Electrotransformation of thermophilic bacterium Caldimonas manganoxidans.

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/µg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.

Photoprotection mechanisms under different CO2 regimes during photosynthesis in a green alga Chlorella variabilis.

Oxygenic photosynthesis converts light energy into chemical energy via electron transport and assimilates CO2 in the Calvin-Benson cycle with the chemical energy. Thus, high light and low CO2 conditions induce the accumulation of electrons in the photosynthetic electron transport system, resulting in the formation of reactive oxygen species. To prevent the accumulation of electrons, oxygenic photosynthetic organisms have developed photoprotection mechanisms, including non-photochemical quenching (NPQ) and alternative electron flow (AEF). There are diverse molecular mechanisms underlying NPQ and AEF, and the corresponding molecular actors have been identified and characterized using a model green alga Chlamydomonas reinhardtii. In contrast, detailed information about the photoprotection mechanisms is lacking for other green algal species. In the current study, we examined the photoprotection mechanisms responsive to CO2 in the green alga Chlorella variabilis by combining the analyses of pulse-amplitude-modulated fluorescence, O2 evolution, and the steady-state and time-resolved fluorescence spectra. Under the CO2-limited condition, ΔpH-dependent NPQ occurred in photosystems I and II. Moreover, O2-dependent AEF was also induced. Under the CO2-limited condition with carbon supplementation, NPQ was relaxed and light-harvesting chlorophyll-protein complex II was isolated from both photosystems. In C. variabilis, the O2-dependent AEF and the mechanisms that instantly convert the light-harvesting functions of both photosystems may be important for maintaining efficient photosynthetic activities under various CO2 conditions.

Phenotypic characterization and comparative genome analysis of two strains of thermophilic, anaerobic, cellulolytic-xylanolytic bacterium Herbivorax saccincola.

The genome sequences of thermophilic, anaerobic, and cellulolytic-xylanolytic bacterium Herbivorax saccincola strains A7 and GGR1 have recently been determined. Although both strains belong to the same species, A7 is alkaliphilic, non-endospore-forming, and ammonium-assimilating, whereas GGR1 is neutrophilic, endospore-forming, and weak-ammonium-assimilating. To better understand the phenotypic diversity among H. saccincola strains, the genome sequences of A7 and GGR1 were compared. A7 contained three additional genes showing similarity to an alkaline stress-associated ABC-transporter but lacked four endospore formation-associated genes, AUG58543 and AUG58618 (encoding SpoVT), AUG57258 (encoding SpoVS), and AUG58614 (encoding YdhD), all of which were present in GGR1. In addition, A7 contained key ammonia assimilation genes PQQ67145 and PQQ66619, encoding ornithine cyclodeaminase and arginase, respectively, which were absent in GGR1. There was no difference in the number and types of cellulosomal-scaffolding proteins and glycosyl hydrolases between the two strains. However, cellulase and xylanase enzymes from A7 demonstrated greater activity and stability at an alkaline pH compared with those from GGR1, and amino acid substitutions were identified in 11 glycosyl hydrolases from A7. This characterization though comparative genomic analysis provides useful information for understanding the genetic basis of the phenotypic differences between H. saccincola strains isolated from distinct areas and environments.

Short-Term Temporal Metabolic Behavior in Halophilic Cyanobacterium Synechococcus sp. Strain PCC 7002 after Salt Shock.

