Liquid Shake Cultures Research Articles

Evaluation of bacterial strategies to promote the bioavailability of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbon (PAHs)-degrading bacteria may enhance the bioavailability of PAHs by excreting biosurfactants, by production of extracellular polymeric substances, or by forming biofilms. We tested these hypotheses in pure cultures of PAHs-degrading bacterial strains. Most of the strains did not substantially reduce the surface tension when grown on PAHs in liquid shaken cultures. Thus, pseudo-solubilization of PAHs in biosurfactant micelles seems not to be a general strategy for these isolates to enhance PAHs-bioavailability. Three semi-colloid Sphingomonas polysaccharides all increased the solubility of PAHs (Gellan 1.3- to 5.4-fold, Welan 1.8- to 6.0-fold and Rhamsan 2.4- to 9.0-fold). The increases were most pronounced for the more hydrophobic PAHs. The polysaccharide-sorbed PAHs were bioavailable. Mineralization rates of 9-[14C]-phenanthrene and 3-[14C]-fluoranthene by Sphingobium EPA505, were similar with and without sphingans, indicating that mass-transfer rates from PAHs crystals to the bulk liquid were unaffected by the polysaccharides. Biofilm formation on PAHs crystals may favor the diffusive mass transfer of PAHs from crystals to the bacterial cells. A majority of the PAHs-degraders tested formed biofilms in microtiter wells coated with PAHs crystals. For strains capable of growing on different PAHs; the more soluble the PAHs, the lower the percentage of cells attached. Biofilm formation on PAHs-sources was the predominant mechanism among the tested bacteria to overcome mass transfer limitations when growing on poorly soluble PAHs.

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of β-glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B5 vitamins, 0.1 mg dm-3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm-3 α-naphthaleneacetic acid, 3 % glucose + 50 mg dm-3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.

Preservation of Nostocacean Hormogone Motility in Desiccated Calcium Carbonate Agar Patches

AbstractFilamentous nitrogen fixing cyanobacteria are resistant to desiccation, nutrient depletion, adverse temperature and challenging photic environments. This is generally thought to account for their role as pioneering forms of life on denuded strata. It has also lent credence to the possibility that cyanobacteria with prolonged viability may have been able to survive conditions of interplanetary space to implement a panspermian hypothesis for the origin of life on earth. Prolonged retention of viability had been noted in our collection of myxotrophically grown strains of nostocaceans cultured in liquid or solid media containing calcium carbonate. Such media permitted satisfactory recovery and regrowth after storage at low light intensities for 2 years or more. Moreover, even the oldest calcium carbonate cultures contained motile hormogonia when observed microscopically. This phenomenon can be used to advantage in sending cultures to other laboratories. Not only are organisms maintained during passage through the postal system but since the cultures formed hormogonia readily, it was possible to produce preservable homogeneous agar lawns that generated hormogonia as needed for demonstration or experiment. A practical technique consists of seeding axenic strains of Nostoc species grown in liquid shake culture under cool‐white fluorescent illumination into solid media containing 0.7% sucrose, 0.05% finely divided calcium carbonate and 1.4% purified agar. The preparations are incubated under red fluorescent illumination to produce several crops of hormogonia in successive cycles of development, within the agar. The heavily grown lawns are cut into disks or squares, then transferred to dishes and dried in desiccators with anhydrous calcium sulfate. The dry patches produce swarms of hormogonia in 2 or 3 h, depending upon the rate of rehydration. The patches are facile tools for purifying cultures through the isolation of hormogonia and studying cyanobacterial physiology by the rapid response of motile hormogonia.

Micropropagation of the rare Eucrosia stricklandii (Amaryllidaceae) by twin-scaling and shake liquid culture

