Toro Nakahara

Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21

Culture conditions of Schizochytrium limacinum SR21 for the purpose of microbial docosahexaenoic acid (DHA) production were investigated. The strain SR21 showed a wide tolerance to salinity; that is, the optimum salinity was between 50% and 200% that of sea water. Monosaccharides (glucose and fructose) and glycerol supported good cell growth and DHA yield. Di- and polysaccharides, oleic acid, and linseed oil gave low DHA yields. A high content of DHA (more than 30% of total fatty acids) was obtained from culture on glucose, fructose, and glycerol, and also the strain had simple polyunsaturated fatty acid profiles. The major polyunsaturated fatty acids other than DHA were n-6 docosapentaenoic acid only, and the contents of icosapentaenoic acid and arachidonic acid were less than 1%. Using corn steep liquor as a nitrogen source, a high total fatty acid content was obtained. The total fatty acid content in the dry cell weight increased as the concentration of the nitrogen source decreased, reached more than 50%. An increase in carbon source concentration led to a high DHA yield. A maximum DHA yield of more than 4 g/l was obtained in both glucose and glycerol media at 9% and 12% respectively. S. limacinum SR21 was thought to be a promising resource for microbial DHA production yielding a good level of productivity as well as a simple polyunsaturated fatty acid profile.

Production of docosahexaenoic and docosapentaenoic acids bySchizochytrium sp. isolated from Yap Islands

A marine microbe (strain SR21) from the coral reef area of the Yap Islands was isolated by a screening test for polyunsaturated fatty acids and was found to accumulate lipid that contained 22:5n-6 docosapentaenoic acid (DPA) as well as 22:6n-3 docosahexaenoic acid (DHA). Strain SR21 was identified as genusSchizochytrium in Labyrinthulomycota, owing to its ultrastructural character and life cycle, which is composed of vegetative cell, zoosporangium, and zoospore stages. After cultural optimization, both in flask and fermenter, the highest DHA and DPA productivities of 2.0 and 0.44 g/L per day, respectively, were obtained in a medium of 60 g/L glucose and corn steep liquor/ammonium sulfate in a half salt concentration of seawater in fermenter culture at 28°C and pH 4. This productivity was almost twice that obtained with flask culture, indicating its high resistance to mechanical stirring. The lipid extracted from the cell was about 50% of the dry cell weight and was composed of 93% triacylglycerol (TG). DHA content of the lipid was 34% of total fatty acids. The TG profile was simple, and the content of the most dominant TG, 1,3-dipalmitoyl-2-DHA-TG, was 27%. TG that contained DHA and n-6-DPA amounted to 57 and 17%, respectively, of total TG molecules. Strain SR21 was revealed to be an excellent source of microbial DHA and n-6 DPA.

Molecular Phylogeny of Labyrinthulids and Thraustochytrids Based On the Sequencing of 18s Ribosomal Rna Gene

ABSTRACT Labyrinthulids and thraustochytrids are unicellular heterotrophs, formerly considered as fungi, but presently are recognized as members in the stramenopiles of the kingdom Protista sensu lato. We determined the 18S ribpsomal RNA gene sequences of 14 strains from different species of the six genera and analyzed the molecular phylogenetic relationships. the results conflict with the current classification based on morphology, at the genus and species levels. These organisms are separated, based on signature sequences and unique inserted sequences, into two major groups, which were named the labyrinthulid phylogenetic group and the thraustochytrid phylogenetic group. Although these groupings are in disagreement with many conventional taxonomic characters, they correlated better with the sugar composition of the cell wall. Thus, the currently used taxonomic criteria need serious reconsideration.

Schizochytrium limacinum sp. nov., a new thraustochytrid from a mangrove area in the west Pacific Ocean

Schizochytrium limacinum sp. nov, a thraustochytrid, is described from sea water from a mangrove area of the Yap Islands in the west Pacific Ocean. It is closest to S. aggregatum , and differs from other Schizochytrium spp. in its limaciform amoeboid cells, the size of zoospores, released without an amoeboid stage, and the assimilation profile of carbon sources.

