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Separation of microalgae of various sizes and shapes is an important process that enables subsequent production of useful compounds. Herein, the separation of microalgae was accomplished using a magnetite-containing gel (42 μm) packed into a column. An algal suspension was injected into the top of the gel bed, after which water was passed through the column. The pressure generated during the process caused the lower domain of the gel bed to deform, resulting in narrowed gaps between the gel beads. When a suspension of Nannochloropsis sp. (0.0069-0.69 g L-1) was loaded and water was passed through the column at an applied pressure of 0.01-0.10 MPa, the majority of microalgae were captured within the upper domain of the gel bed, while only 20% were captured within the lower domain. The amount of Nannochloropsis sp. captured was expressed by an ordinary differential equation to determine the capture coefficient, K, and the maximum capture amount, Qmax. As pressure increased, gel gaps narrowed, K increased, and Qmax decreased because of a reduction in the number of effective capture sites upon compaction of the gel. When a mixed suspension of Anabaena sp., Monoraphidium sp., and Desmodesmus sp. (0.069 g L-1 each) was injected into the gel bed at an applied pressure of 0.01 MPa, only Anabaena sp. was captured at the bottom of the gel bed. This device can be applied for the separation of microalgae in rivers and the sea.

The continuous decline in phosphorus (P) resources is a serious global issue. Therefore, it is important to develop methods to recover P from waste and wastewater. Most P ores are currently used in the phosphate form in the agriculture industry and in detergents, which results in a large release of phosphates into natural aquatic environments. Much attention has been given to measuring phosphate levels and monitoring water quality, survey, and control of algal phytoplankton dynamics. However, phosphite is oxidized from hypophosphite after plating and discharged as waste, so methods to recycle and reuse phosphite should also be developed. Currently, there is no evidence of phosphite utilization by photosynthetic eukaryotes, including eukaryotic algae. Thus, except for the possible utilization by some bacteria when phosphate is unavailable, the fate of the phosphite that is discharged is mostly unknown. Chlorella vulgaris (NIES-2170), Coccomyxa subellipsoidea (NIES-2166), Scenedesmus obliquus (NIES-2280), and Botryococcus braunii (BOT-22) were cultured in phosphite medium under conditions that prevented phosphate contamination and phosphite oxidation. As a result, the number of C. vulgaris and C. subellipsoidea increased in the phosphite medium, demonstrating the availability of phosphite for the growth of these strains. In particular, the growth of C. vulgaris increased as the phosphite concentration increased. After being cultured for 180 days in photosynthetic conditions, phosphite utilization rates were 32–38%. In contrast, S. obliquus and B. braunii strains did not grow in the phosphite medium. In conclusion, C. subellipsoidea and C. vulgaris utilize phosphite as a P resource, which is a novel finding in photosynthetic eukaryotes. The results of this study may have important implications for the phosphorus redox cycle.

Dewatering algal suspensions is an important step in the extraction of oil and other useful substances from algae. In this study, spherical Nannochloropsis sp. and ellipsoidal Monoraphidium sp. suspensions were dewatered in the presence of different amounts of 350-nm magnetite particles using a microfiltration membrane with 360-nm pores in cross-flow mode. Magnetite functions as a filter aid by reducing the deformation of the cake of filtered algae on the membrane and providing paths for water to flow through the filtration cake of algae. In the case of Nannochloropsis sp., the highest dewatering rate was obtained when the number ratio, defined based on the size and ideal density, between Nannochloropsis sp. and magnetite was 1:12.5, but the addition of magnetite had no observable effect on the filtration of ellipsoidal Monoraphidium sp. suspensions through the membrane. After dewatering, magnetite was effectively separated from the concentrated algal suspension by the application of a magnetic field in an open flow system. Magnetite has the potential to enhance dewatering performance using a cross-flow membrane system.

Coastal areas in the Tohoku region were severely damaged by the tsunami that followed the March 2011 earthquake. Consequently, many lands have become non-arable. To assess the use of these non-arable lands for the production of algal biomass, we carried out experimental cultivations of native algal communities in Minamisoma city, Fukushima Prefecture, located in the cool temperate zone of Japan. Productivity of the native algal community was measured using 1-m2 raceway ponds (120 L) and open vessel-type bioreactors (50 L–500 L), with different hydraulic retention times (HRTs) and in the presence or absence of sodium acetate. Maximum productivity in 1-m2 raceway ponds (10-cm water depth) was observed with an HRT of 4 days and sodium acetate supplementation in June 2015 (13.2 g/m2/day); average productivity throughout the year was 10.6 g/m2/day. In the case of deep-water cultivation (80-cm water depth) in vessel-type cultures, the highest productivity (> 29 g/m2/day) was obtained between January and September 2015. The dominant native microalgal species in the cultivations were Desmodesmus sp. and Scenedesmus acuminatus. Heterotrophic organisms, native bacteria, and protista, such as ciliates and amoeba, were also observed. Species composition and abundance varied depending on the medium content and seasonal conditions.

