Microalga-bacteria Community with High Level Carbon Dioxide Acclimation and Nitrogen-fixing Ability.

Microalgal conversion of high-level CO2 in industrial flue gas to value-added products is attractive technology for mitigating global warming. However, reduction of microalgal production costs for medium ingredients, particularly nitrogen salts, is essential. The use of atmospheric nitrogen as a nitrogen source for microalgal cultivation will dramatically reduce its production costs. We attempted to enrich a microalga-bacteria community, which fixes both CO2 and atmospheric nitrogen under high level CO2. By cultivating biofilm recovered from the surface of cobbles in a riverbank, a microalgal flora which grows in a nitrogen salts-free medium under 10% CO2 was enriched, and the coccoid microalgal strain MP5 was isolated from it. Phylogenetic analysis revealed that the strain MP5 belongs to the genus Coelastrella, and the closest known species was C. terrestris. With PCR-DGGE analysis, it was found that the enriched microalgal community includes bacteria, some of which are suggested diazotrophs. The addition of bactericides in culture medium inhibited MP5 growth, even though the strain MP5 is eukaryotic. Growth of bacteria-free MP5 was stimulated by addition of Agrobacterium sp. isolates in nitrogen salts-free medium, suggesting that MP5 and the bacteria have responsibility for photosynthetic carbon fixation and nitrogen fixation, respectively.

Nitrogen-assisted lipid production by biofilms of aerial microalga Coccomyxa subellipsoidea KGU-D001 in the aerial phase.

The aerial microalga (Coccomyxa subellipsoidea KGU-D001) was photoautotrophically cultured under aerial phase conditions, and the influence of the nitrogen source on lipid production was investigated. Coccomyxa biofilms were introduced onto cotton wool using pure water (i.e., nutrient depletion) or a nitrogen-containing solution in a Petri dish, and they were cultured for 14 days under aerial phase conditions. The biomass in the biofilm increased by more than 150% in 14 days under nutrient depleted conditions and then increased further by approximately 30% following the addition of a nitrogen source. The lipid content rose under both nutrient depletion and nitrogen-added conditions, increasing by 170 and 150% in 14 days, respectively. The protein and sugar contents were also monitored and analyzed. In the presence of a nitrogen source, C. subellipsoidea undergo cell division in a relatively short time span, and biomass and lipids can be synthesized under both nutrient depleted and nitrogen-added conditions.

Production of polyglutamic acid-like mucilage protein by Peribacillus simplex strain 8h.

Polyglutamic acid (PGA), a protein in the mucilage of PGA-producing Bacillus spp., has expected applications in medical and biotechnological industries. Although the Bacillaceae family contains over 100 genera, research on bacterial PGA has exclusively focused on the genus Bacillus, especially B. subtilis var. natto and B. licheniformis. In the present study, indigenous Bacillaceae family strains were isolated from withered leaves and soil samples and screened for PGA production. As a result of the screening, the strain 8h was found to produce a mucilage possessing greater viscosity than PGA of B. subtilis var. natto (natto PGA). Biochemical analyses revealed that the 8h mucilage contains 63% protein and 37% polysaccharide, while mucilage of B. subtilis var. natto is composed of 61% protein and 39% polysaccharide. The most plentiful amino acid in 8h mucilage protein was glutamate (43%, mol/mol), which is similar to that of natto PGA, suggesting that it possesses characteristics of PGA. Although natto mucilage contains fructan, glucan was found as the polysaccharide of 8h mucilage. While phylogenetic studies indicated that the strain 8h belongs to Peribacillus simplex, the yield of the viscous mucilage by strain 8h was significantly higher than P. simplex type strain, suggesting that 8h is a mucilage-overproducing strain of P. simplex. Interestingly, 8h mucilage protein was found to contain more hydrophobic amino acid residues than natto PGA, suggesting that its amphiphilicity is suitable as a drug carrier and adjuvant. The present study is the first report of viscous mucilage and PGA-like protein produced by the genus Peribacillus.

Isolation and Characterization of Basidiomycetous Yeasts Capable of Producing Phytase under Oligotrophic Conditions.

