• Korean Journal of Microbiology
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• v.47 no.3
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• pp.179-187
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• 2011

This review describes the characteristics of various unculturable soil bacteria, successfully-cultivating examples of those bacteria, and the diverse factors to be considered for successful cultivation. Most importantly, the selection of proper media is very important because unculturable bacteria demand different types of nutrients at various concentrations of substrates, nitrogens and phosphorus. To develop a new medium to successfully culture unculturable bacteria from soil, molecular ecological studies should be combined together. The inoculum size on a plate is also important: less than 50 bacterial cells are recommended to be plated on a single culture plate. The environmental factors such as pH and salt concentration of the medium need to be adjusted as similar as possible to mimic the original soil environments, and the trial of the various temperatures and extended period of cultivation are better. Since one cannot simply tell about which one was unculturable among a great number of colonies grown on a newly developed medium, some suitable detection methods and fast identification methods are required. Many soil bacteria live with cooperation one another in their communities, so that enrichment such as coculture of using other bacterial metabolites and subsequent pure cultures can also guarantee successful cultivation of the previously uncultured bacteria in soil. Here, this review will discuss for the future perspectives to culture the unculturable soil bacteria.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.6
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• pp.877-884
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• 2004

Although gut microbial functions have been analyzed through cultivation of isolated microbes, molecular analysis without cultivation is becoming a popular approach in recent years. Gene cloning studies have partially revealed the mechanisms involved in fiber digestion of individual microbe. The molecular approach finally made it possible to analyze full genomes of the representative rumen cellulolytic bacteria Fibrobacter and Ruminococcus. The coming database may contain useful information such as regulation of gene expression relating to fiber digestion. Meanwhile, unculturable bacteria are still poorly characterized, even though they are main constituents of gut microbial ecosystem. The molecular analysis is essential to initiating the studies on these unculturable bacteria. The studies dealing with rumen and large intestine are revealing considerable complexity of the microbial ecosystems with many undescribed bacteria. These bacteria are being highlighted as possibly functional members contributing to feed digestion. Manipulation of gut bacteria and gut ecology for improving animal production is still at challenging stage. Bacteria newly introduced in the rumen, whether they are genetically modified or not, suffer from poor survival. In one of these attempts, Butyrivibrio fibrisolvens expressing a foreign dehalogenase was successfully established in sheep rumen to prevent fluoroacetate poisoning. This expands choice of forages in tropics, since many tropic plants are known to contain the toxic fluoroacetate. This example may promise the possible application of molecular breeding of gut bacteria to the host animals with significance in their health and nutrition. When inoculation strategies for such foreign bacteria are considered, it is obvious that we should have more detailed information of the gut microbial ecology.

• 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.

• Proceedings of the Korean Society of Life Science Conference
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• 2007.10a
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• pp.103.1-103.1
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• 2007

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• 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.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.1013-1021
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• 2005

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. The gene encoding an extracellular $\alpha$-amylase from a genomic DNA of cow rumen was cloned in Escherichia coli DH5$\alpha$ and sequenced. The $\alpha$-amylase (amyA) gene was 1,893 bp in length, encoding a protein of 631 amino acid residues with calculated molecular weight of 70,734 Da. The molecular weight of the enzyme was estimated to be about 71,000 Da by active staining of a SDS-PACE. The enzyme was 21 to $59\%$ sequence identical with other amyloyltic enzymes. The AmyA was optimally active at pH 6.0 and $40\%$. The AmyA had a calculated pI of 5.87. AmyA expressed in E. coli DH5$\alpha$ was enhanced in the presence of $Mg^{2+}$ (20 mM) and $Ca^{2+}$ (30 mM) and inhibited in the presence of $Fe^{2+}$ and $Cu^{2+}$. The origin of amyA gene could not be confirmed by PCR using internal primer of amyA gene from extracted genomic DNA of 49 species rumen culturable bacteria so far. An amyh is supposed to obtained from unculturable rumen bacterium in cow rumen environment.

• 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.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.108.1-108
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• 2003

Soil metagenome is untapped total microbial genome including that of the majority of unculturable bacteria present in soil. We constructed soil metagenomic library in Escherichia coli using DNA directly extracted from two different soils, pine tree rhizosphere soil and forest topsoil. Metagenomic libraries constructed from pine tree rhizosphere soil and forest topsoil consisted of approximately 33,700 clones and 112,000 clones with average insert DNA size of 35-kb, respectively. Subsequently, we screened the libraries to select clones with antimicrobial activities against Saccharomyces cerevisiae and Agrobacterium tumefaciens using double agar layer method. So far, we have a clone active against S. cerevisiae and a clone active against A. tumefaciens from the forest topsoil library. In vitro mutagenesis and DNA sequence analysis of the antifungal clone revealed the genes involved in the biosynthesis of antimicrobial secondary metabolite. Metagenomic libraries constructed in this study would be subject to search for diverse genetic resources related with useful microbial products.

• 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 ($2.4{\times}10^6copies/{\mu}l$).