• The Plant Pathology Journal
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• v.21 no.2
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• pp.93-98
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• 2005

Molecular ecological studies of microbial communities revealed that only tiny fraction of total microorganisms in nature have been identified and characterized, because the majority of them have not been cultivated. A concept, metagenome, represents the total microbial genome in natural ecosystem consisting of genomes from both culturable microorganisms and viable but non-culturable bacteria. The construction and screening of metagenomic libraries in culturable bacteria constitute a valuable resource for obtaining novel microbial genes and products. Several novel enzymes and antibiotics have been identified from the metagenomic approaches in many different microbial communities. Phenotypic analysis of the introduced unknown genes in culturable bacteria could be an important way for functional genomics of unculturable bacteria. However, estimation of the number of clones required to uncover the microbial diversity from various environments has been almost impossible due to the enormous microbial diversity and various microbial population structure. Massive construction of metagenomic libraries and development of high throughput screening technology should be necessary to obtain valuable microbial resources. This paper presents the recent progress in metagenomic studies including our results and potential of metagenomics in plant pathology and agriculture.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.94-99
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• 2002

The oceans cover about $71\%$ of the Earth's crust and contain nearly 300,000 described species. Free-living bacteria in the sea and symbiotic bacteria of marine invertebrates are proving to be valuable sources of useful bioactive compounds. Marine sponges, in particular, which contain diverse communities of bacteria, produce many classes of compounds that are unique to the marine environment. Uncultured microorganisms are commonly believed to represent $99.9\%$ of the whole microbial community. They have been investigated for the possibility of isolating and over-expressing genes in viable microorganisms. Strict symbiotic species that have been adapted to the host are candidate unculturable species. With the enormous potential for discovery, development, and market value of marine derived compounds, supply of the products is a major limiting factor for further development.

• Journal of Korean Society on Water Environment
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• v.22 no.2
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• pp.193-202
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• 2006

About 99% of microorganisms in the environment are unculturable. However, advances in molecular biology techniques allow for the analysis of living microorganisms in the environment without any cultivation. With the advent of new technologies and the optimization of previous methods, various approaches to studying the analysis of living microorganisms in the environment are expanding the field of microbiology and molecular biology. In particular, real-time PCR provides methods for detecting and quantifying microorganisms in the environment. Through the use of the methods, researchers can study the influence of environmental factors such as nutrients, oxygen status, pH, pollutants, agro-chemicals, moisture and temperature on the performances of environmental processes and some of the mechanisms involved in the responses of cells to their environment. This review will also address information gaps in the analysis of the microorganisms using real-time PCR in the environmental process and possible future research to develop an understanding of microbial activities in the environment.

• Microbiology and Biotechnology Letters
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• v.39 no.4
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• pp.374-381
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• 2011

The beneficial effects of Eisenia fetida on soil properties have been attributed to their interaction with soil microorganisms. The bacterial diversity of the intestinal tract of E. fetida was investigated by culture-dependent and culture-independent methods including denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analyses. In a pure culture, Lysinibacillus fusiformis (51%), Bacillus cereus (30%), Enterobacter aerogenes (21%), and L. sphaericus (15%) were identified as the dominant microorganisms. In the DGGE analyses, B. cereus (15.1%), Enterobacter sp. (13.6%), an uncultured bacterium (13.1%), and B. stearothermophilus (7.8%) were identified as the dominant microorganisms. In the pyrosequencing analyses, Microbacterium soli (26%), B. cereus (10%), M. esteraromaticum (6%), and Frigoribacterium sp. (6%) were identified as the dominant microorganisms. The other strains identified were Aeromonas sp., Pseudomonas sp., Borrelia sp., Cellulosimicrobium sp., Klebsiella sp., and Leifsonia sp. The results illustrate that culture independent methods are better able to detect unculturable microorganisms and a wider range of species, as opposed to isolation by culture dependent methods.

