Hyun-Joon La

Harvest of Scenedesmus sp. with bioflocculant and reuse of culture medium for subsequent high-density cultures.

The optimal flocculating conditions for harvesting high-density cultures of Scenedesmus sp. were investigated using inorganic coagulants and the bioflocculant produced by Paenibacillus polymyxa AM49. The flocculated medium as nutrients for subsequent algal cultivation was also tested. Consecutive treatment with 8.5 mM CaCl(2) and 0.2 mM FeCl(3) as coagulants and 1% bioflocculant from the culture broth of P. polymyxa AM49 showed the highest flocculating activity of up to 95% for high density algal cultures. The medium flocculated with the coagulants and bioflocculant showed less than 8% decrease in the growth yield in the subsequent algal cultivation. Furthermore, a 20% or 50% fresh BG11 medium supplement allowed the flocculated medium to maintain a high growth yield in subsequent algal cultivation. These results suggest that the flocculation method presented here is efficient and bio-friendly, and allows the reuse of the flocculated medium, thereby contributing to the economic cultivation and harvest of microalgae.

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.

Simple processes for optimized growth and harvest of Ettlia sp. by pH control using CO2 and light irradiation.

Microalgae cultures show wide range of pH depending on the availability of light and CO2 for their strain specific photosynthesis. Thus, the modulation of light irradiation and CO2 supply can be applied for the pH control of microalgae cultures. The optimal pH of Ettlia sp. YC001, for phototrophic growth and auto‐flocculation was investigated by controlling light irradiation and 10% CO2 supply. Ettlia sp. YC001 showed the highest biomass productivity, 96.7 mg L−1 d−1, at pH 8.5. The flocculating activity of Ettlia sp. YC001 showed a sigmoid pattern with pH increase and was above 70% at pH 10.5. Based on these differentiated optimal pH regimes for the growth and flocculation, an integrated process consisting of cultivation and settling vessels was proposed. The integrated process demonstrated that high flocculation activity of Ettlia sp. YC001 could be achieved in the settling vessel with various hydraulic retention times by only irradiation of light to maintain high pH while maintaining the optimal growth in cultivation vessel with the light irradiation and CO2 supply at pH 8.5. Thus, the proposed strategy for pH control would provide a simple, cost‐effective, and flexible design and operation for microalgae cultivation‐harvest systems. Biotechnol. Bioeng. 2015;112: 288–296.

Effective screening of Scenedesmus sp. from environmental microalgae communities using optimal sonication conditions predicted by statistical parameters of fluorescence-activated cell sorting.

The effects of the sonication parameters, including the power and time, were investigated for the effective isolation of Scenedesmus sp. from environmental microalgae communities when using fluorescence-activated cell sorting (FACS). The selectivity, defined as the percentage of Scenedesmus sp. successfully isolated and grown in microplates, appeared as peaks in contour plots spanned by the sonication power and time. For fast screening of the optimal sonication conditions, correlations between the selectivity and the statistical parameters from the FACS analysis were investigated. A graphical comparison analysis of the contour plots showed a pattern similarity of over 82% between the coefficients of variation for the side scatter (SSC-CV) and the selectivity. This predictability of the optimal sonication conditions enabled a Scenedesmus sp. selectivity of ca. 2 times using only one-third of the sonication condition sets arbitrarily chosen around the peaks of the SSC-CV, thereby saving resources and time for subsequent processes.

Evaluation of various techniques for microalgal biomass quantification

Biomass concentration is one of the most important parameters in the biotechnology processes. Its measurement relies on the physical, chemical or biological properties of the cells. Several techniques were applied in this work to measure the cell concentration of four microalgae: Botryococcussp., Botryococcusbraunii, Chlorella vulgaris, and Ettlia sp. The experiments were performed using samples taken from a chemostat for each strain to provide microalgal cell suspensions in a stable physiological state and concentration. The dry cell weight (DCW) was used as the reference method for the evaluation of other methods. The two commercial sensors used to determine optical density and dielectric permittivity showed a broad effective measurement range up to more than 20gl(-1). A Red-Green-Blue model analysis of microalgal digital images in combination with Fourier equation significantly extended the measurements range up to 6gl(-1). Cell count using a flow cytometer showed a broad range of linearity to DCW in washed samples, but other counting methods using hemocytometer and microscopic automated count were limited. Finally, the oxygen production rate, representing the photosynthetic activity, showed a linear regression with DCW at cell concentrations lower than 1gl(-1).

Establishment and maintenance of an axenic culture of Ettlia sp. using a species-specific approach

The establishment of an axenic culture of microalgae is essential step in understanding its physiology, genetics, and ecology. However, culturing of microalgae is usually accompanied by complex and variable associated prokaryotic and eukaryotic microorganisms. Conventional approaches used for obtaining axenic cultures of microalgae are time-consuming and often involve difficulties in maintaining and preserving axenicity. In this study, we developed a procedure for establishing an axenic culture of Ettlia sp. YC001 and demonstrate that we maintained the axenic culture through subculture in the long term. Three sequential treatments, an antibiotic cocktail, serial dilution, and plate spreading, were applied to strain YC001 and we confirmed axenicity using molecular and physiological methods. The bacterial community associated with strain YC001 was investigated to select antibiotics for their specific elimination. The xenic culture (1 × 106 cells/mL) was treated with the antibiotic cocktail-5 (AC-5), carbendazim, chloramphenicol, imipenem, rifampicin, and tetracycline for 3 days, followed by serial dilution up to 1 × 102 cells and spreading on agar plates. The pure colonies were analyzed using denaturing gradient gel electrophoresis (DGGE), fluorescence-activated cell sorting (FACS), and scanning electron microscopy (SEM). The procedure we developed can be applied to other strains of microalgae for the establishment of axenic cultures.

