DNA NUCLEOSIDE COMPOSITION AND METHYLATION IN SEVERAL SPECIES OF MICROALGAE1

Total DNA was isolated from 10 species of microalgae, including representatives of the Chlorophyceae (Chlorella ellipsoidea, Chlamydomonas reinhardtii, and Monoraphidium minutum), Bacillariophyceae (Cyclotella cryptica, Navicula saprophila, Nitzschia pusilla, and Phaeodactylum tricornutum), Charophyceae (Stichococcus sp.), Dinophyceae (Crypthecodinium cohnii), and Prasinophyceae (Tetraselmis suecica). Control samples of Escherichia coli and calf thymus DNA were also analyzed. The nucleoside base composition of each DNA sample was determined by reversed-phase high performance liquid chromatography. All samples contained 5-methyldeoxycytidine, although at widely varying levels. In M. minutum, about one-third of the cytidine residues were methylated. Restriction analysis supported this high degree of methylation in M. minutum and suggested that methylation is biased toward 5′-CG dinucleotides. The guanosine + cytosine (GC) contents of the green algae were, with the exception of Stichococcus sp., consistently higher than those of the diatoms. Monoraphidium minutum exhibited an extremely high GC content of 71%. Such a value is rare among eukaryotic organisms and might indicate an unusual codon usage. This work is important for developing strategies for transformation and gene cloning in these algae.     

Continuous culture with deep seawater of a benthic food diatom Nitzschia sp.

A continuous culture system for a benthic food diatom Nitzschia sp. wasestablished by using properties of high nutrient and clean of deep seawater(DSW). DSW collected from 320 m depth in Muroto City, Japan, was introducedinto a glass-pipe bioreactor (14 cm length, 3 cm diam.) containing glassbeads of 0.5 cm diam. as substrata for the alga, and it was incubated at18°C · 80Em^–2sec^–1 · L:D=14:10. The chlorophyll a yield of benthicdiatoms in a reactor as a unit of surface area of the substratum was only0.001–0.003 g cm^–2 when the flow rate of DSW was 0(batch culture conditions). However, when DSW was supplied continuously to areactor, the yield increased to 1.4 g-chl.a cm^–2 alongwith the increase in flow rate of DSW. Moreover, amounts of chl.a washed outof the system were negligible, 0.0014 to 0.0045%, even though theflow rate of DSW was as much as 25 times h^–1, suggesting thatsloughing of benthic diatoms from the substratum was minimized. Although theyield of diatoms fluctuated significantly at the time that the DSW wascollected, the variation could be minimized by increasing the flow rate ofDSW. These results indicate that the continuous culturing system with DSWsupports the stable and effective mass culture of benthic food diatom.     

Application of statistically-based experimental designs for the optimization of eicosapentaenoic acid production by the diatom Nitzschia laevis

Statistically based experimental designs were applied to the optimization of medium components and environmental factors for eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis in heterotrophic conditions. First, the Plackett-Burman design was used to evaluate the effects of variables including medium components and environmental factors on cell growth and EPA production. Among these variables, NaCl, CaCl(2), PI metal solution, pH, and temperature were identified to have the significant effects (with confidence level > 90 %). Subsequently, the concentrations of NaCl, CaCl(2), PI metal solution as well as the values of pH and temperature were optimized using central composite design. The cell growth and EPA production were found to respectively correlate to NaCl, CaCl(2), pH, and temperature that could be represented by second-order polynomial models. The optimal values of the four parameters were determined by response surface and numerical analyses as 8 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 19.8 degrees C for cell dry weight (DW), and 14 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 18 degrees C for EPA production, respectively. The subsequent verification experiments confirmed the validity of the models. This optimization strategy led to a DW of 9 g/L, an EPA yield of 280 mg/L and an EPA productivity of 28 mg/L/d, respectively, which were considerably higher than those obtained in the previous studies.     

High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development

A fed-batch process was developed for high cell density culture of the diatom Nitzschia laevis for enhanced production of eicosapentaenoic acid (EPA). Firstly, among the various medium components, glucose (Glu) was identified as the limiting substrate while nitrate (NO₃⁻), tryptone (Tr) and yeast extract (Ye) were found to promote cell growth by enhancing specific growth rate. Therefore, these components were considered essential and were included in the feed medium for subsequent fed-batch cultivation. With the optimized ratio of NO₃⁻:Tr:Ye being 1:2.6:1.3 (by weight), the relative proportions of glucose to the nitrogen sources in the feed were investigated. The optimal ratios of Glu:NO₃⁻ for specific growth rate and EPA productivity were both determined to be 32:1 (by weight). Finally, based on the residual glucose concentration in the culture, a continuous medium feeding strategy for fed-batch fermenter cultivation was developed, with which, the maximal cell dry weight and EPA yield obtained were 22.1 g l⁻¹ and 695 mg l⁻¹, respectively, which were great improvements over those of batch cultures.     

Diatoms in Acid Mine Drainage and Their Role in the Formation of Iron-Rich Stromatolites

Adverse conditions in the acid mine drainage (AMD) system at the Green Valley mine, Indiana, limit diatom diversity to one species, Nitzschia tubicola. It is present in three distinct microbial consortia: Euglena mutabilis-dominated biofilm, diatomdominated biofilm, and diatom-exclusive biofilm. E. mutabilis dominates the most extensive biofilm, with lesser numbers of N. tubicola, other eukaryotes, and bacteria. Diatom-dominated biofilm occurs as isolated patches containing N. tubicola with minor fungal hyphae, filamentous algae, E. mutabilis, and bacteria. Diatom-exclusive biofilm is rare, composed entirely of N. tubicola.