SummaryBulbs of E. stricklandii were introduced into in vitro culture by the twin-scaling technique. Different Murashige and Skoog (MS) media supplemented with 0.5% w/v activated charcoal, naphthaleneacetic acid (NAA)/benzyladenine (BA) 0.54/4.44 μM or NAA/Thidiazuron (TDZ) 0.54/0.45 μM were used for shoot induction. Media with different combinations of 2,4-dichlorphenoxyacetic acid (2,4-D) and BA were tested during the secondary multiplication. The shoots obtained in the multiplication step were then transferred to a liquid medium with different sucrose concentrations (from 0.088 to 0.351 M) to increase biomass. Shoot bulbing was induced in 0.263.M sucrose semi-solid medium without growth regulators. Several media containing different concentrations of NAA (ranging from 0 to 5.37 μM) combined with various sucrose concentrations (from 0.044 to 0.263 M) were also tested for root induction and the bulblets were finally transplanted to soil. The best induction of shoots was obtained in the media with activated charcoal or with TDZ, but those treated with activated charcoal showed a better regeneration rate and the shoots were better formed. The best development of new shoots was obtained in the medium containing 0.45 and 4.44 μM of 2,4-D and BA, respectively. 0.175–0.263 M sucrose in the liquid medium appeared to be the most appropriate condition for increase in biomass. Rooting was promoted by low NAA concentrations (0.537–1.343 μM), and 0.175 M sucrose was the best treatment for root development.

The Aspergillus nidulans phsB4 mutation alters colonial growth and development of the mould at acidic pH

The phsB4 mutant of the mould Aspergillus nidulans, identified as showing increased sensitivity to acid pH, is mitotically unstable and its conidia swell and lyse, forming protoplasts during germination and early development in shaken liquid cultures. On solid medium, we observed balloon-shaped hyphal swellings, a phenotype also exhibited by the chitin synthase gene (chsD) disruptants. We also observed that lysis was osmotically remediable with 0.5 M NaCl, but the balloon-shaped hyphal swelling was remedied in a pH-dependent way i.e., this phenotype was remedied only at pH values above 6.5. Based on the nature of our mutant selection, the pH sensitive phenotype of the selected strains, the known occurrence of hyphal swelling in cell wall mutants of A. nidulans, and the transformation with cosmids that hybridize to chsD gene, the phsB and chsD genes are possibly alleles.

Altered regulation of 15-acetyldeoxynivalenol production in Fusarium graminearum.

Most Fusarium graminearum isolates produce low or undetectable levels of trichothecenes in liquid shake cultures, making it difficult to perform biochemical studies of trichothecene biosynthesis. To develop strains with higher levels of trichothecene production under liquid shake conditions we transformed F. graminearum with both a reporter gene containing a homologous trichothecene pathway gene promoter (TRI5) and a gene encoding a heterologous trichothecene pathway transcription factor (TRI6). The TRI5 and TRI6 genes are part of the trichothecene pathway gene clusters of both Fusarium sporotrichioides and F. graminearum. These genes encode trichodiene synthase (encoded by TRI5), the first enzyme in the trichothecene pathway, and a transcription factor (encoded by TRI6) required for pathway gene expression. Transformation of F. graminearum with plasmids containing either an F. graminearum TRI5 promoter fragment (FGTRI5(P)) or FGTRI5(P) coupled with the beta-D-glucuronidase (GUS) reporter gene resulted in the identification of several transformants capable of producing 45 to 200 mg of 15-acetyldeoxynivalenol (15-ADON)/liter in liquid shake culture after 7 days. Increased 15-ADON production was only observed in transformants where plasmid integration occurred through the FGTRI5(P) sequence and was not accompanied by increased GUS expression. 15-ADON production was further increased in liquid culture up to 1,200 mg/liter following introduction of the F. sporotrichioides TRI6 gene (FSTRI16) into F. graminearum. The effects of FSTRI6 on 15-ADON production also depended on plasmid integration via homologous recombination of the FGTRI5(P) fragment and resulted in a 100-fold increase in GUS expression. High-level production of 15-ADON in liquid shake cultures provides a convenient method for large-scale trichothecene preparation. The results suggest that targeting transformation vector integration to FGTRI5(P) alters pathway gene expression and are consistent with the proposed conservation of TRI6 function between Fusarium species.

タイム（Ｔｈｙｍｕｓ ｖｕｌｇａｒｉｓ）の培養によるチモールの生産

The production of thymol was investigated in a bioreactor culture of plantlets of Thymus vulgaris. The plantlets successfully multiplied in a liquid shake culture, but the thymol content was quite low compared to the static culture. When the plantlets were cultured using a non-mechanical agitated type of bioreactor, the plantlets grew well and the productivity of thymol (mol/dry weight of plantlets) was the almost the same as that of the static culture. Most of thymol (approx. 80%) was contained in the medium. When an ebb and flow type of culture system (EFBR type) was used as the bioreactor, the growth was stimulated and the productivity of thymol was increased whereas the medium contained only a small amount of thymol (7% of the total thymol). The EFBR type is thought to be suitable for the multiplication of plantlets and the production of thymol.

