• Journal of Korean Society of Environmental Engineers
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• v.29 no.8
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• pp.895-903
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• 2007

As for the increasing demanding on the development of direct-ecological landfill monitoring methods, it is needed for critically defining the condition of landfills and their influence on the environment, quantifying the amount of enzymes and bacteria mainly concerned with biochemical reaction in the landfills. This study was thus conducted to understand the fates of contaminants in association with groundwater quality parameters. For the study, groundwater was seasonally sampled from four closed unsanitary landfills(i.e. Cheonan(C), Wonju(W), Nonsan(N), Pyeongtaek(P) sites) in which microbial diversity was simultaneously obtained by 16S rDNA methods. Subsequently, a number of primer sets were prepared for quantifying the specific gene of representative bacteria and the gene of encoding enzymes dominantly found in the landfills. The relationship between water quality parameters and gene quantification were compared based on correlation factors. Correlation between DSR(Sulfate reduction bacteria) gene and BOD(Biochemical Oxygen Demand) was greater than 0.8 while NSR(Nitrification bacteria-Nitrospira sp.) gene and nitrate were related more than 0.9. A stabilization indicator(BOD/COD) and MTOT(Methane Oxidation bacteria), MCR(Methyl coenzyme M reductase), Dde(Dechloromonas denitrificans) genes were correlated over 0.8, but ferric iron and Fli(Ferribacterium limineticm) gene were at the lowest of 0.7. For MTOT, it was at the highest related at 100% over BOD/COD. In addition, anaerobic genes(i.e., nirS-Nitrite reductase, MCR. Dde, DSR) and DO were also related more than 0.8, which showing anaerobic reactions generally dependant upon DO. As demonstrated in the study, molecular biological investigation and water quality parameters are highly co-linked, so that quantitative real-time PCR could be cooperatively used for assessing landfill stabilization in association with the conventional monitoring parameters.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.2
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• pp.241-248
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• 2019

Objective: To isolate and identify new methanogens from the rumen of Holstein steers in Korea. Methods: Representative rumen contents were obtained from three ruminally cannulated Holstein steers ($793{\pm}8kg$). Pre-reduced media were used for the growth and isolation of methanogens. Optimum growth temperature, pH, and sodium chloride (NaCl) concentration as well as substrate utilization and antibiotic tolerance were investigated to determine the physiological characteristics of the isolated strain. Furthermore, the isolate was microscopically studied for its morphology. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification. Results: One strain designated as KOR-2 was isolated and found to be a non-motile irregular coccus with a diameter of 0.2 to $0.5{\mu}m$. KOR-2 utilized $H_2+CO_2$ and formate but was unable to metabolize acetate, methanol, trimethylamine, 2-propanol, and isobutanol for growth and methane production. The optimum temperature and pH for the growth of KOR-2 were $38^{\circ}C$ and 6.8 to 7.0, respectively, while the optimum NaCl concentration essential for KOR-2 growth was 1.0% (w/v). KOR-2 tolerated ampicillin, penicillin G, kanamycin, spectromycin, and tetracycline. In contrast, the cell growth was inhibited by chloramphenicol. Phylogenetic analysis of 16S rRNA and mcrA genes revealed the relatedness between KOR-2 and Methanoculleus bourgensis. Conclusion: Based on the physiological and phylogenetic characteristics, KOR-2 was thought to be a new strain within the genus Methanoculleus and named Methanoculleus bourgensis KOR-2.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.8
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• pp.1088-1097
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• 2014

The study aimed to investigate the effects of gynosaponin on in vitro methanogenesis under different forage-concentrate ratios (F:C ratios). Experiment was conducted with two kinds of F:C ratios (F:C = 7:3 and F:C = 3:7) and gynosaponin addition (0 mg and 16 mg) in a $2{\times}2$ double factorial design. In the presence of gynosaponin, methane production and acetate concentration were significantly decreased, whereas concentration of propionate tended to be increased resulting in a significant reduction (p<0.05) of acetate:propionate ratio (A:P ratio), in high-forage substrate. Gynosaponin treatment increased (p<0.05) the butyrate concentration in both F:C ratios. Denaturing gradient gel electrophoresis (DGGE) analysis showed there was no apparent shift in the composition of total bacteria, protozoa and methanogens after treated by gynosaponin under both F:C ratios. The real-time polymerase chain reaction (PCR) analysis indicated that variable F:C ratios significantly affected the abundances of Fibrobacter succinogenes, Rumninococcus flavefaciens, total fungi and counts of protozoa (p<0.05), but did not affect the mcrA gene copies of methanogens and abundance of total bacteria. Counts of protozoa and abundance of F.succinogenes were decreased significantly (p<0.05), whereas mcrA gene copies of methanogens were decreased slightly (p<0.10) in high-forage substrate after treated by gynosaponin. However, gynosaponin treatment under high-concentrate level did not affect the methanogenesis, fermentation characteristics and tested microbes. Accordingly, overall results suggested that gynosaponin supplementation reduced the in vitro methanogenesis and improved rumen fermentation under highforage condition by changing the abundances of related rumen microbes.