Phytic acid is an organic phosphorus source naturally produced by plants as phosphorus stock and can be an alternative to rock phosphate, which is a dwindling resource globally. However, phytic acid is insoluble, owing to its binding to divalent metals and is, thus, not readily bioavailable for plants and monogastric livestock. Therefore, the enzyme phytase is indispensable for hydrolyzing phytic acid to liberate free phosphates for nutritional availability, making the screening of novel phytase-producing microbes an attractive research focus to agriculture and animal feed industries. In the present study, a soil-extract-based culture medium was supplemented with phytic acid as the sole phosphorus source and oligotrophic phytase-producing strains, which had not been previously studied, were isolated. Four fungal strains with phytic acid, assimilation activities were isolated. They were found to produce phytase in the culture supernatants and phylogenetic analysis identified three strains as basidiomycetous yeasts (Saitozyma, Leucosporidium, and Malassezia) and one strain as an ascomycetous fungus (Chaetocapnodium). The optimal pH for phytase activity of the strains was 6.0-7.0, suggesting that they are suitable for industrial applications as feed supplements or fertilizer additives for farmland.

Microflora communities which can convert digested sludge to biogas.

In the present study, we developed several microflora communities that utilize digested sludge (DS), the recalcitrant waste product of anaerobic digestion, as a substrate for biogas production with the aim of their future application to DS recycling. Strict enrichment with DS as the sole nutrient source was introduced to culture microbes from soil and herbivore dung samples; microflora communities promoting stable levels of biogas production were obtained. The average methane and hydrogen yield from soil-derived microflora were 4.86 and 0.94 ml per 1.0 g DS, respectively. Notably, two microflora communities enriched from a riverbank sediment produced 20.79 ml and 14.10 ml methane from 1.0 g DS. By contrast, the methane and hydrogen yield for herbivore dung-derived microfloras were on average 1.31 ml and 1.87 ml per 1.0 g DS, respectively. Potent hydrogen-biogas producers were obtained from rabbit (4.12 ml per 1.0 g DS), goat (3.16 ml per 1.0 g DS), and sheep dung (2.52 ml per 1.0 g DS). The cultured microflora communities included representatives from the eubacterial genera, Clostridiaceae and Eubacteriaceae together with several anaerobic genera. Pseudomonas spp. are found in the riverbank sediment-derived microfloras, suggesting that the floras employ syntrophic acetate oxidation and hydrogentrophic methanogenesis (SAO-HM) pathway for methane production. The methanogenic microflora communities were dominated by bacteria from the Methanobacteriaceae family and unclassified archaea. Moreover, ascomycetous fungi and protists were found, implying that they act as oxygen scavengers and bacterial grazers, respectively. Enzymatic analysis suggested that the microfloras hydrolyze DS via cellulase, chitinase, and protease activities.

Effects of light-emitting diodes (LEDs) on lipid production of the aerial microalga Coccomyxa sp. KGU-D001 under liquid- and aerial-phase conditions.

Algal biofuels are a promising alternative to fossil fuels, but their widespread use is hindered by problems with mass production. Light-emitting diodes (LEDs) with specific light wavelengths could be used as an energy source for algal growth and lipid synthesis. In this study, the effects of light source on the biomass and lipid production of the aerial microalga Coccomyxa sp. KGU-D001 were evaluated using LEDs. The integration of two-phase cultures, including growth and lipid production under the stress of nitrate depletion, was assessed for efficient lipid production under liquid- or aerial-phase conditions. Different wavelengths of light (blue, green, and red) were tested under liquid- and aerial-phase conditions. Under aerial-phase culture, the fatty acid contents in biofilm reached 320 mg g DWC-1 with the red LEDs. In view of these findings, we describe a one-step culture method for growth and lipid accumulation in algal biofilm under aerial-phase culture with red LED irradiation. When Coccomyxa biofilm was cultured on wet cotton wool with BBM in a petri dish under the red LED, it was able to grow and accumulate lipids under the aerial-phase condition. Based on the results of this study, a potential method for a continuous biodiesel production system is proposed.

Production of bioactive oligopeptide hydrolyzed by protease derived from aerial microalga Vischeria helvetica.

This study focused on a culture system of aerial microalgae with the decomposition of casein protein for obtaining bioactive compounds such as peptides with inhibitory activity against angiotensin-converting enzyme (ACE). The aerial microalga Vischeria helvetica exhibited growth in Bold's basal medium supplemented with casein protein as nitrogen source. The algal cells secreted protease and amino oxidase into the medium, and ammonium ions as a nitrogen source was produced by the conjugated-enzyme reaction. Furthermore, a bioactive peptide with ACE-inhibitory activity was efficiently produced from casein protein by the proteases secreted under light conditions. The results presented will facilitate the development of production systems for useful materials from photosynthetic microorganisms and casein protein in a culture medium.

Development of a whole-cell-based screening method for a carotenoid assay using aerial microalgae.

