Byung-Dae Yoon

Rapid method for the determination of lipid from the green alga Botryococcus braunii

Of various methods for lipid recovery in Botryococcus braunii UTEX 572, the most effective method was disruption of the cells with a bead-beater followed by extraction with chloroform/methanol (2:1, v/v). This gave a lipid content of 28.6% of dry wt. There was a significant relationship between in vivo fluorescence of cells stained with Nile Red and lipid content in B. braunii determined gravimetrically (r2 = 0.997). This suggested that the Nile Red staining as a rapid method was as good as the gravimetric method commonly used for lipid determination which requires toxic solvents and considerable time-consuming manipulations.

Microcystin Production by Microcystis aeruginosa in a Phosphorus-Limited Chemostat

ABSTRACT The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C18 cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C18 reverse-phase column. The specific growth rate (μ) ofM. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing μ. The MC-LR and MC-RR contents on a dry weight basis were highest at μ of 0.1/day at 339 and 774 μg g−1, respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower μ, whereas the MC-producing rate was linearly proportional to μ. The C fixation rate at an ambient irradiance (160 microeinsteins m−2s−1) increased with μ. The ratios of the MC-producing rate to the C fixation rate were higher at a lower μ. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.

Harvesting of Chlorella vulgaris using a bioflocculant from Paenibacillus sp. AM49

Microbial flocculants for harvesting mass cultured Chlorella vulgaris were screened and that from Paenibacillussp. AM49 was identified as the best. The flocculation efficiency of this bioflocculant increased with the pH within a range of pH 5–11 and was 83%, which was higher than the 72% and 78% produced by aluminum sulfate and polyacrylamide, respectively. The highest flocculation efficiency was with 6.8 mm CaCl2 as co-flocculant. The bioflocculant from Paenibacillussp. AM49 can be used effectively to harvest C. vulgaris from large-scale cultures.

Production and Properties of a Lipopeptide Biosurfactant from Bacillus subtilis C9

Abstract A lipopeptide biosurfactant, C9-BS produced by Bacillus (B.) subtilis C9, emulsified hydrocarbons, vegetable oil and crude oil. A high yield of C9-BS was obtained from a culture of B. subtilis C9 using a carbohydrate substrate, while a hydrocarbon substrate inhibited the production of the biosurfactant. The optimum medium for the production of C9-BS was as follows (g/l): glucose, 40; NH4HCO3, 13.5; K2HPO4, 10.5; NaH2PO4, 1.5; MgSO4·7H2O, 0.5; MnSO4·4H2O, 0.05; yeast extract, 0.5. The initial pH was 8.0 and the culture temperature was 30°C. The biosurfactant C9-BS was obtained by collection of the foam that overflowed in the fermentor culture. The production of the biosurfactant by B. subtilis C9 was growth-associated. Under O2-limited conditions, C9-BS was produced in a 3-fold higher yield compared to that under O2-sufficient conditions. C9-BS is soluble in ethanol, acetone, methanol, butanol, chloroform, dichloromethane and alkaline water. The biosurfactant C9-BS lowered the surface tension of water to 28.5 dyne/cm, and the CMC (critical micelle concentration) was 40 μM. C9-BS was stable from pH 5.0 to pH 9.5, in incubation at 100°C for 1 h, and at a salt concentration of 1,000 mM for NaCl and 10 mM for CaCl2.

Variation of microcystin content of microcystis aeruginosa relative to medium N:P ratio and growth stage.

Changes in the microcystin content of Microcystis aeruginosa UTEX 2388 were investigated at several N:P ratios of the medium and various growth stages. Under the P‐fixed condition, the microcystin content of the cells changed with different medium N:P ratios, with the highest at 2748 µg g−1 at a N:P ratio of 16 after incubation for 7 d. The microcystin content of M. aeruginosa exhibited a high correlation with the total N content regardless of an N‐fixed or P‐fixed culture. When the N:P ratio of the medium was fixed to 16 : 1, the microcystin content of M. aeruginosa at various growth stages was highest at 2191 µg g−1 after an incubation of 4 d and the chlorophyll‐a content showed a similar tendency. There was a highly significant relationship between the microcystin content of M. aeruginosa and the chlorophyll‐a concentration in the culture during the incubation. Accordingly, the microcystin content of M. aeruginosa during incubation can be easily estimated and monitored by measuring the in vivo fluorescence changes in the culture.