Improvement of Docosahexaenoic Acid Production in a Culture of Thraustochytrium aureum by Medium Optimization

Growth optimization of a docosahexaenoic acid (DHA) producing marine fungus, Thraustochytrium aureum (ATCC 34304), resulted in cell productivity of 5.7 g/l and 460 mg/l of total lipid in 69 h flask culture, almost twice the level of cell productivity obtained for the strain reported previously. The lipid extracted from the cells was primarily composed of triacylglycerol and contained about 40% DHA. The fermentor culture showed a lower growth rate than the flask culture indicating growth inhibition due to mechanical stirring. Cells grown in the fermentor showed a coagulant tendency. ATCC 34304, in culture, seems to reproduce asexually and the life cycle of the cells consists of a zoospore stage, trophic cell stage and sporangium stage. Zoospore release was observed once during the early growth phase throughout growth period both in flask and fermentor culture.

Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18S rRNA genes.

AbstractSeven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids.

Increase in the γ-linolenic acid content by solvent winterization of fungal oil extracted fromMortierella genus

The fungal oil extracted fromMortierella ramanniana var.angulispora (IFO 8187) was solvent winterized in order to raise the content of γ-linolenic acid (GLA). Effects of winterization conditions (solvent, oil concentration in the solvent and temperature) and changes of glyceride compositions were discussed. The fungal oil was separated into four diglycerides and 17 triglycerides (TG) with high performance liquid chromatography. The predominant species were POO, POP and LOP, whose contents were 24.4, 22.9 and 9.4% of the total TG, respectively. Ethanol at 4°C gave the highest GLA content of 10.5% in spite of lower yield than with acetone at −20°C. The highest separation efficiency for GLA (ηGLA) was 0.27 with acetone at −20°C and 10% oil concentration, resulting in 8.3% of GLA from the fungal oil at 5.7% LGA. In case of lower oil concentration at 5–20%, ηGLA showed higher in the following order: acetone (−20°C)>n-hexane (−20°C)>acetone (4°C)>petroleum ether (−20°C). The winterization process also proved to be effective for the separation of TG type, Sa2U (Sa; saturated fatty acid; U, unsaturated fatty acid) into the crystallized fraction and SaU2 into the liquid fraction. Acetone at −20°C showed higher separation efficiency for triunsaturated TG than the other solvents.

Docosahexaenoic Acid Production and Lipid-Body Formation in Schizochytrium limacinum SR21

Schizochytrium limacinum SR21, a thraustochytrid (Labyrinturomycota), is a heterotrophic marine microorganism. SR21 has attracted recent attention because of the production of docosahexaenoic acid (DHA). We obtained highly concentrated SR21 zoospores and successfully observed synchronous growth. We investigated changes of lipid content and fatty acid composition during the growth. The morphological features of the lipid bodies were also described via fluorescent and electron microscopy. The cells developed quickly after zoospore settlement. Lipid bodies developed in accordance with an increase in lipid content during the 8-h synchronous growth. The total lipid was composed mainly of triacylglycerol, sterol esters, and phosphatidylcholine. The proportion of neutral lipids (triacylglycerol and sterol esters) in the total lipid was fairly constant during growth. The fatty acid composition of neutral lipids, primary components of the lipid body, and phospholipids, primary components of the cell membranes, was nearly unchanged during the synchronous growth. However, the DHA content of the phospholipids decreased drastically after a 10-day culture. Electron micrographs prepared using a high-pressure freeze substitution technique revealed a fine structure of light- and dark-staining bands inside the lipid bodies in many stages of the cells.

Optimization and scale-up of γ-linolenic acid production by Mortierella ramanniana MM 15-1, a high γ-linolenic acid producing mutant

Abstract Optimum conditions for cultivation of Mortierella ramanniana mutant MM 15-1 were investigated with an aim of producing a lipid which contains high γ-linolenic acid (GLA). The optimum initial glucose concentration and pH control level were 300 g/ l and 4.0, respectively. Although both pellet and filamentous forms of this mutant were observed during the cultivation, the pellets accumulated lipid with higher GLA content than the filamentous forms. The effects of culture conditions on pellet formation were therefore investigated in a 30- l jar fermentor. The results showed that with an inoculum spore concentration of 5.0 × 10 3 /ml and an agitation speed of 800 rpm, 0.15–0.5 mm pellets were formed and consequently, the GLA content of the lipid increased to a very high value of 18.3%. Furthermore, when this process was scaled up to a 10-kl fermentor, using the impeller tip velocity as the scale-up parameter, both the pellet formation and the lipid production as well as the GLA content of the lipid were consistent with those obtained in the 30- l jar fermentor.

Incorporation of linoleic acid and its conversion to λ-linolenic acid in fungi

The incorporation of [1-14C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6-desaturation of [1-14C]LA. [1-14C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-14C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1-14C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1-14C]LA into λ-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-14C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.