Physical Environment in vitro Affects Laboratory and Nursery Growth of Micropropagated Hostas

Hosta ×hybrid Tratt. `Blue Cadet' and Hosta tokudama Tratt. `Newberry Gold' were micropropagated in shaken liquid culture and on agar media, in conventional vessels and vessels modified for ventilation in vitro. Acclimatization under intermittent mist and growth in an outdoor nursery during the late spring and summer were monitored by dry weight analysis of sample plants every 4 days for a 60-day period (ex vitro growth). Results for `Newberry Gold' were 1) in vitro shoot growth was greater in liquid than agar culture, regardless of vessel; 2) shoots from agar or liquid culture grew at similar rates ex vitro; 3) ex vitro root growth was greater for liquid than agar cultured plants, regardless of vessel type. Results for `Blue Cadet' were 1) in vitro and ex vitro shoot growth was greater in liquid than agar culture regardless of vessel type and 2) ex vitro root growth was greatest for liquid cultured plants from conventional vessels. Ventilated vessels were generally beneficial for agar but not liquid culture. Benefits of liquid culture for micropropagation of Hosta found in vitro are at least maintained and sometimes enhanced during ex vitro growth in the mist bed and nursery.

The effect of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana crosses

The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at 5 weeks after pollination (WAP), significantly improved the germination percentage as compared to 5% sucrose. The germination percentage did not differ between both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained with media containing the full or half of the concentrations of micronutrients and macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or 0.1 mg l-1) had no effect on the germination percentage. The use of liquid-shaken culture resulted in germination percentages which were similar to those on agar-solidified media. Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose. The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice culture, whereas the total concentration of carbohydrates did not decrease remarkably in media for ovule culture.

The Influence of Nutrition on Conidial Production by Rhynchosporium alismatis and on Their Subsequent Infectivity to Alisma lanceolatum

Nutrient composition of liquid-shake cultures significantly influenced conidial production by five isolates of Rhynchosporium alismatis after 6 days at 25 C. Lima bean broth at pH 7.5 produced the most conidia (4.99 x 10 7 /ml). The zwitterionic buffers PIPES and HEPES did not significantly affect broth pH, but did significantly affect yield of conidia. pH per se did not appear to affect yield directly. The medium in which an isolate was grown had a significant effect on the virulence of the resulting conidia as measured by disease severity scores in leaf discs of Alisma lanceolatum after 3 and 13 days. There was a significant difference between isolates, produced in the same medium, in the subsequent rate of disease development. The correlation between image analysis of diseased leaf discs with visual assessment was low, and it was considered that automatic assessment of disease severity was not cost-effective with the equipment used. Isolate DAR 73158 and lima bean broth are considered to be the combination of choice for further studies to explore the fitness of conidia produced in small-scale biofermentors.

Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. A novel type of class II hydrophobin.

A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusiformis. The predicted amino-acid sequence of the protein, dubbed CFTH1, shows a putative signal sequence for secretion, followed by three class II hydrophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cultures showed CFTH1 at the 50-55-kDa position. N-terminal sequencing of both untreated mature CFTH1 and of a fragment obtained by trypsin digestion revealed that CFTH1 is not processed between the hydrophobin domains. Mass spectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post-translational modification but not glycosylation. Purified CFTH1 self-assembled at hydrophilic/hydrophobic interfaces and, after assembly at a water/air interface, it was found to be highly surface active. Antibodies raised against CFTH1 localized the protein in a mucilageous coat surrounding submerged vegetative hyphae in liquid shaken culture and, as a discrete layer of about 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.