In this study, we report a two-dimensional (2D) hexagonal disk obtained by carbonization of Botryococcus braunii (B. braunii) residues. Carbonization at 700℃followed by naturally cooling down to room temperature under a non-inert gas flow atmosphere affords to yield this unique structure. The 2D hexagonal disks consist of more than 52% carbon and more than 25% oxygen. Slight amount of Fe, silicon and magnesium would be the trigger of the formation of hexagonal structure. Treatment of biomass residue is a challenge in the near future accompanied by the achievement of new energy technology in the industrial level. This research points out that efficient use of discharged biomass residue could create a new avenue for material science. The morphology of obtained crystals carbonized in different conditions, especially with the existence of argon flow, was also investigated.

Cryopreservation is useful for long-term maintenance of living strains in microbial culture collections. We applied this technique to environmental specimens from two monitoring sites at Sendai Bay, Japan and compared the microbial diversity of photosynthetic picoeukaryotes in samples before and after cryopreservation. Flow cytometry (FCM) showed no considerable differences between specimens. We used 2500 cells sorted with FCM for next-generation sequencing of 18S rRNA gene amplicons and after removing low-quality sequences obtained 10,088-37,454 reads. Cluster analysis and comparative correlation analysis of observed high-level operational taxonomic units indicated similarity between specimens before and after cryopreservation. The effects of cryopreservation on cells were assessed with representative culture strains, including fragile cryptophyte cells. We confirmed the usefulness of cryopreservation for genetic studies on environmental specimens, and found that small changes in FCM cytograms after cryopreservation may affect biodiversity estimation.

This study investigates the potential of microalgae oil production as an alternative renewable energy source, in a pilot project located at Minamisoma City in the Fukushima Prefecture of Japan. The algal communities used in this research were the locally mixed species, which were mainly composed of Desmodesmus collected from the Minamisoma pilot project. The microalgae oil-production processes in Minamisoma consisted of three stages: cultivation, dewatering, and extraction. The estimated theoretical input-energy requirement for extracting oil was 137.25 MJ to process 50 m3 of microalgae, which was divided into cultivation 15.40 MJ, centrifuge 13.39 MJ, drum filter 14.17 MJ, and hydrothermal liquefaction (HTL) 94.29 MJ. The energy profit ratio (EPR) was 1.41. The total energy requirement was highest in the HTL process (68%) followed by cultivation (11%) and the drum filter (10%). The EPR value increased along with the yield in the cultivation process. Using HTL, the microalgae biomass could be converted to bio-crude oil to increase the oil yield in the extraction process. Therefore, in the long run, the HTL process could help lower production costs, due to the lack of chemical additions, for extracting oil in the downstream estimation of the energy requirements for microalgae oil production.

Microalgae is a promising next-generational energy. In this research, we focus on oil-extracted Botryococcus braunii residues collected by adding polysilicato-iron (PSI) as a flocculant followed by carbonization under argon atmosphere. We conducted carbonization at various temperatures as a first attempt to reveal the fundamental properties of the carbonization process of the microbes. The carbons thus obtained by heat treatment at 900 °C present a unique magnetic behavior due to reduced magnetite (Fe3O4) inclusion, which is produced from polysilicato iron (Fe2O3) during the heating process. Experimental results suggest that this carbonic material can be applied as a heavy metal-capturing carbon and magnetic porous substrate catalyst. The effective use of the waste may open a new avenue for an energy-microbiology-materials system.