Non-destructive approaches based on the application of optical spectroscopy are important for monitoring carotenoid accumulation in a whole cell cultured under various conditions. A simple and rapid assay utilizing aerial microalgae helps to identify stress conditions that can efficiently enhance the carotenogenesis in photosynthetic organisms. The spectra of cell suspensions were characterized in the aerial microalga Coelastrella sp. KGU-Y002, which are unicellular and undifferentiated. Total carotenoid contents could be successfully estimated on the basis of the absorbance values of the cell suspensions and calibration data analyzed by HPLC (high-performance liquid chromatography). A novel screening method, the so-called "whole-cell-based screening method" for carotenoid assays (WCA), was developed based on this procedure. It was possible to investigate the effects of various stresses on carotenoid accumulation in the aerial microalga by adapting this bioassay to a 96-well microtiter plate. When bioactive compounds were screened from our library of plant extracts using this method, an active compound was identified from the plant extract.

Metabolic switching: synergistic induction of carotenogenesis in the aerial microalga, Vischeria helvetica, under environmental stress conditions by inhibitors of fatty acid biosynthesis.

OBJECTIVES: To investigate the roles of fatty acid biosynthesis in carotenogenesis in the high-lipid accumulating aerial microalga Vischeria helvetica KGU-Y001, we cultured algal cells with fatty acid biosynthesis inhibitors. RESULTS: Under nitrogen-deficient, high-light (200 µmol photons m(-2) s(-1)) conditions, the alga accumulated 6.2 mg carotenoids g(-1) dry weight cells (DWC) after 1 week of culture. The total fatty acid content increased gradually, and reached 290 mg g(-1) DWC after 9 weeks. When algal cells were cultured with a fatty acid biosynthesis inhibitor (molinate) under nitrogen-deficient, high-light conditions for 1 week, carotenoid accumulation was synergistically increased to 2.4 times that in algal cells cultured without the inhibitor in nitrogen-deficient, low-light conditions (40 µmol photons m(-2) s(-1)). The synergistic induction of carotenogenesis was suppressed by an inhibitor of c-jun N-terminal kinase, a mitogen-activated protein kinase-like protein. CONCLUSION: In a commercial context, carotenoid production could be increased by using fatty acid biosynthesis inhibitors to redirect metabolic flux to carotenoid biosynthesis instead of fatty acid synthesis.

Fatty acid content and profile of the aerial microalga Coccomyxa sp. isolated from dry environments.

Aerial algae are considered to be highly tolerant of and adaptable to severe conditions including radiation, desiccation, high temperatures, and nutrient deficiency, compared with those from aquatic habitats. There are considerable variations in the fatty acid (FA) composition of aerial microalgae from dry environments. A new species with a high lipid level was found on concrete surfaces and was identified as Coccomyxa sp. KGU-D001 (Trebouxiophyceae). This study characterized its FA content and profile in a bath culture. The alga showed a constant specific growth rate (0.26 day(-1)) ranging in light intensity from 20 to 80 µmol photons m(-2) s(-1). The algal cells started to form oil bodies in the early stationary phase of growth, and oil bodies occupied most of the cells during the late stationary phase when the cells accumulated 27 % total fatty acids (TFA). The process of lipid body formation accumulating large amounts of triacylglycerols (TAG) appeared to be very unusual in response to stress conditions persisting for a relatively long culture time (50 days). This study could indicate that aerial microalgae will be a candidate for biodiesel production when a new cultivation method is developed using extreme stresses such as nutritional deficiency and/or desiccation.

Pisiferdiol restores the growth of a mutant yeast suffering from hyperactivated Ca2+ signalling through calcineurin inhibition.

In the course of our screening program for a new inhibitor of the Ca(2+) signalling pathway using mutant yeast [Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ)], a mouse PP2Cα activator, pisiferdiol, isolated from Chamaecyparis pisifera, was found to alleviate the Ca(2+) signal-mediated growth inhibition. Pisiferdiol showed growth inhibition activity against the mpk1Δ strain compared with the cnb1Δ strain and induced Li(+) sensitivity to the wild-type strain, indicating that it suppresses the calcineurin pathway in the yeast. However, the Li(+) sensitivity to ptc1Δ strain by pisiferdiol was diminished. Pisiferdiol showed growth restored activity in the zds1Δ strain without immunophilins Fkb1p or Cph1p, and in the pmc1Δ strain. It inhibited calcineurin-induced expression in the reporter gene assay and decreased the protein expression (Western blots) of calcineurin (Cnb1p) in addition to a decrease of Swe1p and phosphorylation of Cdc28p in the mutant yeast. These results showed that pisiferdiol could suppress indirectly the action of calcineurin and restored the growth inhibition of the mutant yeast through Ptc1p activation.