In response to salt stress, cyanobacteria increases the gene expression of Na+/H+ antiporter and K+ uptake system proteins and subsequently accumulate compatible solutes. However, alterations in the concentrations of metabolic intermediates functionally related to the early stage of the salt stress response have not been investigated. The halophilic cyanobacterium Synechococcus sp. PCC 7002 was subjected to salt shock with 0.5 and 1 M NaCl, then we performed metabolomics analysis by capillary electrophoresis/mass spectrometry (CE/MS) and gas chromatography/mass spectrometry (GC/MS) after cultivation for 1, 3, 10, and 24 h. Gene expression profiling using a microarray after 1 h of salt shock was also conducted. We observed suppression of the Calvin cycle and activation of glycolysis at both NaCl concentrations. However, there were several differences in the metabolic changes after salt shock following exposure to 0.5 M and 1 M NaCl: (i): the main compatible solute, glucosylglycerol, accumulated quickly at 0.5 M NaCl after 1 h but increased gradually for 10 h at 1 M NaCl; (ii) the oxidative pentose phosphate pathway and the tricarboxylic acid cycle were activated at 0.5 M NaCl; and (iii) the multi-functional compound spermidine greatly accumulated at 1 M NaCl. Our results show that Synechococcus sp. PCC 7002 acclimated to different levels of salt through a salt stress response involving the activation of different metabolic pathways.

Adaptation of light-harvesting functions of unicellular green algae to different light qualities.

Oxygenic photosynthetic organisms perform photosynthesis efficiently by distributing captured light energy to photosystems (PSs) at an appropriate balance. Maintaining photosynthetic efficiency under changing light conditions requires modification of light-harvesting and energy-transfer processes. In the current study, we examined how green algae regulate their light-harvesting functions in response to different light qualities. We measured low-temperature time-resolved fluorescence spectra of unicellular green algae Chlamydomonas reinhardtii and Chlorella variabilis cells grown under different light qualities. By observing the delayed fluorescence spectra, we demonstrated that both types of green algae primarily modified the associations between light-harvesting chlorophyll protein complexes (LHCs) and PSs (PSII and PSI). Under blue light, Chlamydomonas transferred more energy from LHC to chlorophyll (Chl) located far from the PSII reaction center, while energy was transferred from LHC to PSI via different energy-transfer pathways in Chlorella. Under green light, both green algae exhibited enhanced energy transfer from LHCs to both PSs. Red light induced fluorescence quenching within PSs in Chlamydomonas and LHCs in Chlorella. In Chlorella, energy transfer from PSII to PSI appears to play an important role in balancing excitation between PSII and PSI.

Deletion of DNA ligase IV homolog confers higher gene targeting efficiency on homologous recombination in Komagataella phaffii.

The non-conventional yeast Komagataella phaffii, formerly Pichia pastoris, is a popular host for recombinant protein production. The relatively lower gene targeting efficiency observed in this species occurs due to high levels of non-homologous recombination activity. In the current study, we explored the function of the K. phaffii homolog of DNA ligase IV (Dnl4p) by creating a DNL4-disrupted strain. To assess the roles of non-homologous end joining (NHEJ)-related proteins in this species, strains deleted for either or both genes encoding Dnl4p or the telomeric Ku complex subunit (Ku70p) were generated. These deletions were constructed by either of two distinct marker-recycling methods (yielding either a seamless gene deletion or a Cre-loxP-mediated gene deletion). The resulting dnl4- and/or ku70-deleted K. phaffii strains were used to evaluate gene targeting efficiency in gene knock-out and gene knock-in experiments. The Dnl4p-defective strain showed improved gene targeting efficiency for homologous recombination compared to the wild-type and Ku70p-deffective strains. The dnl4 ku70 double knock-out strain exhibited a further improvement in gene targeting efficiency. Thus, the K. phaffii dnl4 and dnl4 ku70 deletion strains are expected to serve as useful platforms for functional analysis and strain development in this species.

A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA.

The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

Direct and highly productive conversion of cyanobacteria Arthrospira platensis to ethanol with CaCl2 addition.

