Phylogeography and pigment type diversity of Synechococcus cyanobacteria in surface waters of the northwestern pacific ocean

The widespread unicellular cyanobacteria Synechococcus are major contributors to global marine primary production. Here, we report their abundance, phylogenetic diversity (as assessed using the RNA polymerase gamma subunit gene rpoC1) and pigment diversity (as indirectly assessed using the laterally transferred cpeBA genes, encoding phycoerythrin‐I) in surface waters of the northwestern Pacific Ocean, sampled over nine distinct cruises (2008–2015). Abundance of Synechococcus was low in the subarctic ocean and South China Sea, intermediate in the western subtropical Pacific Ocean, and the highest in the Japan and East China seas. Clades I and II were by far the most abundant Synechococcus lineages, the former dominating in temperate cold waters and the latter in (sub)tropical waters. Clades III and VI were also fairly abundant in warm waters, but with a narrower distribution than clade II. One type of chromatic acclimater (3dA) largely dominated the Synechococcus communities in the subarctic ocean, while another (3dB) and/or cells with a fixed high phycourobilin to phycoerythrobilin ratio (pigment type 3c) predominated at mid and low latitudes. Altogether, our results suggest that the variety of pigment content found in most Synechococcus clades considerably extends the niches that they can colonize and therefore the whole genus habitat.     

Microcolony formation by single-cell Synechococcus strains as a fast response to UV radiation

UV radiation (UVR) has different effects on prokaryotic cells, such as, for instance, filamentation and aggregation in bacteria. Here we studied the effect of UVR on microcolony formation in two freshwater Synechococcus strains of different ribotypes (group B and group I) and phycobiliprotein compositions (phycoerythrin [PE] and phycocyanin [PC]). Each strain was photoacclimated at two light intensities, low light (LL) (10 μmol m⁻² s⁻¹) and moderate light (ML) (100 μmol m⁻² s⁻¹). The cultures were exposed for 6 days to treatments with UVR or without UVR. PE-rich Synechococcus acclimated to LL had a low carotenoid/chlorophyll a (car/chl) ratio but responded faster to UVR treatment, producing the highest percentages of microcolonies and of cells in microcolonies. Conversely, the same strain acclimated to ML, with a higher car/chl ratio, did not aggregate significantly. These results suggest that microcolony formation by PE-rich Synechococcus is induced by UVR if carotenoid levels are low. PC-rich Synechococcus formed a very low percentage of microcolonies in both acclimations even with low car/chl ratio. The different responses of the two Synechococcus strains to UVR depend on their pigment compositions. On the other hand, this study does not exclude that UVR-induced microcolony formation could also be related to specific ribotypes.     

EFFECT OF SPECTRAL QUALITY ON GROWTH AND PIGMENTATION OF PICOCYANOBACTERIA1

The influence of spectral quality on growth and pigmentation was compared among five strains of marine and freshwater picocyanobacteria grown under the same photon flux density (28 μE · m^?2·s^?1). Growth and phycoerythrin (PE) concentration per unit carbon increased when marine Synechococcus WH7803 was grown under green light as compared to red light, but no change in phycocyanin concentration occurred. Marine Synechococcus strain 48B66 also showed greater levels of PE when grown under green light than under red light, but no concomitant growth increase occurred. Both strains thus exhibited Group II chromatic adaptation. Additionally, strain 48B66 increased the relative level of phycourobilin compared to phycoerythrobilin when grown under red light. In contrast, both marine and freshwater Synechococcus strains containing no PE showed decreased growth under green light. Chlorophyll a concentrations were greatest or among the greatest in all strains grown under green light. These results suggest that light quality, through its effects on growth rate, may be an important factor controlling the distribution and abundance of the various pigment types of Synechococcus.     