• Journal of Microbiology and Biotechnology
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• v.21 no.9
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• pp.885-892
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• 2011

The bacterial communities in the intestinal tracts of earthworm were investigated by culture-dependent and -independent approaches. In total, 72 and 55 pure cultures were isolated from the intestinal tracts of earthworms under aerobic and anaerobic conditions, respectively. Aerobic bacteria were classified as Aeromonas (40%), Bacillus (37%), Photobacterium (10%), Pseudomonas (7%), and Shewanella (6%). Anaerobic bacteria were classified as Aeromonas (52%), Bacillus (27%), Shewanella (12%), Paenibacillus (5%), Clostridium (2%), and Cellulosimicrobium (2%). The dominant microorganisms were Aeromonas and Bacillus species under both aerobic and anaerobic conditions. In all, 39 DNA fragments were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Aeromonas sp. was the dominant microorganism in feeds, intestinal tracts, and casts of earthworms. The DGGE band intensity of Aeromonas from feeds, intestinal tracts, and casts of earthworms was 12.8%, 14.7%, and 15.1%, respectively. The other strains identified were Bacillus, Clostridium, Enterobacter, Photobacterium, Pseudomonas, Shewanella, Streptomyces, uncultured Chloroflexi bacterium, and uncultured bacterium. These results suggest that PCR-DGGE analysis was more efficient than the culturedependent approach for the investigation of bacterial diversity and the identification of unculturable microorganisms.

• Journal of the Korean Society of Food Science and Nutrition
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• v.42 no.7
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• pp.1115-1124
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• 2013

The present study was conducted to ensure the diversity of domestic solar salt by analyzing the composition and microbiological characteristics of solar salt (from Docho island: DS) and the flower of salt produced in different Korean salt flats (Sinui island: SF, Bigum island: BF, and Docho island: DF). The analyses showed that the moisture content of the three types of flower of salt and solar salt ranged from 10.54~13.82% and NaCl content ranged from 78.81~84.61%. The mineral content of those salts ranged from 3.57~5.51%. The content of insoluble matter in these salts was $0.01{\pm}0.00{\sim}0.05{\pm}0.00%$. The sand content of these salts was $0.01{\pm}0.01{\sim}0.03{\pm}0.01%$. By Hunter's color value analysis, the color of the flower of salt was brighter and whiter than solar salt. The salinity of the flower of salt was a little higher than solar salt as well. The magnesium and potassium ion content of DF was $9,886.72{\pm}104.78mg/kg$ and $2,975.23{\pm}79.73mg/kg$, respectively, which was lower than the content in SF, BF, and DS. The heavy metal content of all salts was acceptable under the Korean Food Sanitation Law. The flower of salt was confirmed to be sweeter and preferable to solar salt. More than 80% of the solar salt crystals were 2~3 mm in size, whereas crystals from the flower of salt were 0.5~2 mm in size. The bacterial diversity of DF and DS were investigated by culture and denaturing gradient gel electrophoresis (DGGE) methods. The number of cultured bacteria in flower of salt was approximately three times more than solar salt. By DGGE analysis, major microbes of DF were Maritimibacter sp., Cupriavidus sp., and unculturable bacteria, and those of DS were Cupriavidus sp., Dunalidella salina and unculturable bacteria. The results of DGGE analysis showed that major microorganisms in solar salts were composed of unidentified and unculturable bacteria and only a few microorganisms were culturable.

• Korean Journal of Microbiology
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• v.45 no.1
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• pp.1-9
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• 2009

Microbes within complex communities show quite different physiology from pure cultured microbes. However, historically the study of microbes has focused on single species in pure culture and most of microbes are unculturable in our labs, so understanding of complex communities lags behind understanding of pure cultured cells. Methodologies including stable isotope probing (SIP), a combination of fluorescence in situ hybridization (FISH) and microautoradiography (MAR), isotope micrarray, and metagenomics have given insights into the uncultivated majority to link phylogenetic and functional information. Here, we review some of the most recent literatures, with an emphasis on methodological improvements to the sensitivity and utilities of these methods to link phylogeny and function in complex microbial communities.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1057-1062
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• 2016