BACKGROUND: The cyanobacterium Arthrospira platensis shows promise as a carbohydrate feedstock for biofuel production. The glycogen accumulated in A. platensis can be extracted by lysozyme-degrading the peptidoglycan layer of the bacterial cell walls. The extracted glycogen can be converted to ethanol through hydrolysis by amylolytic enzymes and fermentation by the yeast Saccharomyces cerevisiae. Thus, in the presence of lysozyme, a recombinant yeast expressing α-amylase and glucoamylase can convert A. platensis directly to ethanol, which would simplify the procedure for ethanol production. However, the ethanol titer and productivity in this process are lower than in ethanol production from cyanobacteria and green algae in previous reports. RESULTS: To increase the ethanol titer, a high concentration of A. platensis biomass was employed as the carbon source for the ethanol production using a recombinant amylase-expressing yeast. The addition of lysozyme to the fermentation medium increased the ethanol titer, but not the ethanol productivity. The addition of CaCl2 increased both the ethanol titer and productivity by causing the delamination of polysaccharide layer on the cell surface of A. platensis. In the presence of lysozyme and CaCl2, ethanol titer, yield, and productivity improved to 48 g L-1, 93% of theoretical yield, and 1.0 g L-1 h-1 from A. platensis, corresponding to 90 g L-1 of glycogen. CONCLUSIONS: We developed an ethanol conversion process using a recombinant amylase-expressing yeast from A. platensis with a high titer, yield, and productivity by adding both lysozyme and CaCl2. The direct and highly productive conversion process from A. platensis via yeast fermentation could be applied to multiple industrial bulk chemicals.

Characterization and high-quality draft genome sequence of Herbivorax saccincola A7, an anaerobic, alkaliphilic, thermophilic, cellulolytic, and xylanolytic bacterium.

An anaerobic, cellulolytic-xylanolytic bacterium, designated strain A7, was isolated from a cellulose-degrading bacterial community inhabiting bovine manure compost on Ishigaki Island, Japan, by enrichment culture using unpretreated corn stover as the sole carbon source. The strain was Gram-positive, non-endospore forming, non-motile, and formed orange colonies on solid medium. Strain A7 was identified as Herbivorax saccincola by DNA-DNA hybridization, and phylogenetic analysis based on 16S rRNA gene sequences showed that it was closely related to H. saccincola GGR1 (= DSM 101079T). H. saccincola A7 (= JCM 31827=DSM 104321) had quite similar phenotypic characteristics to those of strain GGR1. However, the optimum growth of A7 was at alkaline pH (9.0) and 55°C, compared to pH 7.0 at 60°C for GGR1, and the fatty acid profile of A7 contained 1.7-times more C17:0 iso than GGR1. The draft genome sequence revealed that H. saccincola A7 possessed a cellulosome-like extracellular macromolecular complex, which has also been found for Clostridium thermocellum and C. clariflavum. H. saccincola A7 contained more glycoside hydrolases (GHs) belonging to GH families-11 and -2, and more diversity of xylanolytic enzymes, than C. thermocellum and C. clariflavum. H. saccincola A7 could grow on xylan because it encoded essential genes for xylose metabolism, such as a xylose transporter, xylose isomerase, xylulokinase, and ribulose-phosphate 3-epimerase, which are absent from C. thermocellum. These results indicated that H. saccincola A7 has great potential as a microorganism that can effectively degrade lignocellulosic biomass.

Energy transfer in Anabaena variabilis filaments adapted to nitrogen-depleted and nitrogen-enriched conditions studied by time-resolved fluorescence.

Nitrogen is among the most important nutritious elements for photosynthetic organisms such as plants, algae, and cyanobacteria. Therefore, nitrogen depletion severely compromises the growth, development, and photosynthesis of these organisms. To preserve their integrity under nitrogen-depleted conditions, filamentous nitrogen-fixing cyanobacteria reduce atmospheric nitrogen to ammonia, and self-adapt by regulating their light-harvesting and excitation energy-transfer processes. To investigate the changes in the primary processes of photosynthesis, we measured the steady-state absorption and fluorescence spectra and time-resolved fluorescence spectra (TRFS) of whole filaments of the nitrogen-fixing cyanobacterium Anabaena variabilis at 77 K. The filaments were grown in standard and nitrogen-free media for 6 months. The TRFS were measured with a picosecond time-correlated single photon counting system. Despite the phycobilisome degradation, the energy-transfer paths within phycobilisome and from phycobilisome to both photosystems were maintained. However, the energy transfer from photosystem II to photosystem I was suppressed and a specific red chlorophyll band appeared under the nitrogen-depleted condition.

