Hee-Mock Oh

Comparison of several methods for effective lipid extraction from microalgae

Various methods, including autoclaving, bead-beating, microwaves, sonication, and a 10% NaCl solution, were tested to identify the most effective cell disruption method. The total lipids from Botryococcus sp., Chlorella vulgaris, and Scenedesmus sp. were extracted using a mixture of chloroform and methanol (1:1). The lipid contents from the three species were 5.4-11.9, 7.9-8.1, 10.0-28.6, 6.1-8.8, and 6.8-10.9 g L(-1) when using autoclaving, bead-beating, microwaves, sonication, and a 10% NaCl solution, respectively. Botryococcus sp. showed the highest oleic acid productivity at 5.7 mg L(-1)d(-1) when the cells were disrupted using the microwave oven method. Thus, among the tested methods, the microwave oven method was identified as the most simple, easy, and effective for lipid extraction from microalgae.

Selection of microalgae for lipid production under high levels carbon dioxide

To select microalgae with a high biomass and lipid productivity, Botryococcus braunii, Chlorella vulgaris, and Scenedesmus sp. were cultivated with ambient air containing 10% CO(2) and flue gas. The biomass and lipid productivity for Scenedesmus sp. with 10% CO(2) were 217.50 and 20.65 mg L(-1)d(-1) (9% of biomass), while those for B. braunii were 26.55 and 5.51 mg L(-1)d(-1) (21% of biomass). With flue gas, the lipid productivity for Scenedesmus sp. and B. braunii was increased 1.9-fold (39.44 mg L(-1)d(-1)) and 3.7-fold (20.65 mg L(-1)d(-1)), respectively. Oleic acid, a main component of biodiesel, occupied 55% among the fatty acids in B. braunii. Therefore, the present results suggested that Scenedesmus sp. is appropriate for mitigating CO(2), due to its high biomass productivity and C-fixation ability, whereas B. braunii is appropriate for producing biodiesel, due to its high lipid content and oleic acid proportion.

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.

Algae–bacteria interactions: Evolution, ecology and emerging applications

Algae and bacteria have coexisted ever since the early stages of evolution. This coevolution has revolutionized life on earth in many aspects. Algae and bacteria together influence ecosystems as varied as deep seas to lichens and represent all conceivable modes of interactions - from mutualism to parasitism. Several studies have shown that algae and bacteria synergistically affect each others physiology and metabolism, a classic case being algae-roseobacter interaction. These interactions are ubiquitous and define the primary productivity in most ecosystems. In recent years, algae have received much attention for industrial exploitation but their interaction with bacteria is often considered a contamination during commercialization. A few recent studies have shown that bacteria not only enhance algal growth but also help in flocculation, both essential processes in algal biotechnology. Hence, there is a need to understand these interactions from an evolutionary and ecological standpoint, and integrate this understanding for industrial use. Here we reflect on the diversity of such relationships and their associated mechanisms, as well as the habitats that they mutually influence. This review also outlines the role of these interactions in key evolutionary events such as endosymbiosis, besides their ecological role in biogeochemical cycles. Finally, we focus on extending such studies on algal-bacterial interactions to various environmental and bio-technological applications.

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.

Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil

ABSTRACT Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with φ29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

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.