Kimchi is a traditional Korean fermented vegetable food, the production of which involves brining of Korean cabbage, blending with various other ingredients (red pepper powder, garlic, ginger, salt-pickled seafood, etc.), and fermentation. Recently, kimchi has also become popular in the Western world because of its unique taste and beneficial properties such as antioxidant and antimutagenic activities, which are derived from the various raw materials and secondary metabolites of the fermentative microorganisms used during production. Despite these useful activities, analysis of the microbial community present in kimchi has received relatively little attention. The objective of this study was to evaluate the bacterial community structure from the raw materials, additives, and final kimchi product using the culture-independent method. Specifically, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the 16S rRNA partial sequences of the microflora. One primer set for bacteria, 341F^GC-518R, reliably produced amplicons from kimchi and its raw materials, and these bands were clearly separated on a 35-65% denaturing gradient gel. Overall, 117 16S rRNA fragments were identified by PCR-DGGE analysis. Pediococcus pentosaceus, Leuconostoc citreum, Leuconostoc gelidum, and Leuconostoc mesenteroides were the dominant bacteria in kimchi. The other strains identified were Tetragenococcus, Pseudomonas, Weissella, and uncultured bacterium. Comprehensive analysis of these microorganisms could provide a more detailed understanding of the biologically active components of kimchi and help improve its quality. PCR-DGGE analysis can be successfully applied to a fermented food to detect unculturable or other species.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1655-1660
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• 2007

A metagenome is a unique resource to search for novel microbial enzymes from the unculturable microorganisms in soil. A forest soil metagenomic library using a fosmid and soil microbial DNA from Gwangneung forest, Korea, was constructed in Escherichia coli and screened to select lipolytic genes. A total of seven unique lipolytic clones were selected by screening of the 31,000-member forest soil metagenome library based on tributyrin hydrolysis. The ORFs for lipolytic activity were subcloned in a high copy number plasmid by screening the secondary shortgun libraries from the seven clones. Since the lipolytic enzymes were well secreted in E. coli into the culture broth, the lipolytic activity of the subclones was confirmed by the hydrolysis of p-nitrophenyl butyrate using culture supernatant. Deduced amino acid sequence analysis of the identified ORFs for lipolytic activity revealed that 4 genes encode hormone-sensitive lipase (HSL) in lipase family IV. Phylogenetic analysis indicated that 4 proteins were clustered with HSL in the database and other metagenomic HSLs. The other 2 genes and 1 gene encode non-heme peroxidase-like enzymes of lipase family V and a GDSL family esterase/lipase in family II, respectively. The gene for the GDSL enzyme is the first description of the enzyme from metagenomic screening.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1193-1201
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• 2012

The analysis and quantification of ammonia-oxidizing bacteria (AOB) is crucial, as they initiate the biological removal of ammonia-nitrogen from sewage. Previous methods for analyzing the microbial community structure, which involve the plating of samples or culture media over agar plates, have been inadequate because many microorganisms found in a sewage plant are unculturable. In this study, to exclusively detect AOB, the analysis was carried out via denaturing gradient gel electrophoresis using a primer specific to the amoA gene, which is one of the functional genes known as ammonia monooxygenase. An AOB consortium (S1 sample) that could oxidize an unprecedented 100% of ammonia in 24 h was obtained from sewage sludge. In addition, real-time PCR was used to quantify the AOB. Results of the microbial community analysis in terms of carbon utilization ability of samples showed that the aeration tank water sample (S2), influent water sample (S3), and effluent water sample (S4) used all the 31 substrates considered, whereas the AOB consortium (S1) used only Tween 80, D-galacturonic acid, itaconic acid, D-malic acid, and $_L$-serine after 192 h. The largest concentration of AOB was detected in S1 ($7.6{\times}10^6copies/{\mu}l$), followed by S2 ($3.2{\times}10^6copies/{\mu}l$), S4 ($2.8{\times}10^6copies/{\mu}l$), and S3 ($2.4{\times}10^6copies/{\mu}l$).