K. Ohki

THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE)1

Cultures of Trichodesmium NIBB 1067 were grown in the synthetic medium AQUIL with a range of iron added from none to 5 × 10−7 M Fe for 15 days. Chlorophyll‐a, cell counts, and total cell volume were two or three times higher in medium with 10−7 M Fe than with no added Fe. Oxygen production rate per chlorophyll‐a was over 60% higher with higher iron. Increased iron stimulated photosynthesis at all irradiances from about 12–250 μE · m−2· s−1. Nitrogen fixation rate, estimated from acetylene reduction, for 10−7 and 10−8 M Fe cultures was approximately twice that of the cultures with no added Fe. The range of rates of O2 production and N2 fixation in cultures at the iron concentrations we used were similar to the rates from natural samples of Trichodesmium from both the Atlantic, and the Pacific oceans. This similarity may allow this clone to be used, with some caution, for future physiological ecology studies. This study demonstrates the importance of iron to photosynthesis and nitrogen fixation and suggests that Trichodesmium plays a central role in the biogeochemical cycles of iron, carbon and nitrogen.

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N2 as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N2 was not suppressed after 7 h incubation with 2 mM NaNO3 or 0.02 mM NH4Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO3, 0.5 mM urea or 0.02 mM NH4Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo−Fe protein, revealed the following: (1) both the Fe-protein and the Mo−Fe protein were synthesized in cells grown with N2 as well as in cells grown with NaNO3 or low concentration of NH4Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO3 or low concentration of NH4Cl; (4) neither the Fe-protein nor the Mo−Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.

Aerobic nitrogenase activity measured as acetylene reduction in the marine non-heterocystous cyanobacterium Trichodesmium spp. grown under artificial conditions

Aerobic nitrogenase activity in the marine non-heterocystous cyanobacterium Trichodesmium spp. NIBB 1067, isolated off the Izu Peninsula, Japan in 1983 and grown under artificial conditions, was assayed by the acetylene reduction method. This strain exhibited acetylene reduction activity under aerobic conditions when cells had been grown in the medium free of combined nitrogen. Activity was markedly enhanced by light, and dependent on the growth phase being higher during the exponential growth phase and lower during the late linear and stationary growth phases. Since typical colony formation occurred during the last growth phase, the present results contradict the idea that N2-fixation depends on colony formation. The photosynthesis inhibitor DCMU at 10-6M inhibited light-dependent acetylene reduction completely. Acetylene reduction by Trichodesmium spp. was tolerant of O2 as strongly as that in the heterocystous cyanobacteria. Even at a partial pressure of oxygen (pO2) of ∼3 atm, the activity still remained as high as half of the maximum. It was almost under anaerobic conditions. Maximum activity was obtained at pO2 of ca. 0.1 atm.

Cultures of the pelagic cyanophytes Trichodesmium erythraeum and T. thiebautii in synthetic medium

Trials for determination of culture conditions for the marine cyanophytes of Trichodesmium erythraeum and T. thiebautii were made with use of a synthetic medium. The “Aquil” medium, either with or without combined nitrogen, brought about stable growth of the two strains, T. erythraeum and T. thiebautii. However, they failed to grow in an ASP7 medium. The failure was found to be due to the toxic effect of Tris-aminomethane, the pH-buffer in this medium. Two important chemical conditions for the stable growth of Trichodesmium spp. were revealed. (1) Stable growth was supported by Ca2+ at high concentrations; in a concentration lower than 0.9 mM, cell-lysis promptly occurred, while the cells could grow without cell-lysis at Ca2+ concentrations higher than 7.5 mM even at a salinity as low as 19‰ S. Ca2+ is probably essential for the osmotic regulation in this organism. (2) Phosphate-toxicity at high concentrations was at least partly due to heavy metal(s) contaminating the reagent of inorganic phosphate. After treatment with a Chelex-100 column, phosphate concentration could be increased up to four times the previous concentrations without toxicity.

Determination of photosynthetic pigment composition in an individual phytoplankton cell in seas and lakes using fluorescence microscopy; properties of the fluorescence emitted from picophytoplankton cells

To develop a method for the determination of photosynthetic pigment species in individual phytoplankton cells, especially natural picophytoplankton cells, the fluorescence spectra of intact cells were studied with cultured phytoplankton. The study was made mainly with phycoerythrin-containing picophytoplankton collected off Japan in 1982 with reference to diatomal species and phycoerythrin-free cyanophycean species. The spectra were measured for cell suspensions with an ordinary spectrofluorometer, and for individual cells with a microscope spectrofluorometer, paying special attention to the effect of cell-fixation. Results indicated that: (1) the cell-fixation with the glutaraldehyde and paraformaldehyde mixture modified phycoerythrin emission from picophytoplankton markedly in its wavelength location and intensity, but (2) the emission from phycocyanin was affected far less, and (3) the emission from chlorophyll a was not altered. However, the phycoerythrin emission modified by the fixation was found to be easily distinguished from other emissions, and kept its intensity high enough for detection with a fluorescence microscope. The fluorescence properties after the fixation were kept unaltered for a long period of time. Based on the results, we propose a simple method for the determination of photosynthetic pigments in individual phytoplankton cells in seas and lakes using fluorescence microscopy. Results of our tests with natural samples of phytoplankton are presented, and problems for further improvement are also discussed.