• Journal of Korean Society of Environmental Engineers
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• v.28 no.2
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• pp.128-136
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• 2006

This study was conducted for developing the stability parameter in uncontrolled landfill by using a biomolecular investigation on the microbial community growing through leachate plume. Landfill J(which is in Cheonan) and landfill T(which is in Wonju) were chosen for this study among a total of 244 closed uncontrolled landfills. It addressed the genetic diversity of the microbial community in the leachate by 165 rDNA gene cloning using PCR and compared quantitative analysis of denitrifiers and methanotrophs with the conventional water quality parameters. From the BLAST search, genes of 47.6% in landfill J, and 32.5% in landfill T, respectively, showed more than 97% of the similarity where Proteobacteria phylum was most significantly observed. It showed that the numbers of denitrification genes, i.e. nirS gene and cnorB gene in the J site are 7 and 4 times higher than those in T site, which is well reflecting from a difference of site closure showing 7 and 13 years after being closed, respectively. In addition, the quantitative analysis on methane formation gene showed that J1 spot immediately bordering with the sources has the greatest number of methane formation bacteria, and it was decreased rapidly according to distribute toward the outer boundary of landfill. The comparative investigation between the number of genes, i.e. nirS gene, cnorB gene and MCR gene, md the conventional monitoring parameters, i.e. TOC, $NH_3-N,\;NO_3-N,\;NO_2-N,\;Cl^-$, alkalinity, addressed that more than 99% of the correlation was observed except for the $NO_3-N$. It was concluded that biomolecular investigation was well consistent with the conventional monitoring parameters to interpret their influences and stability made by leachate plume formed in downgradient around the uncontrolled sites.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1464-1472
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• 2014

The microbial community structures of two continuous stirred tank reactors digesting turkey manure with pine wood shavings as well as chicken and swine manure were investigated. The reactor fed with chicken/swine wastes displayed the highest organic acids concentration (up to 15.2 g/l) and ammonia concentration (up to 3.7 g/l ammonium nitrogen) and generated a higher biogas yield (up to $366ml/g_{VS}$) compared with the reactor supplied with turkey wastes (1.5-1.8 g/l of organic acids and 1.6-1.7 g/l of ammonium levels; biogas yield was up to $195ml/g_{VS}$). The microbial community diversity was assessed using both sequencing and profiling terminal restriction fragment length polymorphisms of 16S rRNA genes. Additionally, methanogens were analyzed using methyl coenzyme M reductase alpha subunit (mcrA) genes. The bacterial community was dominated by members of unclassified Clostridiales with the prevalence of specific clostridial phylotypes in each reactor, indicating the effect of the substrate type on the community structure. Of the methanogenic archaea, methanogens of the genus Methanosarcina were found in high proportions in both reactors with specific methanosarcinas in each reactor, whereas the strict hydrogenotrophic methanogens of Methanoculleus sp. were found at significant levels only in the reactor fed with chicken/swine manure (based on the analyses of 16S rRNA gene). This suggests that among methanogenic archaea, Methanosarcina species which have different metabolic capabilities, including aceticlastic and hydrogenotrophic methanogenesis, were mainly involved in anaerobic digestion of turkey wastes.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.319-325
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• 2010