UV-induced phycobilisome dismantling in the marine picocyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. WH8102

The marine picocyanobacterium Synechococcus sp. WH8102 was submitted to ultraviolet (UV-A and B) radiations and the effects of this stress on reaction center II and phycobilisome integrity were studied using a combination of biochemical, biophysical and molecular biology techniques. Under the UV conditions that were applied (4.3 W m⁻² UV-A and 0.86 W m⁻² UV-B), no significant cell mortality and little chlorophyll degradation occurred during the 5 h time course experiment. However, pulse amplitude modulated (PAM) fluorimetry analyses revealed a rapid photoinactivation of reaction centers II. Indeed, a dramatic decrease of the D1 protein amount was observed, despite a large and rapid increase in the expression level of the psbA gene pool. Our results suggest that D1 protein degradation was accompanied (or followed) by the disruption of the N-terminal domain of the anchor linker polypeptide L_CM, which in turn led to the disconnection of the phycobilisome complex from the thylakoid membrane. Furthermore, time course analyses of in vivo fluorescence emission spectra suggested a partial dismantling of phycobilisome rods. This was confirmed by characterization of isolated antenna complexes by SDS-PAGE and immunoblotting analyses which allowed us to locate the disruption site of the rods near the phycoerythrin I—phycoerythrin II junction. In addition, genes encoding phycobilisome components, including α-subunits of all phycobiliproteins and phycoerythrin linker polypeptides were all down regulated in response to UV stress. Phycobilisome alteration could be the consequence of direct UV-induced photodamages and/or the result of a protease-mediated process.     

GROWTH REGULATION OF rRNA CONTENT IN PROCHLOROCOCCUS AND SYNECHOCOCCUS (MARINE CYANOBACTERIA) MEASURED BY WHOLE‐CELL HYBRIDIZATION OF rRNA‐TARGETED PEPTIDE NUCLEIC ACIDS1

The cyanobacteria Synechococcus and Prochlorococcus are important primary producers in marine ecosystems. Because currently available approaches for estimating microbial growth rates can be difficult to apply in the field, we have been exploring the feasibility of using quantitative rRNA measurements as the basis for making such estimates. In this study we examined the relationship between rRNA and growth rate in several Synechococcus and Prochlorococcus strains over a range of light‐regulated growth rates. Whole‐cell hybridization with fluorescently labeled peptide nucleic acid (PNA) probes was used in conjunction with flow cytometry to quantify rRNA on a per cell basis. This PNA probing technique allowed rRNA analysis in a phycoerythrin‐containing Synechococcus strain (WH7803) and in a non–phycoerythrin‐containing strain and in Prochlorococcus. All the strains showed a qualitatively similar tri‐phasic relationship between rRNA·cell^?1 and growth rate, involving relatively little change in rRNA·cell^?1 at low growth rates, linear increase at intermediate growth rates, and a plateau and/or decrease at the highest growth rates. The onset of each phase was associated with the relative, rather than absolute, growth rate of each strain. In the Synechococcus strains, rRNA normalized to flow cytometrically measured forward angle light scatter (an indicator of size) was well‐correlated with growth rate across strains. These findings support the idea that cellular rRNA may be useful as an indicator of in situ growth rate in natural Synechococcus and Prochlorococcus populations.     

UNIQUE LOCATION OF THE PHYCOBILIPROTEIN LIGHT-HARVESTING PIGMENT IN THE CRYPTOPHYCEAE1

The cryptophyte algae, or cryptomonads, comprise a small algal group with a unique photosynthetic apparatus. Both a chlorophyll a/c₂ light-harvesting complex and a phycobiliprotein antenna (which can be either phycoerythrin or phycocyanin) are present, with the phycobiliprotein playing the major role in harvesting light for photosynthesis. Longstanding circumstantial evidence suggested that, in cryptophytes, the phycobiliprotein is located in the intrathylakoid space (thylakoid lumen) rather than on the outer surface of the thylakoid as part of a phycobilisome as in other algae. We used immunogold labeling to show conclusively that 1) the phycoerythrin (PE) of the cryptophyte Rhodomonas lens Pascher and Ruttner is located within the intrathylakoid space, 2) the PE is not exclusively bound to the thylakoid membrane but instead is distributed across the thylakoid lumen and 3) a fraction of this PE is tightly associated with the thylakoid membrane. The thylakoids are not everted to compensate for this unusual arrangement. The location of the major light-harvesting pigment on the “wrong” side of the otherwise very normal photo-synthetic membrane is unexpected, unique to the cryptophytes, and, remarkably, does not impair the photosynthetic abilities of this organism. A model is presented which incorporates these results -with previous information to give a complete structural picture of the cryptophyte light-harvesting apparatus.     

