Gang-Guk Choi

Recent trends in development of biosensors for detection of microcystin.

Increased cyanobacterial blooms, a source of cyanotoxins are linked with climate change and eutrophication in aquatic bodies, a major concern worldwide. Microcystins are potently hepatotoxic, nephrotoxic as well as carcinogenic. Thus microcystins are threat to tourism, agriculture and animals health. However, there is a still lacuna in the knowledge of regulation of microcystins production. Presence of toxic and non-toxic cyanobacterial strains together and occurrence of various microcystin variants in aquatic bodies compounded the problem. Although several analytical techniques for microcystin detection such as bioassay, ELISA, HPLC and LC-MS etc. have been already prevalent, the development of biosensors offered rapid and accurate detection, high reproducibility and portability. Sequencing of Microcystis spp., opened the new vistas towards the development of biosensor at molecular and genetic level. This review incorporates the current trends in the development of biosensors for microcystin detection in the light of state-of-the-art techniques.

Ettlia sp. YC001 showing high growth rate and lipid content under high CO2.

Over 100 green-colored colonies were isolated from environmental samples when cultivating on a BG11 agar medium, and 4 strains showing different morphologies were selected based on light microscopic observation. Among these strains, the microalgal species with the highest growth rate under 10% CO(2) was identified as Ettlia sp. YC001 using an 18S rDNA-based phylogenetic analysis and morphological comparison. The highest cell density of 3.10 g/L (based on dry cell weight) and biomass productivity of 0.19 g/L/d were obtained under 5% CO(2) after 16 days. The lipid content and productivity were also up to 42% of the dry cell weight and 80.0mg/L/d, respectively. The color of the Ettlia sp. YC001 culture changed from green to red after a month due to the accumulation of certain carotenoids. Therefore, it would seem that Ettlia sp. YC001 is appropriate for mitigating CO(2) due to its high biomass productivity, and a suitable candidate for producing biodiesel and high-value products.

Determination of Cyanobacterial Diversity during Algal Blooms in Daechung Reservoir, Korea, on the Basis of cpcBA Intergenic Spacer Region Analysis

ABSTRACT The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes.

Dynamics of microcystin production and quantification of potentially toxigenic Microcystis sp. using real-time PCR

Cyanobacterial blooms in eutrophied water body are generally composed of various genotypes with or without microcystin-producing genes (mcy gene cluster). Thus there is a need for quantification of potent toxin producing strains. The present study aimed at identifying microcystin variants and its producer strains in Durgakund pond, Varanasi, India, based on quantification of cpcBA-IGS and mcyA (condensation domain) genes using real-time PCR and LC-MS. Increase in microcystin concentrations was correlated with increase in mcyA copy number and the level of pigments (chlorophyll a, phycocyanin and carotenoids). Also, selected environmental factors (water temperature, light irradiance, rainfall, pH, N and P) and the concentration of microcystin variants (MC-LR, -RR and -YR) were also assessed in samples during May 2010 to April 2011 to establish the possible correlation among these parameters. Nutrients favored cyanobacterial bloom but it could not be correlated with the levels of microcystin variants and seemed to be geographically specific. Microcystis sp. dominant in the pond comprised potentially toxigenic cells. The ratio of potentially toxigenic Microcystis sp. to that of total Microcystis sp. ranged from 0% to 14%. Such studies paved the way to identify and quantify the most potent microcystin producer in the tropical aquatic body.

Simple, rapid and cost-effective method for high quality nucleic acids extraction from different strains of Botryococcus braunii.

This study deals with an effective nucleic acids extraction method from various strains of Botryococcus braunii which possesses an extensive extracellular matrix. A method combining freeze/thaw and bead-beating with heterogeneous diameter of silica/zirconia beads was optimized to isolate DNA and RNA from microalgae, especially from B. braunii. Eukaryotic Microalgal Nucleic Acids Extraction (EMNE) method developed in this study showed at least 300 times higher DNA yield in all strains of B. braunii with high integrity and 50 times reduced working volume compared to commercially available DNA extraction kits. High quality RNA was also extracted using this method and more than two times the yield compared to existing methods. Real-time experiments confirmed the quality and quantity of the input DNA and RNA extracted using EMNE method. The method was also applied to other eukaryotic microalgae, such as diatoms, Chlamydomonas sp., Chlorella sp., and Scenedesmus sp. resulting in higher efficiencies. Cost-effectiveness analysis of DNA extraction by various methods revealed that EMNE method was superior to commercial kits and other reported methods by >15%. This method would immensely contribute to area of microalgal genomics.