Technical and Strategic Approach for the Control of Cyanobacterial Bloom in Fresh Waters

Cyanobacteria (blue-green algae) are not only the first oxygenic organisms on earth but also the foremost primary producers in aquatic environment. Massive growth of cyanobacteria, in eutrophic waters, usually changes the water colour to green and is called as algal (cyanobacterial) bloom or green tide. Cyanobacterial blooms are a result of high levels of primary production by certain species such as Microcystis sp., Anabaena sp., Oscillatoria sp., Aphanizomenon sp. and Phormidium sp. These cyanobacterial species can produce hepatotoxins or neurotoxins as well as malodorous compounds like geosmin and 2-methylisoborneol (MIB). In order to solve the nationwide problem of hazardous cyanobacterial blooms in Korea, the following technically and strategically sound approaches need to be developed. 1) As a long-term strategy, reduction of the nutrients such as phosphorus and nitrogen in our water bodies to below permitted levels. 2) As a short term strategy, field application of combination of already established bloom remediation techniques. 3) Development of emerging convergence technologies based on information and communication technology (ICT), environmental technology (ET) and biotechnology (BT). 4) Finally, strengthening education and creating awareness among students, public and industry for effective reduction of pollution discharge. Considering their ecological roles, a complete elimination of cyanobacteria is not desirable. Hence a holistic approach mentioned above in combination to addressing the issue from a social perspective with cooperation from public, government, industry, academic and research institutions is more pragmatic and desirable management strategy.

Light intensity as major factor to maximize biomass and lipid productivity of Ettlia sp. in CO2-controlled photoautotrophic chemostat

The optimal culture conditions are critical factors for high microalgal biomass and lipid productivity. To optimize the photoautotrophic culture conditions, combination of the pH (regulated by CO2 supply), dilution rate, and light intensity was systematically investigated for Ettlia sp. YC001 cultivation in a chemostat during 143days. The biomass productivity increased with the increase in dilution rate and light intensity, but decreased with increasing pH. The average lipid content was 19.8% and statistically non-variable among the tested conditions. The highest biomass and lipid productivities were 1.48gL-1d-1 and 291.4mgL-1d-1 with a pH of 6.5, dilution rate of 0.78d-1, and light intensity of 1500μmolphotonsm-2s-1. With a sufficient supply of CO2 and nutrients, the light intensity was the main determinant of the photosynthetic rate. Therefore, the surface-to-volume ratio of a photobioreactor should enable efficient light distribution to enhance microalgal growth.

Unique mitochondrial genome structure of the green algal strain YC001 (Sphaeropleales, Chlorophyta), with morphological observations

Abstract: We characterized the complete sequence of the mitochondrial genome of the freshwater green algal strain YC001, known as ‘Ettlia sp.’, which is of great interest for biofuel production. The size of the mitogenome was 52,489 base pairs (bp), with 49.0% GC content. It consisted of 49 mitochondrial genes typical of the Chlorophyceae, including 16 protein-coding genes, 27 transfer RNA genes and six ribosomal RNA genes as well as three introns within cox1 (6832 bp), rnl4 (1805 bp) and cob (1107 bp). We determined that YC001 is a species of Coelastrella on the basis of molecular phylogenetic analyses and microscopic observations. A unique feature of this mitogenome is the presence of three different types of inverted repeats between trnL and trnI and between nad4 and cox1 as well as the presence of orf215, orf755 and one additional copy of the trnM gene, all of which enable a comparison with an available sister taxon, Acutodesmus obliquus.

Aeration effects on metabolic events during sporulation of Bacillus thuringiensis

The metabolism of Bacillus thuringiensis during its sporulation process was investigated under different concentrations of oxygen. At the beginning of sporulation, the aeration conditions were regulated to obtain different oxygen transfer rates (OTR) in four separate fermentations, representing interrupted, limited, non-limited, and saturated oxygenation, respectively. A higher OTR resulted in a higher pH, up to about 9 in the case of saturated oxygenation, while the interrupted oxygenation resulted in a significantly acidic culture. In contrast, the absence of oxygen resulted in rapid sporangia lysis and caused acidification of the medium, indicating a distinctly different sporangia composition and different metabolism. The bacterium also showed different CO2 production rates during sporulation, although amaximum point was observed in every case.With a higher OTR, the maximal value was observed after a longer time and at a lower value (40, 26, and 13 mmol/L/h for limited, non-limited, and saturated cases, respectively). Despite the exhaustion of glucose prior to the sporulation phase, the interrupted oxygenation resulted in acetate, lactate, and citrate in the medium with a maximum concentration of 4.8, 1.3, and 5.0 g/L, respectively. Notwithstanding, while the metabolic events differed visibly in the absence of oxygen, once sporulation was triggered, it was completed, even in the case of an interrupted oxygen supply.