Changes in the cyanobacterial photosynthetic apparatus during acclimation to macronutrient deprivation

When the cyanobacterium Synechococcus sp. Strain PCC 7942 is deprived of an essential macronutrient such as nitrogen, sulfur or phosphorus, cellular phycobiliprotein and chlorophyll contents decline. The level of -carotene declines proportionately to chlorophyll, but the level of zeaxanthin increases relative to chlorophyll. In nitrogen- or sulfur-deprived cells there is a net degradation of phycobiliproteins. Otherwise, the declines in cellular pigmentation are due largely to the diluting effect of continued cell division after new pigment synthesis ceases and not to net pigment degradation. There was also a rapid decrease in O₂ evolution when Synechococcus sp. Strain PCC 7942 was deprived of macronutrients. The rate of O₂ evolution declined by more than 90% in nitrogen- or sulfur-deprived cells, and by approximately 40% in phosphorus-deprived cells. In addition, in all three cases the fluorescence emissions from Photosystem II and its antennae were reduced relative to that of Photosystem I and the remaining phycobilisomes. Furthermore, state transitions were not observed in cells deprived of sulfur or nitrogen and were greatly reduced in cells deprived of phosphorus. Photoacoustic measurements of the energy storage capacity of photosynthesis also showed that Photosystem II activity declined in nutrient-deprived cells. In contrast, energy storage by Photosystem I was unaffected, suggesting that Photosystem I-driven cyclic electron flow persisted in nutrient-deprived cells. These results indicate that in the modified photosynthetic apparatus of nutrient-deprived cells, a much larger fraction of the photosynthetic activity is driven by Photosystem I than in nutrient-replete cells.Abbreviations ES energy storage - N nitrogen - P phosphorus - PBS phycobilisomes - S sulfur     

Light-Harvesting System of the Red Alga Gracilaria tikvahiae: I. Biochemical Analyses of Pigment Mutations

Wild type Gracilaria tikvahiae, a macrophytic red alga, and fourteen genetically characterized pigment mutants were analyzed for their biliprotein and chlorophyll contents. The same three biliproteins, phycoerythrin, phycocyanin, and allophycocyanin, which are found in the wild type are found in all the Mendelian and non-Mendelian mutants examined. Some mutants overproduce R-phycoerythrin while others possess only traces of phycobiliprotein; however, no phycoerythrin minus mutants were found. Two of the mutants are unique; one overproduces phycocyanin relative to allophycocyanin while the nuclear mutant obr synthesizes a phycoerythrin which is spectroscopically distinct from the R-phycoerythrin of the wild type. The phycoerythrin of obr lacks the typical absorption peak at 545 nanometers characteristic of R-phycoerythrin and possesses a phycoerythrobilin to phycourobilin chromophore ratio of 2.6 in contrast to a ratio of 4.2 found in the wild type. Such a lesion provides evidence for the role of nuclear genes in phycoerythrin synthesis. In addition, comparisons are made of the pigment compositions of the Gracilaria strains with those of Neoagardhiella bailyei, a macrophytic red alga which has a high phycoerythrin content, and Anacystis nidulans, a cyanobacterium which lacks phycoerythrin. The mutants described here should prove useful in the study of the genetic control of phycobiliprotein synthesis and phycobilisome structure and assembly.     