Algae have been recognized as important resources providing functional components due to their capacity to exert beneficial effects on health. Therefore, there is increasing interest in investigating the biological activity of algae. In this study, we evaluated the antidepressant-like effect of the administration of 100 mg/kg/day of the ethanol extract of colonial green alga Botryococcus braunii (EEB) for 14 consecutive days in the forced swimming test (FST)-induced depression in imprinting control region (ICR) mice. Imipramine, a commercial antidepressant drug, was used as a positive control. In addition, we investigated the molecular mechanisms underlying the effect of EEB by measuring ATP production and by assessing any change in gene expression at the end of the treatment using real-time polymerase chain reaction (PCR) and microarray assays. We showed that the immobility time in the water-administered control (FST stress) group gradually increased from day 1 to day 14. However, treatment with EEB caused a significant decrease of immobility time in the FST compared with that in the FST stress group. Microarray and real-time PCR results revealed that EEB treatment induced variation in the expression of several genes associated with neurogenesis, energy metabolism, and dopamine synthesis. Interestingly, we revealed that only EEB treatment enhanced the promotion of energy production, while treatment with imipramine was ineffective. Our study provides the first evidence that B. braunii enhances energy production, which may contribute to the modulation of neurogenesis and to the enhancement of dopaminergic function, in turn potentially underlying the antistress- and antidepressant-like effects that we observed.

The relationship between hydrocarbons and the molecular phylogeny of Botryococcus braunii was investigated using 31 axenic strains isolated in Japan. The resulting 18S rRNA tree, which includes additional sequences from the gene bank, revealed the existence of three major pylogenetic clades (I, II, and III) and four subclades (II1, II2, III1, and III2) with high bootstrap values. By gas chromatography/mass spectrometry analysis, nine hydrocarbon species were detected and categorized as 4 types: the three known races A, B, and L and the tentatively named race S comprising epoxy-n-alkane and saturated n-alkane chains with carbon numbers 18 and 20, respectively. Strains of clade I were coincident with race A. Clade II contained race B, and clade III contained race L and its derivatives, and race S. Clade III was further categorized into subclade III1 containing the typical race L strains and subclade III2 containing race S with high bootstrap values. The phylogenetic relationship of Botryococcus strains appeared to be in considerable agreement with unique hydrocarbon synthesis pathways. Maximum divergence values of 18S rRNA indicated large speciation among the clades at almost the species level, suggesting the utility of biochemical properties, e.g., hydrocarbon types, in determining the taxonomy of Botryococcus .

Summary Red tides of Chattonella spp. have caused continuous damage to Japanese aquaculture, however, the life cycle of this organism remains incompletely understood. To further investigate this matter, we assessed genotypes at 14 microsatellite markers in three varieties of Chattonella marina, viz., C. marina var. antiqua, C. marina var. marina, and C. marina var. ovata, to establish whether Chattonella undergoes asexual diploidization or sexual reproduction. After genotyping 287 strains of C. marina, all but one of these strains was shown to be heterozygous for at least some loci, and thus, in the diploid state, suggesting that Chattonella strains undergo sexual reproduction. In addition, we performed single-cell amplification on ‘small cells’ that are derived from vegetative cells under dark and low-nutrient conditions. The results indicated the existence of two types of small cells. The ‘Small cell Type 1’ was found to be heterozygous, genotypically equivalent to the vegetative cells, and is therefore diploid. These small cells may change to resting cells (cysts) directly. The ‘Small cell Type 2’ was homozygous at all analyzed loci, suggesting that these small cells are haploid and may be derived by meiosis. As fusion between small cells has previously been observed, the ‘Small cell Type 2’ may be the gamete of Chattonella. We present a construct of the full life cycle of Chattonella marina based on our own and previous results.

Botryococcus braunii is a green colonial alga that produces large volumes of liquid hydrocarbon. Therefore, B. braunii is expected to be useful as an alternative fuel resource. Natural blooms of B. braunii have been recorded in several lakes and reservoirs. Elucidation of natural B. braunii blooming would provide important information for the development of an open-pond cultivation system. In this study, we periodically assessed the genetic diversity and colony density of B. braunii populations, along with several environmental parameters, in two Japanese reservoirs (provisionally called “N” and “S”) from December 2008 to December 2009. Reservoir N had low numbers of B. braunii colonies whereas Reservoir S was characterized by periodic density increases that occurred in December 2008, and in March, September, and December 2009. Population genetics analysis using specific environmental sequences (PGA-SES method) was conducted for B. braunii populations for the first time. Among the B. braunii -dominated samples of Reservoir S, high levels of genetic diversity were observed in December 2008 and March 2009, whereas the diversity levels in September and December 2009 were low. The results suggest that B. braunii periodicity can be categorized into a high genetic diversity type and a low genetic diversity type. The high genetic diversity type may be caused by simultaneous growth of many genotypes, whereas the low genetic diversity type seems to be explained by increases in the cell density of only a few adapted genotypes.

