Efficient separation of phycocyanin of Nostoc commune by multistep diafiltration using ultra-filtration membrane modules.

Diafiltration (DF) is a separation method used to separate and concentrate macromolecules, such as polysaccharides and proteins. To obtain high-purity target molecules by DF, appropriate conditions should be used. In this study, a mathematical model was developed to suggest appropriate ultra-filtration (UF) membrane modules for the separation of phycocyanin (PC) by multistep DF. PC is a protein produced by microalgae. The contribution of each UF membrane module to PC productivity and purity at each stage of the multistep DF process was quantified by the proposed model. The parameters required as model inputs (k, Fα1, and Fα2) were experimentally determined by permeating PC-containing solution through UF membrane modules (150, 30, and 10 kDa cutoffs). The resulting analytical solutions and those predicted by the model were in close agreement. The PC purity increased from 0.20 to 0.30 when a 10 kDa UF membrane module was used in two-step DF. An orthogonal table was used to determine the combination of UF membrane modules needed to achieve higher purity of PC. The model predicted that the 30 kDa UF membrane module would have the highest contribution to PC productivity and purity at any position in a three-step DF. The developed model can help identify appropriate conditions for separating macromolecules by DF.

Separation of microalgae using a compacted magnetite-containing gel bed.

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.

A Novel Polyculture Growth Model of Native Microalgal Communities to Estimate Biomass Productivity for Biofuel Production.

Native polyculture microalgae is a promising scheme to produce microalgal biomass as biofuel feedstock in an open raceway pond. However, predicting biomass productivity of native polycultures microalgae is incredibly complicated. Therefore, developing polyculture growth model to forecast biomass yield is indispensable for commercial-scale production. This research aims to develop a polyculture growth model for native microalgal communities in the Minamisoma algae plant and to estimate biomass and biocrude oil productivity in a semi-continuous open raceway pond. The model was built based on monoculture growth of polyculture species and it is later formulated using species growth, polyculture factor (k value ), initial concentration, light intensity, and temperature. In order to calculate species growth, a simplified Monod model was applied. In the simulation, 115 samples of the 2014-2015 field dataset were used for model training, and 70 samples of the 2017 field dataset were used for model validation. The model simulation on biomass concentration showed that the polyculture growth model with k value had a root-mean-square error of 0.12, whereas model validation provided a better result with a root-mean-square error of 0.08. Biomass productivity forecast showed maximum productivity of 18.87 g/m2 /d in June with an annual average of 13.59 g/m2 /d. Biocrude oil yield forecast indicated that hydrothermal liquefaction process was more suitable with a maximum productivity of 0.59 g/m2 /d compared with solvent extraction which was only 0.19 g/m2 /d. With satisfactory root mean square errors less than 0.3, this polyculture growth model can be applied to forecast the productivity of native microalgae. This article is protected by copyright. All rights reserved.

Dewatering of algal suspension using microfiltration with cross flow in the presence of magnetite as a filter aid.

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.

Ethanol Extract of Aurantiochytrium mangrovei 18W-13a Strain Possesses Anti-inflammatory Effects on Murine Macrophage RAW264 Cells.

In this study, the effects of an ethanolic extract of Aurantiochytrium mangrovei 18W-13a strain (AM18W-13a) on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264 murine macrophages were studied. Pre-treatment with the AM18W-13a extract significantly suppressed the LPS-induced production of nitric oxide and pro-inflammatory cytokines. RAW264 cells treated with the AM18W-13a extract for 1 and 24 h were subjected to DNA microarray analyses for detecting the differentially expressed genes. The treatment of RAW264 cells with the AM18W-13a extract for 24 h significantly suppressed the expression of several genes associated with inflammation or chemotaxis. Furthermore, treatment with the AM18W-13a extract for 1 h suppressed the expression of Pde4b, but induced the expression of Egr2 and Egr3 in RAW264 cells. Additionally, the AM18W-13a extract significantly enhanced the expression of certain anti-inflammatory mediators. This study is the first report of the anti-inflammatory effects of the AM18W-13a extract and its mechanism of action in LPS-stimulated murine macrophages.

Modulation of Neurogenesis through the Promotion of Energy Production Activity Is behind the Antidepressant-Like Effect of Colonial Green Alga, Botryococcus braunii.

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.

Application of cryopreservation to genetic analyses of a photosynthetic picoeukaryote community.

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.

Changes in microbial communities, including both uncultured and culturable bacteria, with mid-ocean ballast-water exchange during a voyage from Japan to Australia.

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.

Desiccation tolerance of Botryococcus braunii (Trebouxiophyceae, Chlorophyta) and extreme temperature tolerance of dehydrated cells.

Botryococcus braunii Kützing, a green colonial microalga, occurs worldwide in both freshwater and brackish water environments. Despite considerable attention to B. braunii as a potential source of renewable fuel, many ecophysiological properties of this alga remain unknown. Here, we examined the desiccation and temperature tolerances of B. braunii using two newly isolated strains BOD-NG17 and BOD-GJ2. Both strains survived through 6- and 8-month desiccation treatments but not through a 12-month treatment. Interestingly, the desiccation-treated cells of B. braunii gained tolerance to extreme temperature shifts, i.e., high temperature (40 °C) and freezing (-20 °C). Both strains survived for at least 4 and 10 days at 40 and -20 °C, respectively, while the untreated cells barely survived at these temperatures. These traits would enable long-distance dispersal of B. braunii cells and may account for the worldwide distribution of this algal species. Extracellular substances such as polysaccharides and hydrocarbons seem to confer the desiccation tolerance.

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations.

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.

Denaturing gradient gel electrophoresis shows that bacterial communities change with mid-ocean ballast water exchange.

Ships carry ballast water for better stability and to control trim. However, the discharge of ballast water near ports is known to transport invasive species from one coastal area to another. The exchange of ballast water on the high seas is supposed to reduce such invasions of exotic species. In this study, we used denaturing gradient gel electrophoresis (DGGE) to analyze the composition of the bacterial community in ballast water before and after such a mid-ocean exchange, and we also measured total bacterial counts. Our findings confirmed that the ballast water was replaced by the mid-ocean exchange, as indicated by the marked change in the composition of the bacterial community. There was also a significant decrease in bacterial abundance after the mid-ocean exchange. Finally, our findings support the incubation hypothesis, because the composition of the bacterial communities changed over time within the same ballast water.

Mitochondrial group II introns in the raphidophycean flagellate Chattonella spp. suggest a diatom-to-Chattonella lateral group II intron transfer.

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.