Seasonal Variation and Indirect Monitoring of Microcystin Concentrations in Daechung Reservoir, Korea

ABSTRACT Physicochemical and biological water quality, including the microcystin concentration, was investigated from spring to autumn 1999 in the Daechung Reservoir, Korea. The dominant genus in the cyanobacterial blooming season was Microcystis. The microcystin concentration in particulate form increased dramatically from August up to a level of 200 ng liter−1 in early October and thereafter tended to decrease. The microcystin concentration in dissolved form was about 28% of that of the particulate form. The microcystins detected using a protein phosphatase (PP) inhibition assay were highly correlated with those microcystins detected by a high-performance liquid chromatograph (r= 0.973; P < 0.01). Therefore, the effectiveness of a PP inhibition assay for microcystin detection in a high number of water samples was confirmed as easy, quick, and convenient. The microcystin concentration was highly correlated with the phytoplankton number (r = 0.650; P < 0.01) and chlorophyll-a concentration (r = 0.591;P < 0.01). When the microcystin concentration exceeded about 100 ng liter−1, the ratio of particulate to dissolved total nitrogen (TN) or total phosphorus (TP) converged at a value of 0.6. Furthermore, the microcystin concentration was lower than 50 ng liter−1 at a particulate N/P ratio below 8, whereas the microcystin concentration varied quite substantially from 50 to 240 ng liter−1 at a particulate N/P ratio of >8. Therefore, it seems that the microcystin concentration in water can be estimated and indirectly monitored by analyzing the following: the phytoplankton number and chlorophyll-a concentration, the ratio of the particulate and the dissolved forms of N and P, and the particulate N/P ratio when the dominant genus is toxigenic Microcystis.

Characterization of bioflocculant produced by Bacillus sp. DP-152

A strain (designated DP-152) which produces an excellent flocculating substance was isolated from soil samples and identified as Bacillus species. The major flocculating substance (bioflocculant DP-152) produced by Bacillus sp. DP-152 was purified by ethanol precipitation and cetylpyridinium chloride (CPC) precipitation and gel permeation chromatography. In kaolin suspension, the highest flocculating activity was obtained at the bioflocculant concentration of 1 mg/l. The bioflocculant DP-152, which has an estimated molecular weight of over 2 × 106 daltons, is a novel bioflocculant derived from sugar components consisting of glucose, mannose, galactose, and fucose in an approximate molar ratio of 8 : 4 : 2 : 1. Some of its physico-chemical properties were also determined.

Characterization of a biosurfactant, mannosylerythritol lipid produced from Candida sp. SY16.

Abstract One yeast strain, SY16, was selected as a potential producer of a biosurfactant, and identified as a Candida species. A biosurfactant produced from Candida sp. SY16 was purified and confirmed to be a glycolipid. This glycolipid-type biosurfactant lowered the surface tension of water to 29 dyne/cm at critical micelle concentration of 10 mg/l (1.5 × 10−5 M), and the minimum interfacial tension was 0.1 dyne/cm against kerosene. Thin-layer and high-pressure liquid chromatography studies demonstrated that the glycolipid contained mannosylerythritol as a hydrophilic moiety. The hydrophilic sugar moiety of the biosurfactant was determined to be β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by nuclear magnetic resonance (NMR) and fast atom bombardment mass–spectroscopy analyses. The hydrophobic moiety, fatty acids, of the biosurfactant was determined to be hexanoic, dodecanoic, tetradecanoic, and tetradecenoic acid by gas chromatography–mass spectroscopy. The structure of the native biosurfactant was determined to be 6-O-acetyl-2,3- di-O-alkanoyl-β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by NMR analyses. We newly determined that an acetyl group was linked to the C-6 position of the d-mannose unit in the hydrophilic sugar moiety.

Effects of Crude Oil, Oil Components, and Bioremediation on Plant Growth

Abstract The phytotoxic effects of crude oil and oil components on the growth of red beans (Phaseolus nipponesis OWH1) and corn (Zea mays) was investigated. In addition, the beneficial effects of bioremediation with the oil-degrading microorganism, Nocardia sp. H17-1, on corn and red bean growth in oil-contaminated soil was also determined. It was found that crude oil-contaminated soil (10,000 mg/kg) was phytotoxic to corn and red beans. In contrast, obvious phytotoxicity was not observed in soils contaminated with 0–1000 mg/kg of aliphatic hydrocarbons such as decane (C10) and eicosane (C20). Phytotoxicity was observed in soils contaminated with 10–1000 mg/kg of the poly aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, and pyrene. It was observed that phytotoxicity increased with the number of aromatic rings, and that corn was more sensitive than red beans to PAH-contaminated soil. Bioremediation with Nocardia sp. H17-1 reduced phytotoxicity more in corn than in red bean, suggesting that this microbial species might degrade PAHs to some degree.

Growth inhibition of bloom‐forming cyanobacterium Microcystis aeruginosa by rice straw extract

Aims:  To inhibit the growth of the bloom‐forming cyanobacterium Microcystis aeruginosa using a rice straw extract.