Variety in excitation energy transfer processes from phycobilisomes to photosystems I and II.

The light-harvesting antennas of oxygenic photosynthetic organisms capture light energy and transfer it to the reaction centers of their photosystems. The light-harvesting antennas of cyanobacteria and red algae, called phycobilisomes (PBSs), supply light energy to both photosystem I (PSI) and photosystem II (PSII). However, the excitation energy transfer processes from PBS to PSI and PSII are not understood in detail. In the present study, the energy transfer processes from PBS to PSs in various cyanobacteria and red algae were examined in vivo by selectively exciting their PSs or PBSs, and measuring the resulting picosecond to nanosecond time-resolved fluorescences. By observing the delayed fluorescence spectrum of PBS-selective excitation in Arthrospira platensis, we demonstrated that energy transfer from PBS to PSI via PSII (PBS→PSII→PSI transfer) occurs even for PSI trimers. The contribution of PBS→PSII→PSI transfer was species dependent, being largest in the wild-type of red alga Pyropia yezoensis (formerly Porphyra yezoensis) and smallest in Synechococcus sp. PCC 7002. Comparing the time-resolved fluorescence after PSs- and PBS-selective excitation, we revealed that light energy flows from CP43 to CP47 by energy transfer between the neighboring PSII monomers in PBS-PSII supercomplexes. We also suggest two pathways of energy transfer: direct energy transfer from PBS to PSI (PBS→PSI transfer) and indirect transfer through PSII (PBS→PSII→PSI transfer). We also infer that PBS→PSI transfer conveys light energy to a lower-energy red chlorophyll than PBS→PSII→PSI transfer.

Energy Transfer in Cyanobacteria and Red Algae: Confirmation of Spillover in Intact Megacomplexes of Phycobilisome and Both Photosystems.

Cyanobacteria and red algae control the energy distributions of two photosystems (PSI and PSII) by changing the energy transfer among phycobilisome (PBS), PSI, and PSII. However, whether PSII → PSI energy transfer (spillover) occurs in the intact megacomplexes composed of PBS, PSI, and PSII (PBS-PSII-PSI megacomplexes) in vivo remains controversial. In this study, we measured the delayed fluorescence spectra of PBS-selective excitation in cyanobacterial and red algal cells. In the absence of spillover, 7% of the PBS (at most) would combine with PSII, inconsistent with the PBSs' function as the antenna pigment-protein complexes of PSII. Therefore, we conclude that spillover occurs in vivo in PBS-PSII-PSI megacomplexes of both cyanobacteria and red algae.

Construction of a Genome-Scale Metabolic Model of Arthrospira platensis NIES-39 and Metabolic Design for Cyanobacterial Bioproduction.

Arthrospira (Spirulina) platensis is a promising feedstock and host strain for bioproduction because of its high accumulation of glycogen and superior characteristics for industrial production. Metabolic simulation using a genome-scale metabolic model and flux balance analysis is a powerful method that can be used to design metabolic engineering strategies for the improvement of target molecule production. In this study, we constructed a genome-scale metabolic model of A. platensis NIES-39 including 746 metabolic reactions and 673 metabolites, and developed novel strategies to improve the production of valuable metabolites, such as glycogen and ethanol. The simulation results obtained using the metabolic model showed high consistency with experimental results for growth rates under several trophic conditions and growth capabilities on various organic substrates. The metabolic model was further applied to design a metabolic network to improve the autotrophic production of glycogen and ethanol. Decreased flux of reactions related to the TCA cycle and phosphoenolpyruvate reaction were found to improve glycogen production. Furthermore, in silico knockout simulation indicated that deletion of genes related to the respiratory chain, such as NAD(P)H dehydrogenase and cytochrome-c oxidase, could enhance ethanol production by using ammonium as a nitrogen source.

