1.24、1.45、1.43和1.46倍,为EPA高效合成积累提供前驱物和能量。富里酸作用效果协同强化了三角褐指藻EPA合成积累效率。     

富里酸诱导对三角褐指藻EPA合成积累的影响

探究不同富里酸质量浓度对三角褐指藻二十碳五烯酸(EPA)合成积累的影响。结果表明，在质量浓度20 mg/L富里酸诱导下，三角褐指藻EPA含量和产量均达到最大值，为19.81 g/100 g和738.91 mg/L，较无富里酸诱导EPA含量和产量分别提高1.12和1.50倍。外源富里酸作用提高了三角褐指藻胞内过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的酶活，使色素(叶绿素、类胡萝卜素)和总多酚抗氧化组分比例增加，活性氧(ROS)和丙二醛(MDA)水平降低，整体抗氧化能力获得显著提升，有效阻抑EPA的氧化分解；富里酸作用同时还上调了EPA合成通路去饱和酶(Δ⁵-FADS、Δ⁶-FADS、Δ¹²-FADS、Δ¹⁵-FADS、Δ¹⁷-FADS)和延长酶(Δ⁶-ELOVL)等系列关键酶，使其上调表达量较无富里酸作用分别增加0.33、0.94、0.33、0.58、1.15和0.70倍，为EPA高效合成积累提供前驱物和能量。     

Autoxidation of synthetic isomers of triacylglycerol containing eicosapentaenoic acid

Several triacylglycerols (TAG) that contained eicosapentaenoic acid (EPA) were chemically synthesized and stored at 25°C to assess the influence of TAG structure on oxidative stability and formation of oxidation products. Oxidative stability was evaluated by oxygen consumption during storage of the TAG. Autoxidation products of TAG were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Results showed that a 2:1 (mole/mole) mixture of trieicosapentaenoylglycerol (EEE) and tripalmitoylglycerol (PPP) was most susceptible to autoxidation. The oxidative stability of TAG that contained EPA and palmitic acid was negatively correlated with the moles of EPA in a single TAG molecule. When TAG with one EPA and two other fatty acids were oxidized, chainlength of constituent fatty acids hardly affected the oxidative stability of EPA-containing TAG molecules, except for stearic acid. HPLC and LC-MS analyses showed that monohydroperoxides were major oxidation products regardless of type of TAG. Bis- and tris-hydroperoxides were formed during autoxidation of EEE and dieicos-apentaenoylpalmitoylglycerol. Monohydroperoxy epidioxides were found in all autoxidized TAG. These observations suggested that TAG structure affected the oxidation of TAG with highly unsaturated fatty acids.     

Conversion of oleic acid to 10-ketostearic acid by Sphingobacterium sp. strain O22

The conversion of oleic acid by a bacterium, tentatively identified as Sphingobacterium thalpophilum strain O22, was investigated. The microorganism was isolated as a stable culture from compost that was enriched with soybean oil outdoors and subsequently with oleic acid in the laboratory. Strain O22 converted oleic acid to products identified as 10-ketostearic acid (95% of the total conversion product) and 10-hydroxystearic acid (5%). This is in contrast to S. thalpophilum strain B-14797, which produces solely 10-hydroxystearic acid. Maximal conversion was reached in about 36 h after the addition of oleic acid to the fermentation broth. The yield of 10-ketostearic acid was approximately 75% from 0.26 g of oleic acid in 30 mL fermentation broth at 28°C and 200 rpm for 48 h. This is the first report on the major production of 10-ketostearic acid by a microorganism in the genus Sphingobacterium.     

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.     

Production and structural features of a water-soluble polysaccharide from a mutant strain of Agrobacterium sp.

We have developed a mutant strain derived from Agrobacterium sp. ATCC 31750, which produces a water-soluble polysaccharide having potential utility to the food, feed, pharmaceutical and cosmetic industries. A high concentration of product (15 g/L) is obtained by 48 h cultivation of the mutant strain under optimized fermentation conditions. The water-soluble polysaccharide obtained from cultures of the mutant strain beta82 has Glc:Man:Gal in approximate molar ratios of 5.8:6.7:1.0. The molecular weight of the polysaccharide was determined to be approximately 1000 kDa by HPSEC analysis. Linkage analysis contained 3-Glcp, 3-Manp, terminal Glcp and terminal Manp, as well as a small proportion of 3- and 3,4-Galp, and 4,6-Manp residues. Based on analyses using FT-IR and ¹³C NMR spectrometers, most glycosidic bonds joining these sugar residues are of the α-type, and acetyl groups are apparently attached to the polymer chain at random.