Cell morphology and growth characteristics ofPorphyridium aerugineum (Rhodophyta)

Four unialgal strains of the freshwater coccoid red algaPorphyridium aerugineum Geitler were cultivated under laboratory conditions. Cell morphology was studied with the light microscope. The cell surface was examined by means of electron microscopy in order to contribute to the knowledge of polysaccharide sheaths and cytoplasmic membranes. Optimum growth conditions were determined. The range of cell sizes and the average dry masses of single cells were compared in all four strains cultivated at exactly defined temperatures and irradiances. Photosynthetic pigment maxima were measured in intact cells. The red-coloured phycobiliprotein phycoerythrin was not found in any of the examined strains.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

Evaluation of<Emphasis Type="Italic">Tolypothrix</Emphasis> germplasm for phycobiliprotein content

Twenty Tolypothrix strains, including 15 strains of T. tenuis, three strains of T. ceylonica and one strain each of T. nodosa and T. bouteillei, were evaluated for their phycobiliprotein content and composition. Significant differences among the Tolypothrix strains were found at both inter- and intra-specific levels in the production of phycobiliprotein constituents--phycocyanin (PC), allophycocyanin (APC) and phycoerythrin (PE). Four specific parameters, viz. PC or PE content, total phycobiliprotein and total protein content, and percentage of phycobiliproteins, in a mixture of total proteins were used to select four T. tenuis and one T. ceylonica strain as useful for phycobiliproteins production.     

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.     

A Comparative Study of Quantitative Relationship between Phycobiliproteins and Photosystem II in Cyanobacteria and Red Algae

Quantitative relationship between phycobiliprotein (PBP) andPS II contents was compared for 10 cyanobacterial and 3 redalgal strains. Following results were obtained: (1) contentof PBP per PS II was always equal to that of one phycobilisome(PBS) when PBS was hemidiscoidal type, (2) unusually high ratiosbetween antenna PBP [phycoerythrin (PE) and phycocyanin (PC)]and allophycocyanin contents were found in the PE-containingcyanobacteria Phormidium persicinum, Phormidium sp. NIBB 1052and Synechococcus sp. NIBB 1059 and the red alga Porphyra yezoensissuggesting that PBS in these organisms is not ordinary hemidiscoidaltype, and (3) the PBP content per PS II in Synechococcus NIBB1059 and P. yezoensis was as small as 14 to 13 of that of onePBS similarly to the case of Porphyridium cruentum. Resultssuggest that (1) hemidiscoidal PBS is the antenna for only onePS II, but (2) in some of organisms containing non-hemidiscoidalPBS, one PBS becomes a common antenna for plural PS II (around4), and (3) such PBS occurs not only in red algae but also incyanobacteria. ¹ Present address: Department of Biology, Faculty of Science,Toho University, Funabashi, Chiba 274, Japan. 1219 (Received April 15, 1987; Accepted July 11, 1987)     

PROTOPLAST FUSION AND GENETIC COMPLEMENTATION OF PIGMENT MUTATIONS IN THE RED MICROALGA PORPHYRZDZUM SP.1

Protoplasts from two green pigment mutants of Porphyridium sp. (UTEX 637) containing a low phycoerythrin level were fused by exposure to polyethylene glycol (MW 6000) combined with a short heat shock (45° C, 5 min). Following regeneration on agar plates, red colonies arose in which complementation of the phycoerythrin deficiency had occurred. The complementation frequency was estimated to be 0.2%. Eight progeny showing red pigmentation were isolated and purified by consecutive transfers on agar plates. Characterization of the fusion progeny revealed that their phycobiliprotein and chlorophyll contents per cell were higher than those of their parental mutant strains and, in most strains, similar to that of the wild type. The fusion products proved to be stable over many growth cycles. The DNA content of the wild type and of the parental mutant strains was about 0.05 pg-cell^?1. Fusion progeny strains showed a variable DNA content: a few fusants contained the same amount of DNA as the wild type and the parental strains, while others had about 50% more DNA per cell. The DNA content of one of the progeny strains (CF1c) was double that of the wild type (0.1 pg. cell^?1). Cells of this fusion progeny contained one nucleus per cell, which suggests that nuclear fusion and the formation of a stable diploid followed cell fusion. Analysis of phycobilisome components of CF1c revealed complementation of linker polypeptides associated with phycoerythrin (γ subunits). CF1c contained, like the wild-type strain, four linker polypeptides; all of these were absent in one parental strain and one was absent in the second. To the best of our knowledge, this is the first report of protoplast fusion, formation of somatic hybrids, and the apparent completion of a parasexual cycle in a red microalga.     