Diatom distribution is influenced by seasonal and intraseasonal changes in water temperature and chemistry. Diatoms are absent in winter due to cool water temperatures. In summer, isolated patchy communities are present due to warmer water temperatures. In 2001, the diatom community expanded its distribution following a major rainfall that temporarily diluted the effluent, creating hospitable conditions for diatom growth. After several weeks when effluent returned to preexisting conditions, the diatom biofilm retreated to isolated patches, and E. mutabilis biofilm flourished.

Iron-rich stromatolites underlie the biofilms and consist of distinct laminae, recording spatial and temporal oscillations in physicochemical conditions and microbial activity. The stromatolites are composed of thin, wavy laminae with partially decayed E. mutabilis biofilm, representing microbial activity and iron precipitation under normal AMD conditions. Alternating with the wavy layers are thicker, porous, spongelike laminae composed of iron precipitated on and incorporated into radiating colonies of diatoms. These layers indicate episodic changes in water chemistry, allowing diatoms to temporarily dominate the system.     

Glutamate agonists from marine algae

The occurrence of three glutamate agonists — glutamic acid, D-homocysteic acid and kainic acid — in a spontaneous mutant of Palmaria palmata is reported. Glutamic acid and D-homocysteic acid, but not kainic acid, were found in the wild-type plant. The closely related glutamate agonist, domoic acid, was found in the red alga Chondria baileyana and in the diatom Nitzschia pungens forma multiseries. In the diatom, domoic acid can build up to high levels in excess of 3% (dry wt.), making N. pungens a potential commercial source of this neuroactive amino acid. Information is also presented on the distribution, chemistry and biological activity of neuroactive amino acids from algae, and a possible biogenic relationship among kainoid metabolites is discussed. author for correspondence     

STUDIES OF NITRATE TRANSPORT BY MARINE PHYTOPLANKTON USING 36Cl-ClO3− AS A TRANSPORT ANALOGUE. I. PHYSIOLOGICAL FINDINGS1

Transport of a nitrate analogue, ³⁶Cl-ClO₃⁻, was examined in two diatoms, Skeletonema costatum (Greve.) Cleve and Nitzschia closterium (Ehrenb) W. Sm. A dinoflagellate, Gonyaulax polyedra did not transport ClO₃⁻. Transport of ³⁶Cl-ClO₃⁻ by diatoms appeared to be active and showed saturation kinetics. The data were fitted by Michaelis-Menten equation at all but the lowest chlorate concentrations (where plots of S vs. v showed a slight concave bend). Affinity of cells for nitrate was considerably higher than for chlorate. The K_i for nitrate inhibition of chlorate transport was calculated assuming competitive inhibition. Light had little or no effect on chlorate transport. Pulse-chase experiments demonstrated that (1) ClO₃⁻ (hence NO₃⁻) was stored in two intracellular compartments of equal size, (2) internal ClO₃⁻ was exchangeable with external ClO₃⁻ (rates of efflux and influx were measured), and (3) efflux of intracellular ClO₃⁻ showed transient states following a chase of ClO₃⁻ or NO₃⁻ which stabilized after 10–20 min. Transport of chlorate was a function of growth phase.     

Relationship between growth irradiance and the xanthophyll cycle pool in the diatom Nitzschia palea

The size and de-epoxidation state of the xanthophyll cycle pool was measured in cultures of Nitzschia palea grown at six fluence rates in continuous light or with a 12 h photoperiod. In both series the size of the pool increased with increasing irradiance. The de-epoxidized form, diatoxanthin, was only present at fluence rates saturating for growth. The portion of diadinoxanthin, which was not readily de-epoxidized in saturating light, was constant and not related to the size of the pool. In the culture grown in a light-dark cycle at 300 μmol photons m^-2 s^-1 (PAR) increasing de-epoxidation took place in the latter half of the photoperiod, when the rate of photosynthesis was decreasing. A rapid, spectrophotometric method for measuring the extent of de-epoxidation of the xanthophyll cycle pool in a culture of diatoms is described. Upon addition of a small volume of hydrochloric acid to an extract of pigments in 90% acetone, the absorbance at 480 nm is reduced. The size of the reduction is a measure of the state of the xanthophyll cycle pool, since the absorbance of diatoxanthin is reduced by 5%, but the absorbance of diadinoxanthin by 87% due to an epoxide-furanoid rearrangement, which causes the absorption spectrum to be shifted by ca 20 nm towards shorter wavelengths.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

Changes in eicosapentaenoic acid content of Navicula saprophila, Rhodomonas salina and Nitzschia sp. under mixotrophic conditions

Three species of microalgae able to produce eicosapentaenoic acid(EPA) were collected from brackish and sea water around Japan. The species were identified as Navicula saprophila, Rhodomonassalina and Nitzschia sp. EPA as a proportion of total fatty acids increased in the presence of acetic acid for Rhodomonas salina and Nitzschia sp. However, Navicula saprophila displayed the greatest productivity of EPA and the EPA content of its biomass was enhanced under mixotrophic conditions in the presence of acetic acid. This revised version was published online in August 2006 with corrections to the Cover Date.