Hanbyul Lee

A simple method for DNA extraction from marine bacteria that produce extracellular materials

We present a simple method for extracting DNA from the marine bacteria Hahella chejuensis, a Streptomyces sp., and a Cytophaga sp. Previously, DNA purification from these strains was hindered by the presence of extracellular materials. In our extraction method, the marine bacteria are lysed by freezing and grinding in liquid nitrogen, and treated with SDS. The extracted DNA is purified using a phenol/chloroform mixture, and precipitated in isopropanol. The extracted DNA is of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion, genomic DNA blot hybridization, and genomic DNA library construction. We used this method to extract genomic DNA from several other marine bacteria. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from marine bacteria. Furthermore, the low cost of this method makes it attractive for large-scale studies.

Biotechnological procedures to select white rot fungi for the degradation of PAHs

White rot fungi are essential in forest ecology and are deeply involved in wood decomposition and the biodegradation of various xenobiotics. The fungal ligninolytic enzymes involved in these processes have recently become the focus of much attention for their possible biotechnological applications. Successful bioremediation requires the selection of species with desirable characteristics. In this study, 150 taxonomically and physiologically diverse white rot fungi, including 55 species, were investigated for their performance in a variety of biotechnological procedures, such as dye decolorization, gallic acid reaction, ligninolytic enzymes, and tolerance to four PAHs, phenanthrene, anthracene, fluoranthene, and pyrene. Among these fungi, six isolates showed the highest (>90%) tolerance to both individual PAH and mixed PAHs. And six isolates oxidized gallic acid with dark brown color and they rapidly decolorized RBBR within ten days. These fungi revealed various profiles when evaluated for their biotechnological performance to compare the capability of degradation of PAHs between two groups selected. As the results demonstrated the six best species selected from gallic acid more greatly degraded four PAHs than the other isolates selected via tolerance test. It provided that gallic acid reaction test can be performed to rank the fungi by their ability to degrade the PAHs. Most of all, Peniophora incarnata KUC8836 and Phlebia brevispora KUC9033 significantly degraded the four PAHs and can be considered prime candidates for the degradation of xenobiotic compounds in environmental settings.

Investigation of Marine-Derived Fungal Diversity and Their Exploitable Biological Activities

Marine fungi are potential producers of bioactive compounds that may have pharmacological and medicinal applications. Fungi were cultured from marine brown algae and identified using multiple target genes to confirm phylogenetic placement. These target genes included the internal transcribed spacer (ITS), the nuclear large subunit (LSU), and the β-tubulin region. Various biological activities of marine-derived fungi were evaluated, including their antifungal, antioxidant and cellulolytic enzyme activities. As a result, a total of 50 fungi was isolated from the brown algae Sargassum sp. Among the 50 isolated fungi, Corollospora angusta was the dominant species in this study. The genus Arthrinium showed a relatively strong antifungal activity to all of the target plant pathogenic fungi. In particular, Arthrinium saccharicola KUC21221 showed high radical scavenging activity and the highest activities in terms of filter paper units (0.39 U/mL), endoglucanase activity (0.38 U/mL), and β-glucosidase activity (1.04 U/mL).

Diversity of Wood-Inhabiting Polyporoid and Corticioid Fungi in Odaesan National Park, Korea

Abstract Polyporoid and corticioid fungi are among the most important wood-decay fungi. Not only do they contribute to nutrient cycling by decomposing wood debris, but they are also valuable sources for natural products. Polyporoid and corticioid wood-inhabiting fungi were investigated in Odaesan National Park. Fruit bodies were collected and identified based on morphological and molecular analyses using 28S and internal transcribed spacer regions of DNA sequences. As a result, a total of 149 species, 69 genera, 22 families, and 11 orders were recognized. Half (74 species) of the species were polypores, and the other half (75 species) were corticioid fungi. Most of the species belonged to Polyporales (92 species) followed by Hymenochaetales (33 species) and Russulales (11 species). At the genus level, a high number of species was observed from Steccherinum, Hyphodontia, Phanerochaete, Postia, and Trametes. Concerning distribution, almost all the species could be found below 1,000 m, and only 20% of the species were observed from above 1,000 m. Stereum subtomentosum, Trametes versicolor, T. hirsuta, T. pubescens, Bjerkandera adusta, and Ganoderma applanatum had wide distribution areas. Deciduous wood was the preferred substrate for the collected species. Sixty-three species were new to this region, and 21 species were new to Korea, of which 17 species were described and illustrated.