A pilot-scale floating closed culture system for the multicellular cyanobacterium Arthrospira platensis NIES-39.

Microalgae are considered to be efficient bio-resources for biofuels and bio-based chemicals because they generally have high productivity. The filamentous cyanobacterium Arthrospira (Spirulina) platensis has been widely used for food, feed, and nutrient supplements and is usually cultivated in open ponds. In order to extend the surface area for growing this alga, we designed a pilot-scale floating closed culture system for cultivating A. platensis on open water and compared the growth and quality of the alga harvested at both subtropical and temperate regions. The biomass productivity of A. platensis NIES-39 was ca. 9 g dry biomass m-2 day-1 in summer at Awaji Island (warm temperature region) and ca. 10 and 6 g dry biomass m-2 day-1 in autumn and winter, respectively, at Ishigaki Island, (subtropical region) in Japan. If seawater can be used for culture media, culture cost can be reduced; therefore, we examined the influence of seawater salt concentrations on the growth of A. platensis NIES-39. Growth rates of A. platensis NIES-39 in diluted seawater with enrichment of 2.5 g L-1 NaNO3, 0.01 g L-1 FeSO4·7H2O, and 0.08 g L-1 Na2EDTA were considerably lower than SOT medium, but the biomass productivity (dry weight) was comparable to SOT medium. This is explained by the heavier cell weight of the alga grown in modified seawater media compared to the alga grown in SOT medium. Furthermore, A. platensis grown in modified seawater-based medium exhibited self-flocculation and had more loosely coiled trichomes.

Changes in Lignin and Polysaccharide Components in 13 Cultivars of Rice Straw following Dilute Acid Pretreatment as Studied by Solution-State 2D 1H-13C NMR.

A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) 1H-13 C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45-0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethylfurfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = -0.95 to -0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate.

Improving polyglucan production in cyanobacteria and microalgae via cultivation design and metabolic engineering.

Photosynthetic microorganisms, such as cyanobacteria and microalgae, are currently being investigated as alternative biomass resources for bioethanol production, owing to their benefits, including high-photosynthetic activity and whole-year cultivation without utilization of arable land. Polyglucans comprise the major carbohydrate content of these organisms. Polyglucans can be utilized as a carbon source for microbial fermentation. Although polyglucan production has so far been promoted by nutrient limitation, it must be further enhanced to accommodate market demand. This review focuses on the recent progress in the production of α-polyglucans such asglycogen and starch in cyanobacteria and green microalgae via cultivation design, including modifying the nutrient supply and replacing the growth medium. The control and manipulation of polyglucan metabolism necessitates the elucidation of the polyglucan production mechanism. We reviewed gene expression and metabolite accumulation profiles of cyanobacteria and green microalgae during nutrient limitation-stimulated α-polyglucan accumulation. We also focus on the enhancement in cyanobacterial glycogen production via the genetic engineering of glycolysis, CO2 concentration mechanism, and photosynthetic light-harvesting protein based on the polyglucan accumulation mechanism. The combined strategies of cultivation design and genetic engineering should be considered for further enhancement of polyglucan productivity for bioethanol production.

Energy transfer in Anabaena variabilis filaments under nitrogen depletion, studied by time-resolved fluorescence.