Sequence comparison of two highly homologous phycoerythrins differing in bilin composition

Genes encoding the and subunits of class II phycoerythrin from Synechococcus sp. strain WH8103 were cloned and sequenced. The deduced amino acid sequences were compared to class II phycoerythrin from Synechococcus sp. strain WH8020 and found to share 92% identity, yet the proteins differ in the bilin isomer (phycoerythrobilin versus phycourobilin) bound to two of the six chromophore attachment sites. Amino acid residues which might contact the bilin at each of the two variable sites were inferred by sequence alignment with phycocyanins. Putative bilin-contacting residues differing between the two phycocrythrins were identified which may determine bilin specificity.     

Energy transfers from Photosystem II to Photosystem I in Cryptomonas rufescens (Cryptophyceae)

In Cryptomonas rufescens (Cryptophyceae), phycoerythrin located in the thylakoid lumen is the major accessory pigment. Oxygen action spectra prove phycoerythrin to be efficient in trapping light energy.The fluorescence excitation spectra at ?196°C obtained by the method of Butler and Kitajima (Butler, W.L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85) indicate that like in Rhodophycease, chlorophyll a is the exclusive light-harvesting pigment for Photosystem I.For Photosystem II we can observe two types of antennae: (1) a light-harvesting chlorophyll complex connected to Photosystem II reaction centers, which transfers excitation energy to Photosystem I reaction centers when all the Photosystem II traps are closed. (2) A light-harvesting phycoerythrin complex, which transfers excitation energy exclusively to the Photosystem II reaction complexes responsible for fluorescence at 690 nm.We conclude that in Cryptophyceae, phycoerythrin is an efficient light-harvesting pigment, organized as an antenna connected to Photosystem II centers, antenna situated in the lumen of the thylakoid. However, we cannot afford to exclude that a few parts of phycobilin pigments could be connected to inactive chlorophylls fluorescing at 690 nm.     

The structure of cyanobacterial phycobilisomes: a model

Phycobilisomes, supramolecular complexes of water-soluble accessory pigments, serve as the major light-harvesting antennae in cyanobacteria and red algae. Regular arrays of these organelles are found on the surface of the thylakoid membranes of these organisms. In the present study, the hemi-discoidal phycobilisomes of several species of cyanobacteria were examined in thin sections of cells and by negative staining after isolation and fixation. Their fundamental structures were found to be the same. Isolated phycobilisomes possessed a triangular core assembled from three stacks of disc-shaped subunits. Each stack contained two discs which were 12 nm in diameter and 6–7 nm thick. Each of these discs was probably subdivided into halves 3–3.5 nm thick. Radiating from each of two sides of the triangular core were three rods 12 nm in diameter. Each rod consisted of stacks of 2 to 6 disc-shaped subunits 6 nm thick. These discs were subdivided into halves 3 nm thick.The average number of discs of 6 nm thickness forming the peripheral rods varied among the strains studied. For certain chromatically adapting strains, the average rod length was dependent upon the wavelength of light to which cells were exposed during growth. Analyses of phycobilisomes by spectroscopic techniques, polyacrylamide gel electrophoresis, and electron microscopy were compared. These analyses suggested that the triangular core was composed of allophycocyanin and that the peripheral rods contained phycocyanin and phycoerythrin (when present). A detailed model of the hemi-discoidal phycobilisome is proposed. This model can account for many aspects of phycobiliprotein assembly and energy transfer.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin - AP-B allophycocyanin B - C- cyanobacterial - R- rhodophytan - B- Bangiophycean - SDS sodium dodecyl sulfate - LPP Lyngbya-Plectonema-Phormidium group - Na-KPO₄ buffers NaH₂PO₄ titrated with a solution of KH₂PO₄ of equivalent molarity to a given pH     

Diversity of Synechococcus at the Martha’s Vineyard Coastal Observatory: Insights from Culture Isolations,Clone Libraries,and Flow Cytometry