Metagenomics approaches have been developing rapidly in marine sciences. However, the application of these approaches to marine eukaryotes, and in particular to the smallest ones, is challenging because marine microbial communities are dominated by prokaryotes. One way to circumvent this problem is to separate eukaryotic cells using techniques such as single-cell pipetting or flow cytometry sorting. However, the number of cells that can be recovered by such techniques remains low and genetic material needs to be amplified before metagenomic sequencing can be undertaken. In this methodological study, we tested the application of whole-genome amplification (WGA) to photosynthetic eukaryotes. We performed various optimization steps both on a mixture of known microalgal strains and on natural photosynthetic eukaryote populations sorted by flow cytometry. rRNA genes were used as markers for assessing the efficiency of different protocols. Our data indicate that WGA is suitable for the amplification of photosynthetic eukaryote genomes, but that biases are induced, reducing the diversity of the initial population. Nonetheless, this approach appears to be suitable for obtaining metagenomics data on microbial eukaryotic communities.

Demura M., Noel M.-H., Kasai F., Watanabe M.M. and Kawachi M. 2009. Taxonomic revision of Chattonella antiqua, C. marina and C. ovata (Raphidophyceae) based on their morphological characteristics and genetic diversity. Phycologia 48: 518–535. DOI: 10.2216/08-98.1. Three raphidophyte species, Chattonella antiqua, C. marina and C. ovata, are red tide–forming harmful phytoplankton species, causing mass mortality at fish farms. These species were originally distinguished from each other solely based on their morphological characters. Recent genetic diversity analyses showed that the three species were extremely similar. It was therefore necessary to re-examine these three species to determine if they were independent single species. We compared 104 strains of the three species using morphological characters, nuclear ITS rDNA regions, the chloroplast rbcL gene, the mitochondrion COI gene and selected microsatellite regions. The morphological characters for the three species formed a continuum of varia...

We assessed changes in the microbial communities in ballast water during a trans-Pacific voyage from Japan to Australia that included a mid-ocean ballast-water exchange. Uncultured (i.e., total) and culturable bacteria were counted and were characterized by using denaturing gradient gel electrophoresis (DGGE). There was a clear decrease over time in numbers of uncultured microorganisms, except for heterotrophic nanoflagellates, whereas the abundance of culturable bacteria initially decreased after the ballast-water exchange but then increased. The increase, however, was only up to 5.34% of the total number of uncultured bacteria. Cluster analysis showed that the DGGE profiles of uncultured bacteria clearly changed after the exchange. In contrast, there was no clear change in the DGGE profiles of culturable bacteria after the exchange. Multidimensional scaling analysis showed changes in microbial communities over the course of the voyage. Although indicator microbes as defined by the International Convention for the Control and Management of Ships' Ballast Water and Sediments were occasionally detected, no coliform bacteria were detected after the exchange.

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide-forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.

In the cytochrome c oxidase subunit I (cox1) gene of four raphidophycean flagellates Chattonella antiqua, C. marina, C. ovata, and C. minima we found two group II introns described here as Chattonella cox1-i1 and Chattonella cox1-i2 encoding an open reading frame (ORF) comprised of three domains: reverse transcriptase (RT), RNA maturase (Ma) and zinc finger (H-N-H) endonuclease domains. The secondary structures show both Chattonella cox1-i1 and Chattonella cox1-i2 belong to group IIA1, albeit the former possesses a group IIB-like secondary structural character in the epsilon' region of arm I. Our phylogenetic analysis inferred from RT domain sequences of the intronic ORF, comparison of the insertion sites, and the secondary structures of the introns suggests that Chattonella cox1-i1 likely shares an evolutionary origin with the group II introns inserted in cox1 genes of five phylogenetically diverged eukaryotes. In contrast, Chattonella cox1-i2 was suggested to bear a close evolutionary affinity to the group II introns found in diatom cox1 genes. The RT domain-based phylogeny shows a tree topology in which Chattonella cox1-i2 is nested in the diatom sequences suggesting that a diatom-to-Chattonella intron transfer has taken place. Finally, we found no intron in cox1 genes from deeper-branching raphidophyceans. Based on parsimonious discussion, Chattonella cox1-i1 and Chattonella cox1-i2 have invaded into the cox1 gene of an ancestral Chattonella cell after diverging from C. subsalsa.