Cultivation of the marine basidiomycete Nia vibrissa (Moore & Meyers)

An isolate of Nia vibrissa was fermented in liquid shake cultures and on agar. Suitable cultivation conditions are a prerequisite for the continuous synthesis of biological active metabolites. The growth response of N. vibrissa to four selected media and several environmental factors, such as salinity, pH and light, was studied. The amount of produced mycelia and the quantity and activity of the organic extracts were parameters for the optimal cultivation method. The ethanolic extracts of the mycelia of N. vibrissa grown in all the investigated nutrient media, showed an influence of the duration of cultivation on the biological activity. A synthetic medium with a pH of 7.5 was the preferred nutrient medium. The addition of wood and incubation under continuous light had no effect on growth but increased the activity of the ethanolic extract. The optimal agar medium salinity for colony growth was in the range between 5 and 25‰. At a salinity of 150‰ growth was not observed.

ササユリ小球根の液体振とう培養における培地からの糖および無機イオンの吸収に及ぼす照明の影響とグルタミン添加による球根肥大促進

The enlargement and nutrient absorption from a liquid-shaking culture with or without glucose by Lilium japonicum bulbs were investigated. Promotion of NH4+, NO3- and H2PO4- absorption by illumination occurred after one or two weeks in the medium, independent of glucose. Glucose absorption from the medium was promoted by illumination three or four weeks after treatments were started ; a concomitant promotion of bulb enlargement was noted. These results indicate that nitrogen uptake was accelerated by illumination as well as bulb enlargement through the increase in glucose absorption. The effects of glutamine supplement on the bulb enlargement and nitrogen uptake were investigated because it is absorbed more readily than inorganic nitrogen ions are. Bulb enlargement and absorption of total N were promoted by the presence of glutamine in the medium even in the dark. Thus, adding glutamine to the medium could substitute for illumination in promoting bulb enlargement ; it would eliminate the need for light and, thereby, reduce the cost of bulb production by liquid-shaking culture.

Simplified clonal multiplication of mulberry using liquid shake culture

Shoot apex and axillary bud explants of mulberry (Morus indica) were cultured in liquid Murashige and Skoog medium containing N-(2-chloro-4-pyridyl)-N′-phenylurea (a urea-type cytokinin) at 0.5–2 mg l-1. The cultures were maintained at 120 rpm up to 6 weeks. During the first 4 weeks, shoot induction and shoot elongation were observed, followed by adequate rooting during the latter 2 weeks in both explants. A single-step liquid medium which induces consecutive development of shoots and roots in a same medium is useful for effective multiplication of mulberry plantlets with reduced labor and cost.

Purification and characterization of laccase from Chaetomium thermophilium and its role in humification.

Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45 degreesC both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70 degreesC and had half-lives of 24 and 12 h at 40 and 50 degreesC, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.

液体振とう培養におけるササユリ小球根の養分吸収と肥大に及ぼす照明の影響

The effects of illuminating the liquid-shaking culture on the absorption of nutrients by enlarging miniature bulbs of Lilium japonicum Thunb. were investigated. When bulbs (0.3 to 0.4g FW) were cultured for 165 days under the light or dark condition in the three kinds of liquid media containing different types and concentrations of sugars, light promoted enlargement. A media with 3% glucose under light was optimum for bulb enlargement ; fresh bulb weight reached about 5.3 g by the end of the experiment. Cumulative absorption of sugars, NH4+, NO3-, H2PO4- and K+ by bulbs exposed to light and sucrose was significantly higher than those grown in the dark ; Light and sucrose had no effect on Ca2+ and Mg2+ absorption. The gain in fresh bulb weight in the light almost equalled that of sugar absorption by the bulb, which suggests that bulb enlargement was not related to photosynthesis but to promotion of sugar absorption from the medium. Thus, light illuminating the liquid-shaking culture enabled us to produce 5 g bulbs (about 9 cm in circumference) in 5-6 months.

Production and purification of an extracellular β-galactosidase from the Dutch elm disease fungusOphiostoma novo-ulmi