Cleavage mechanism and anti-tumor activity of 3,6-epidioxy-1,10-bisaboladiene isolated from edible wild plants.

A bisabolane sesquiterpene endoperoxide compound, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from edible wild plants grown in the northern area of Japan, Cacalia delphiniifolia and Cacalia hastata, using a mutant yeast (cdc2-1 rad9Δ). It showed cytotoxicity at IC(50) = 3.4 µM and induced apoptosis against the human promyelocytic leukemia cell line HL60 through a new stable rearrangement product (1) when in the presence of FeSO(4). This conversion mechanism is different from another sesquiterpene endoperoxide lactone compound, dihydroartemisinin (DHA), which is an anti-malarial drug. The cytotoxicity of EDBD decreased in the presence of the ferrous ion chelating drug deferoxamine mesylate (DFOM), and this suggested that the structural change of the drug caused by Fe(2+) may be responsible for its biological activities. EDBD induced apoptosis via phosphorylation of p38 mitogen-activated protein kinase (MAPK) in HL60 cells, and was detected by Western blot. EDBD resulted in an immediate increase in DCF fluorescence intensity in HL60 cells using DCFH-DA (2',7'-dichlorofluorescin diacetate) assay. The in vitro reaction of EDBD with FeSO(4) also increased DCF fluorescence intensity in a dose dependent manner. These results showed that the biological activity of EDBD involves an unstable carbon-centered radical intermediate. Furthermore, there was no similarity between the JFCR39 fingerprints of EDBD and DHA (correlation coefficient on COMPARE Analysis γ = 0.158). EDBD showed anti-tumor effects against a xenograft of Lox-IMVI cells in vivo.

Isopimarane diterpene glycosides, isolated from endophytic fungus Paraconiothyrium sp. MY-42.

Six isopimarane diterpenes, compounds 1-6, were isolated from the endophytic fungus Paraconiothyrium sp. MY-42. Compound 1 possesses a 19-glucopyranosyloxy group. Its structure was first elucidated by spectroscopic data analysis and finally confirmed by X-ray crystallography, whereas structures 2-6 were mainly elucidated based on the analysis of spectroscopic evidence. Compounds 2 and 3 showed moderate cytotoxicities against the human promyelocytic leukemia cell line HL60 (IC50 6.7 µM value for 2 and 9.8 µM for 3).

Sanguinarine as a potent and specific inhibitor of protein phosphatase 2C in vitro and induces apoptosis via phosphorylation of p38 in HL60 cells.

Sanguinarine, a plant alkaloid, was identified as a potent and specific protein phosphatase (PP) 2C inhibitor. It inhibited PP2C competitively with respect to alpha-casein (Ki=0.68 microM) and showed selectivity for PP2C as compared with PP1, PP2A, and PP2B in vitro. In vivo, sanguinarine showed cytotoxicity toward human promyelocytic leukemia cell line HL60, with an IC(50) value of 0.37 microM, and induced apoptosis through a caspase-3/7-dependent mechanism involving the phosphorylation of p38, a PP2Calpha substrate. The apoptosis activity induced by sanguinarine was partially inhibited by a p38 inhibitor, SB203580, and was involved in the phospho-p38 protein in HL60 cells.

The bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene, isolated from Cacalia delphiniifolia inhibits the growth of human cancer cells and induces apoptosis.

In the course of our screening for a new anti-tumor substance, the bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from the edible wild-plant, Cacalia delphiniifolia. EDBD showed cytotoxicity toward human chronic myelogenous leukemia K562 and human prostate carcinoma LNCaP cell lines with IC50 values of 9.1 microM and 23.4 microM, respectively. DNA fragmentation and condensation of chromatin, the hallmarks of apoptosis, appeared in K562 cells after an 18-h treatment with EDBD. alpha-Curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD, also showed cytotoxicity toward both K562 and LNCaP cell lines at over a 10-times higher dose than that of EDBD. The results indicate the importance of the endoperoxide structure within EDBD to its anti-tumor activity in vitro.

Inhibitory activity of linoleic acid isolated from proso and Japanese millet toward histone deacetylase.

Linoleic acid was isolated from both the methanol extracts of proso and Japanese millet as a histone deacetylase inhibitor. It showed uncompetitive inhibitory activity toward histone deacetylase (IC(50)=0.51 mM) and potent cytotoxicity toward human leukemia K562 (IC(50)=68 microM) and prostate cancer LNCaP cells (IC(50)=193 microM). Millet containing linoleic acid might have anti-tumor activity.