Comparison of the Diversity of Basidiomycetes from Dead Wood of the Manchurian fir (Abies holophylla) as Evaluated by Fruiting Body Collection, Mycelial Isolation, and 454 Sequencing

In this study, three different methods (fruiting body collection, mycelial isolation, and 454 sequencing) were implemented to determine the diversity of wood-inhabiting basidiomycetes from dead Manchurian fir (Abies holophylla). The three methods recovered similar species richness (26 species from fruiting bodies, 32 species from mycelia, and 32 species from 454 sequencing), but Fisher’s alpha, Shannon-Wiener, Simpson’s diversity indices of fungal communities indicated fruiting body collection and mycelial isolation displayed higher diversity compared with 454 sequencing. In total, 75 wood-inhabiting basidiomycetes were detected. The most frequently observed species were Heterobasidion orientale (fruiting body collection), Bjerkandera adusta (mycelial isolation), and Trichaptum fusco-violaceum (454 sequencing). Only two species, Hymenochaete yasudae and Hypochnicium karstenii, were detected by all three methods. This result indicated that Manchurian fir harbors a diverse basidiomycetous fungal community and for complete estimation of fungal diversity, multiple methods should be used. Further studies are required to understand their ecology in the context of forest ecosystems.

Screening for xylanase and β-xylosidase production from wood-inhabiting Penicillium strains for potential use in biotechnological applications.

Abstract Experiments were performed to find potential sources for enzyme production for the pulp and paper industry and for biological ethanol production by screening the cellulase, xylanase and β-xylosidase activities of 36 species of Penicillium isolated from various wood materials in Korea. Rice straw powder (RiceP), birchwood xylan (BirchX), and beechwood xylan (BeechX) were supplied as individual carbon sources for the Penicillium species. All Penicillium species tested in this study showed little cellulase activity, but some species exhibited remarkably high xylanase and β-xylosidase activities, as determined by a filter paper assay. P. oxalicum showed the greatest xylanase activity on RiceP (158.70 U ml-1). On the other hand, P. brevicompactum produced the highest active β-xylosidase on BirchX (6.25 U ml-1).

Miniaturized enzyme production and development of micro-assays for cellulolytic and xylanolytic enzymes

Miniaturized fungal cultivation and enzyme assays were developed. Cultivation for enzyme production was performed in 50mL conical tubes. In addition, the miniaturized enzyme assays reduced the amount of enzymes and reagents necessary. These procedures can be adopted in screening fungi to determine if they produce cellulolytic and xylanolytic enzymes.

Microbial community composition and PAHs removal potential of indigenous bacteria in oil contaminated sediment of Taean coast, Korea

The tidal flats near Sinduri beach in Taean, Korea, have been severely contaminated by heavy crude oils due to the Koreas worst oil spill accident, say the Hebei Spirit Oil Spill, in 2007. Crude oil compounds, including polycyclic aromatic hydrocarbons (PAHs), pose significant environmental damages due to their wide distribution, persistence, high toxicity, mutagenicity, and carcinogenicity. Microbial community of Sinduri beach sediments samples was analyzed by metagenomic data with 16S rRNA gene amplicons. Three phyla (Proteobacteria, Firmicutes, and Bacteroidetes) accounted for approximately ≥93.0% of the total phyla based on metagenomic analysis. Proteobacteria was the dominant phylum in Sinduri beach sediments. Cultivable bacteria were isolated from PAH-enriched cultures, and bacterial diversity was investigated through performing culture characterization followed by molecular biology methods. Sixty-seven isolates were obtained, comprising representatives of Actinobacteria, Firmicutes, α- and γ-Proteobacteria, and Bacteroidetes. PAH catabolism genes, such as naphthalene dioxygenase (NDO) and aromatic ring hydroxylating dioxygenase (ARHDO), were used as genetic markers to assess biodegradation of PAHs in the cultivable bacteria. The ability to degrade PAHs was demonstrated by monitoring the removal of PAHs using a gas chromatography mass spectrometer. Overall, various PAH-degrading bacteria were widely present in Sinduri beach sediments and generally reflected the restored microbial community. Among them, Cobetia marina, Rhodococcus soli, and Pseudoalteromonas agarivorans were found to be significant in degradation of PAHs. This large collection of PAH-degrading strains represents a valuable resource for studies investigating mechanisms of PAH degradation and bioremediation in oil contaminated coastal environment, elsewhere.

Isolation and Analysis of the Enzymatic Properties of Thermophilic Fungi from Compost

Abstract To the best of our knowledge, this is the first report on thermophilic fungi isolated in Korea. Three species of thermophiles were isolated from compost and were identified as Myriococcum thermophilum, Thermoascus aurantiacus, and Thermomyces lanuginosus. They can grow at temperatures above 50°C and produce high levels of cellulolytic and xylanolytic enzymes at high temperatures. Notably, the considerable thermostability of the endo-glucanase produced by T. aurantiacus has made the fungus an attractive source of industrial enzymes.

Wood decay fungi in South Korea: Polypores from seoul

Abstract In Seoul, a majority of plant communities have undergone significant changes over the last few decades; however, how wood decay fungi have responded and adapted to the changes in vegetation remains unknown. Through an ongoing investigation of Korean indigenous fungi, ca. 300 specimens with poroid basidiocarp were collected in Seoul during 2008~2012. Morphological examination and molecular analysis using the internal transcribed spacer and nuclear large subunit ribosomal DNA region sequences helped identify 38 species belonging to 28 genera, 10 families, and 5 orders in this area. Among them, three polypores, Abundisporus pubertatis, Coriolopsis strumosa, and Perenniporia maackiae were found to be new to South Korea.