Enhancing lipid productivity of Chlorella vulgaris using oxidative stress by TiO2 nanoparticles

Ability to increase the lipid production in microalgae is one of the heavily sought-after ideas to improve the economic feasibility of microalgae-derived transportation fuels for commercial applications. We used the oxidative stress by TiO2 nanoparticles, a well-known photocatalyst, to induce lipid production in microalgae. Chlorella vulgaris UTEX 265 was cultivated under various concentrations of TiO2 ranging from 0.1 to 5 g/L under UV-A illumination. Maximum specific growth rate was affected in responding to TiO2 concentrations. In the presence of UV-A, chlorophyll concentration was decreased at the highest concentration of TiO2 (5 g/L TiO2) by oxidative stress. The fatty acid ethyl ester (FAME) composition analysis suggested that oxidative stress causes the accumulation and decomposition of lipids. The highest FAME productivity was 18.2 g/L/d under low concentrations of TiO2 (0.1 g/L) and a short induction time (two days). The controlled condition of TiO2/UV-A inducing oxidative stress (0.1 g/L TiO2 and two days induction) could be used to increase the lipid productivity of C. vulgaris UTEX 265. Our results show the possibility of modulating the lipid induction process through oxidative stress with TiO2/UV-A.

Novel approach for the development of axenic microalgal cultures from environmental samples.

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence‐activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.

Use of orange peel extract for mixotrophic cultivation of Chlorella vulgaris: increased production of biomass and FAMEs.

Mass cultivation of microalgae is necessary to achieve economically feasible production of microalgal biodiesel, but the high cost of nutrients is a major limitation. In this study, orange peel extract (OPE) was used as an inorganic and organic nutrient source for the cultivation of Chlorella vulgaris OW-01. Chemical composition analysis of the OPE indicated that it contains sufficient nutrients for mixotrophic cultivation of C. vulgaris OW-01. Analysis of biomass and FAME production showed that microalgae grown in OPE medium produced 3.4-times more biomass and 4.5-times more fatty acid methyl esters (FAMEs) than cells cultured in glucose-supplemented BG 11 medium (BG-G). These results suggest that growth of microalgae in an OPE-supplemented medium increases lipid production and that OPE has potential for use in the mass cultivation of microalgae.

Enhancement of lipid productivity by ethyl methane sulfonate-mediated random mutagenesis and proteomic analysis in Chlamydomonas reinhardtii

Microalgae-derived biomass has been considered as the most promising candidate for next generation biofuel due to its sustainability and biodegradability. In this study, microalgal strain Chlamydmonas reinhardtii was randomly mutagenized by using a chemical mutagen, ethyl methane sulfonate (EMS) to create mutants showing enhanced lipid production. We identified three random mutants that displayed high lipid production in the screening using Nile red staining. Among those, mutant #128 was selected as candidate for further studies. Our flow cytometry and confocal microscopy analysis revealed that mutant #128 contains larger and more abundant lipid bodies than that of wild-type. Moreover, mutant #128 showed 1.4-fold increased fatty acid methyl ester (FAME) content compared to wild-type under nitrogen depleted condition. In addition, mutant #128 grew faster and accumulated more biomass, resulting in high lipid production. 2D gel electrophoresis and MALDI-TOF analysis used for gene targeting revealed that β-subunit of mitochondrial ATP Synthase and two-component response regulator PilR may be involved in enhanced characteristics of mutant #128. These results show the possibilities of EMS mediated random mutagenesis in generation of mutants to produce high amount of lipid as well as further study for molecular mechanism of mutants.

Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina

Highlights • Heterologous sfCherry protein was expressed in N. salina for the first time.• N. salina was transformed by particle bombardment.• Integration site of the transgene on the genome was determined by RESDA PCR.• Expression of sfCherry was confirmed by a western blotting and confocal microscopy.