PHOTOREGULATION OF PHYCOBILISOME STRUCTURE DURING COMPLEMENTARY CHROMATIC ADAPTATION IN THE MARINE CYANOPHYTE PHORMIDIUM SP. C861

Changes in the molecular structure of phycobilisomes during complementary chromatic adaptation were studied in the marine cyanophyte Phormidium sp. C86. This strain forms phycoerythrin (PE)‐less phycobilisomes under red light but synthesizes PE‐rich phycobilisomes under green light. Analysis of phycobiliprotein composition and electron microscopic examination of phycobilisomes in ultra‐thin sections of cells and of isolated phycobilisomes were performed for cells acclimated to red and green light, respectively. The structure of phycobilisomes formed under red light conditions was typically hemidiscoidal. Phycobilisomes in cells acclimated to green light were twice as large in size as those in cells acclimated to red light. This increase in phycobilisome size was a result of the increase in the molar ratio of antenna pigment (PE and phycocyanin) to allophycocyanin, from 3.5 to 11.3. Pigment composition and fine structure of phycobilisomes formed under green light were similar to those of “nonhemidiscoidal” phycobilisomes reported in Phormidium persicinum. These results suggest that changes occur not only in the molecular species of peripheral rods but also in the structure of rods and probably of cores in relation to their connection with rods during chromatic adaptation of Phormidium sp. C86.

Trichodesmium: Establishment of Culture and Characteristics of N2-Fixation

Details of techniques for isolation and culture of Trichodesmium from Kuroshio waters, including special care for maintaining a stable culture, are explained. Properties of this isolate grown under artificial conditions indicate that the organism in culture is similar to Trichodesmium in the natural habitat. Some characteristic features of N2-fixation in this organism are also reviewed.

Photosynthetic characteristics of marine Synechococcus spp. with special reference to light environments near the bottom of the euphotic zone of the open ocean

The present study aimed to resolve the question why marine Synechococcus spp. abundantly occur even at the bottom of the euphotic zone in the Kuroshio are. Photosynthesis under such conditions was examined using simulated blue-green model light (BGL). Results indicated that photosynthesis of marine Synechococcus spp. under BGL is as active enough to support growth of these organisms. Examination of light-harvesting under BGL indicated that active photosynthesis is permitted by an unusually high abundance of phycoerythrin (PE), which is the main light-harvesting pigment for photosystem II (PSII), due to a phycobilisome (PBS) structure which is different from ordinary hemidiscoidals. Although the absorption maximum of PE is located at longer wavelengths than the energy maximum of BGL, PE was found to absorb BGL significantly. Thus, BGL cannot be a typical photosystem I (PSI) light. PSII is also significantly excited by BGL. Carotenoids, which largely absorb BGL, were found to be effective in light-harvesting for PSI. Based on the results obtained, possible reasons why marine Synechococcus spp. commonly occur in warm waters were discussed. Two strains of Synechococcus spp. isolated from the Gulf Stream in 1981 and from Kuroshio, Japan in 1983 were used in the present study.

Photosynthetic pigment system of picophytoplankton of cyanophytes isolated from subsurface water in the Kuroshio area

AbstractsPhotosynthetic pigment system of picophytoplankton of cyanophytes was examined with five strains isolated from the Kuroshio water at the depth of 70 m. Examination was made for the absorption spectra of intact cells of each strain. Analysis of pigment composition was also made withSynechococcus NIBB 1059 and 1071, which were isolated from surface waters of the Gulf Stream and Kuroshio area, respectively. Results indicated that (1) all strains contain phycoerythrin with a very high concentration, and (2) the phycoerythrin in these strains contains two chromophores, phycoerythrobilin and phycourobilin, and (3) a large abundance of phycoerythrin and phycourobilin in the phycoerythrin enablesSynechococcus picophytoplankton to absorb effectively the light in the blue-green region at the subsurface depth. These characteristics suggest that cyanophytes in the subsurface water can collectt the blue-green light and perform actively photosynthesis even at the bottom of euphotic layer.

Non-Hemidiscoidal Phycobilisome in Cyanophytes

Phycobilisome (PBS) is a supramolecular unit of phycobiliproteins (PBSs) functioning as a light harvesting system for the photosynthesis of cyanophyte and rhodophyte. At least three structural types have been known for PBS: [1] rodshaped, [II] hemidiscoidal and [III] hemiellipsoidal or hemispherical (1). The type [I] has been found only in Glaeobacter violaseus (2). The type [II] is common in cyanophytes and also found in rhodophyte (3) while the type [III] has been found only in rhodophytes. from comparison of PBS in various cyanophytes, we found that two strains in Phormidium genus and one in Synechococcus genus contain a PBS different from the type [II], presumably similar to the type [III]. Quantitative relationship between PBS and photosystem II (PS II) indicate that PBS of Synechococcus sp. NIBB1059 is a common antenna for plural PS II while PBS of two Phormidium strains is the antenna for only one PS II.