The cyanobacterium Synechococcus is a ubiquitous, important phytoplankter across the world’s oceans. A high degree of genetic diversity exists within the marine group, which likely contributes to its global success. Over 20 clades with different distribution patterns have been identified. However, we do not fully understand the environmental factors that control clade distributions. These factors are likely to change seasonally, especially in dynamic coastal systems. To investigate how coastal Synechococcus assemblages change temporally, we assessed the diversity of Synechococcus at the Martha’s Vineyard Coastal Observatory (MVCO) over three annual cycles with culture-dependent and independent approaches. We further investigated the abundance of both phycoerythrin (PE)-containing and phycocyanin (PC)-only Synechococcus with a flow cytometric setup that distinguishes PC-only Synechococcus from picoeukaryotes. We found that the Synechococcus assemblage at MVCO is diverse (13 different clades identified), but dominated by clade I representatives. Many clades were only isolated during late summer and fall, suggesting more favorable conditions for isolation at this time. PC-only strains from four different clades were isolated, but these cells were only detected by flow cytometry in a few samples over the time series, suggesting they are rare at this site. Within clade I, we identified four distinct subclades. The relative abundances of each subclade varied over the seasonal cycle, and the high Synechococcus cell concentration at MVCO may be maintained by the diversity found within this clade. This study highlights the need to understand how temporal aspects of the environment affect Synechococcus community structure and cell abundance.     

Characterization of the lipopolysaccharides from eight strains of the cyanobacterium Synechococcus

Lipopolysaccharides have been isolated from eight strains of the unicellular cyanobacterium Synechococcus. Fucose, mannose, galactose, glucose and glucosamine were found in all of the lipopolysaccharides investigated. Additionally, strain-specific sugars are present and permit the chemotyping of lipopolysaccharide. Chemotype I, comprising three strains with a high G+C content of DNA (71-66 mol%), is characterized by a high rhamnose portion and by 3,6-dideoxy-d-arabino-hexose (tyvelose). Chemotype III, represented by three strains with a low G+C content of DNA (55-48 mol%), contains a mannose-polymer with small amounts of 3-O-methyl-mannose, 4-O-methyl-mannose, 2-keto-3-deoxyoctonate and mannosamine. Lipopolysaccharides of the two strains of chemotype II contain 2,3,4-tri-O-methyl-arabinose.Lipid A is difficult to split off from the polysaccharide moiety, but is present in all lipopolysaccharides from the Synechococcus strains. The presence of Lipid A is supported by the finding of -hydroxy fatty acids, predominantly -hydroxypalmitic acid. The distribution of branched -hydroxy fatty acids, detected in small amounts, parallels chemotyping of lipopolysaccharide based on the sugar composition. The phosphorus content of the lipopolysaccharides is low.The pyrogenicity of lipopolysaccharides from two strains is low. Synechococcus lipopolysaccharides have little reactivity in antisera raised in rabbits against homologous cells. As far as tested they do not migrate in immunoelectrophoresis. This confirms the neutral character or low negative charge of Synechococcus lipopolysaccharides.Dedicated to Professor Otto Kandler on occasion of his 60th birthday     

Examination of Genetic Relatedness of Marine Synechococcus spp. by Using Restriction Fragment Length Polymorphisms

The relatedness of several marine Synechococcus spp. was estimated by DNA hybridization. Strains isolated from various geographical locations and representing a diversity of DNA base compositions and phycobiliprotein profiles were compared by restriction fragment length polymorphisms for a number of genes. DNAs from two marine red algae and a cryptomonad alga (which exhibit a phycobiliprotein composition similar to that of the marine Synechococcus spp.) and Synechococcus strain PCC6301 (Anacystis nidulans) were also included in the comparison. Strains WH8008, WH8018, and WH7805 were shown to be very similar to one another, as were strains WH7802 and WH7803. Strains WH8110 and WH5701 were clearly unrelated to any of the other strains, and no marine Synechococcus isolate showed any similarity to the freshwater Synechococcus strain PCC6301 or the eucaryotic algae. The method is relatively straightforward and sensitive and uses a variety of basic molecular biology techniques. Its utility in ascertaining the genetic relatedness and diversity of marine Synechococcus spp. and possible extension to field studies are discussed.     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.