Some filamentous cyanobacteria (including Anabaena) differentiate into heterocysts under nitrogen-depleted conditions. During differentiation, the phycobiliproteins and photosystem II in the heterocysts are gradually degraded. Nitrogen depletion induces changes in the pigment composition of both vegetative cells and heterocysts, which affect the excitation energy transfer processes. To investigate the changes in excitation energy transfer processes of Anabaena variabilis filaments grown in standard medium (BG11) and a nitrogen-free medium (BG110), we measured their steady-state absorption spectra, steady-state fluorescence spectra, and time-resolved fluorescence spectra (TRFS) at 77 K. TRFS were measured with a picosecond time-correlated single photon counting system. The pigment compositions of the filaments grown in BG110 changed throughout the growth period; the relative phycocyanin levels monotonically decreased, whereas the relative carotenoid (Car) levels decreased and then recovered to their initial value (at day 0), with formation of lower-energy Cars. Nitrogen starvation also altered the fluorescence kinetics of PSI; the fluorescence maximum of TRFS immediately after excitation occurred at 735, 740, and 730 nm after 4, 8, and 15 days growth in BG110, respectively. Based on these results, we discuss the excitation energy transfer dynamics of A. variabilis filaments under the nitrogen-depleted condition throughout the growth period.

Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed.

Differences in energy transfer of a cyanobacterium, Synechococcus sp. PCC 7002, grown in different cultivation media.

Currently, cyanobacteria are regarded as potential biofuel sources. Large-scale cultivation of cyanobacteria in seawater is of particular interest because seawater is a low-cost medium. In the present study, we examined differences in light-harvesting and energy transfer processes in the cyanobacterium Synechococcus sp. PCC 7002 grown in different cultivation media, namely modified A medium (the optimal growth medium for Synechococcus sp. PCC 7002) and f/2 (a seawater medium). The concentrations of nitrate and phosphate ions were varied in both media. Higher nitrate ion and/or phosphate ion concentrations yielded high relative content of phycobilisome. The cultivation medium influenced the energy transfers within phycobilisome, from phycobilisome to photosystems, within photosystem II, and from photosystem II to photosystem I. We suggest that the medium also affects charge recombination at the photosystem II reaction center and formation of a chlorophyll-containing complex.

Glycogen production for biofuels by the euryhaline cyanobacteria Synechococcus sp. strain PCC 7002 from an oceanic environment.

BACKGROUND: Oxygenic photosynthetic microorganisms such as cyanobacteria and microalgae have attracted attention as an alternative carbon source for the next generation of biofuels. Glycogen abundantly accumulated in cyanobacteria is a promising feedstock which can be converted to ethanol through saccharification and fermentation processes. In addition, the utilization of marine cyanobacteria as a glycogen producer can eliminate the need for a freshwater supply. Synechococcus sp. strain PCC 7002 is a fast-growing marine coastal euryhaline cyanobacteria, however, the glycogen yield has not yet been determined. In the present study, the effects of light intensity, CO2 concentration, and salinity on the cell growth and glycogen content were investigated in order to maximize glycogen production in Synechococcus sp. strain PCC 7002. RESULTS: The optimal culture conditions for glycogen production in Synechococcus sp. strain PCC 7002 were investigated. The maximum glycogen production of 3.5 g L(-1) for 7 days (a glycogen productivity of 0.5 g L(-1) d(-1)) was obtained under a high light intensity, a high CO2 level, and a nitrogen-depleted condition in brackish water. The glycogen production performance in Synechococcus sp. strain PCC 7002 was the best ever reported in the α-polyglucan (glycogen or starch) production of cyanobacteria and microalgae. In addition, the robustness of glycogen production in Synechococcus sp. strain PCC 7002 to salinity was evaluated in seawater and freshwater. The peak of glycogen production of Synechococcus sp. strain PCC 7002 in seawater and freshwater were 3.0 and 1.8 g L(-1) in 7 days, respectively. Glycogen production in Synechococcus sp. strain PCC 7002 maintained the same level in seawater and half of the level in freshwater compared with the optimal result obtained in brackish water. CONCLUSIONS: We conclude that Synechococcus sp. strain PCC 7002 has high glycogen production activity and glycogen can be provided from coastal water accompanied by a fluctuation of salinity. This work supports Synechococcus sp. strain PCC 7002 as a promising carbohydrate source for biofuel production.