The causal agents of Dutch elm disease, Ophiostoma ulmi (isolate H200) and Ophiostoma novo-ulmi (isolate CKT-11), secreted similar amounts of β-galactosidase in liquid shake cultures when grown on galacturonic acid or sodium pectate (1.45 ± 0.16 and 1.03 ± 0.24 nkat∙mL−1for O. ulmi, respectively, and 1.30 ± 0.08 and 1.28 ± 0.26 nkat∙mL−1for O. novo-ulmi, respectively). Rhamnose and pectin also stimulated secretion but to a lesser extent, whereas on glucose, enzyme activity was barely detectable (≤0.01 nkat∙mL−1). Ophiostoma novo-ulmi was shown by Q-Sepharose chromatography to form two β-galactosidases, named β-galactosidases I and II. In cultures grown on galacturonic acid β-galactosidase I accounted for approximately 75% of the total activity in the culture filtrate. β-Galactosidase I was further purified to apparent electrophoretic homogeneity by means of Sephacryl gel filtration chromatography, chromatofocusing, and Superdex75 gel filtration. The molecular mass of the enzyme was 135 kDa by SDS–PAGE and 123 kDa by gel filtration. Its isoelectric point, determined by chromatofocusing, was 4.9. The optimal pH for enzyme activity was 5.8 and the optimal temperature was 50 °C. The Kmvalues for p-nitrophenyl β-D-galactopyranoside and lactose were 7.52 and 14.23 mM, respectively, and the maximum velocities for these substrates were 1733 and 355 nkat∙mg protein−1, respectively. The Kivalue for D(−)-galactonic acid γ-lactone was 2.29 mM.Key words: Dutch elm disease, β-galactosidase, Ophiostoma ulmi, Ophiostoma novo-ulmi.

Expression of a plant protein by Neurospora crassa.

Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.

Impact of Temperature, Osmotic Potential, and Osmoregulant on the Growth of Three Ectotrophic Root-Infecting Fungi of Kentucky Bluegrass.

Two isolates each of Magnaporthe poae, Gaeumannomyces incrustans, and Leptosphaeria korrae were grown at 25°C in liquid shake culture in minimal salts medium (-0.12 MPa) or minimal salts medium adjusted to -0.5 MPa with KCl, MgCl2, or polyethylene glycol (PEG). Fungal dry weight of all three species was greater in minimal salts medium amended to -0.5 MPa with MgCl2 than in nonamended medium, and dry weight in medium amended with PEG was not different from dry weights in nonamended medium or medium amended with KCl. Fungi were incubated at varying temperatures on a minimal salts solid-agar medium (-0.12 MPa) adjusted to osmotic potentials ranging from -0.5 to -5.0 MPa with KCl or MgCl2. Optimum growth of M. poae, G. incrustans, and L. korrae on nonamended medium occurred at 30, 30, and 25°C, respectively. At optimum temperatures for each species, fungal growth was greatest at the higher osmotic potentials tested (-0.5 to -1.0 MPa) and decreased in a linear manner as osmotic potential decreased. In most cases, growth was detected at the lowest osmotic potential measured (-5.0 MPa). The relationship of fungal growth to osmotic potential depended on both temperature and osmoregulant. At temperatures optimal or nearly optimal for fungal development, the growth of all three fungi declined more rapidly with decreasing osmotic potential when grown on medium amended with MgCl2 than on medium amended with KCl. At the highest temperature evaluated for growth of M. poae and L. korrae (35 and 30°C, respectively), growth on medium amended with KCl was curvilinear and peaked at osmotic potentials of -2.5 to -3.0 MPa. Furthermore, between osmotic potentials of -2.0 and -5.0 MPa, M. poae grew best at 35°C. When maintained on nonamended minimal salts medium (-0.12 MPa) in liquid culture at 25°C or on nonamended solid-agar medium at temperatures optimal for growth, M. poae grew at a faster daily rate than L. korrae.

Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185.

As a representative of a genus with species considered to be potential commercial producers of the nutritionally important polyunsaturated fatty acid docosahexaenoic acid (DHA), Thraustochytrium sp. ATCC 26185 was investigated to determine its potential for DHA production and lipid composition. Cells from liquid shake cultures contained 32% (w/w) lipid, 18% of which was nonsaponifiable lipid. The major saturated fatty acids (14:0 and 16:0) comprised up to 59% of the total fatty acids, and DHA was up to 25% after 6 d incubation. Squalene represented 63% of the nonsaponifiable lipid, and cholesterol composed 41% of the total sterols. The phospholipids expected for eucaryotic microbes were detected with phosphatidylcholine as the major phospholipid at 76% of the total. The ultrastructure of this species was similar to other Thraustochytrium species except that the cells did not have surface scales and they contained unusual membrane-like structures that appeared to be associated with oil formation.