The purpose of this study is to investigate the soil compositions, methane production, the number of methanogens, and the community structure of methanogens in rice paddy soils and dry field farming soils in the summer and autumn seasons. As a result of the analysis of soil compositions, any regular tendency to increase or decrease has not been found in most soil samples due to the change of seasons. It has also been found that more methanogens exist in the rice paddy soil that utilize organic farming practices and emptiness farming practices than in the dry field farming soil. The fewer numbers of methanogens utilizing the acetate have been found than those of the methanogens utilizing the hydrogen or the formate. In an experiment of methane production, the methanes increased for two weeks when the acetate was added, but they continued to increase for seven weeks more when the formate and the hydrogen were added. In the phylogenetic analysis using the mcrA gene, the methanogens had diverse clusters in the rice paddy soil, whereas the methanogens were concentrated only in a few clusters in the dry field farming soil.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1198-1205
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• 2017

Objective: An experiment was conducted to isolate and identify new methanogens in Korea from an anaerobic digester that uses pig slurry. Methods: An anaerobic digestate sample was collected from an anaerobic digester using pig slurry. Pre-reduced media were used for the growth and isolation of methanogens. Growth temperature range, pH range, NaCl concentration range, substrate utilization, and antibiotic tolerance were investigated to determine the physiological characteristics of isolated methanogens. The isolates were also examined microscopically for their morphology and Gram-stained. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification purpose. Results: Four strains, designated KOR-3, -4, -5, and -6, were isolated and were non-motile, irregular coccoid, and 0.5 to $1.5{\mu}m$ in diameter. Moreover, the cell walls of isolated strains were Gram-negative. KOR-3 and KOR-4 strains used acetate for methane production but did not use $H_2+CO_2$, formate, or methanol as a growth substrate KOR-5 and KOR-6 strains utilized acetate, methanol, and trimethylamine for methanogenesis but did not use $H_2+CO_2$ or formate as a growth substrate. The optimum temperature and pH for growth of four strains were $39^{\circ}C$ and 6.8 to 7.2, respectively. The optimum concentration of NaCl for growth of KOR-3, KOR-5, and KOR-6 were 1.0% (w/v). The optimum NaCl concentration for KOR-4 was 0.5% (w/v). All of the strains tolerated ampicillin, penicillin G, kanamycin, streptomycin, and tetracycline; however, chloramphenicol inhibited cell growth. Phylogenetic analysis of 16S rRNA and mcrA genes demonstrated that strains KOR-3, -4, -5, and -6 are related to Methanosarcina mazei (M. mazei, 99% sequence similarity). Conclusion: On the basis of physiological and phylogenetic characteristics, strains KOR-3, -4, -5, and -6 are proposed to be new strains within the genus Methanosarcina, named M. mazei KOR-3, -4, -5, and -6.

• Journal of Dairy Science and Biotechnology
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• v.39 no.1
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• pp.1-8
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• 2021

The surroundings of livestock farms, including dairy farms, are known to be a major source of development and transmission of antibiotic-resistant bacteria. To control antibioticresistant bacteria in the livestock breeding environment, farms have installed livestock wastewater treatment facilities to treat wastewater before discharging the final effluent in nearby rivers or streams. These facilities have been known to serve as hotspots for inter-bacterial antibiotic-resistance gene transfer and extensively antibiotic-resistant bacteria, owing to the accumulation of various antibiotic-resistant bacteria from the livestock breeding environment. This review discusses antibiotic usage in livestock farming, including dairy farms, livestock wastewater treatment plants as hotspots for antibiotic resistant bacteria, and nonenteric gram-negative bacteria from wastewater treatment plants, and previous findings in literature.

• Korean Journal of Microbiology
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• v.45 no.1
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• pp.17-25
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• 2009

We studied soil components, methane production, the number of methanogens, and T-RFLP patterns dependent on agricultural methods with the change of seasons. There is no regular increase or decrease tendency of the most soil components followed by sampling period. And the water content in soil was higher in October than May. Also a lot of methanogens existed in soil, and acetotrophs were relatively of smaller number than hydogenotrophs and formate utilizing methanogens using MPN (most probable number) enumeration. In the experiment using the formate, it was used from the first week, and only a minute amount was detecte after four weeks. However in the acetate, it was increased until the third week, and after that was consumed. And there was higher methane production for all soil samples which administered with the hydrogen spike. The activity of methanogens was higher in the organic and low-agrichemical agricultural method samples, and the organic agricultural method had high methanogen activity among the other samples. A result of T-RFLP pattern of mcrA gene digested with Sau96I, methanogen community have a little relation with agricultural methods and seasons. This results also agreed to no critical difference the soil components dependent on agricultural methods, but some analytical data have a positive relationship with a agricultural methods. Therefor we could concluded that the comparison study of community for soil bacteria sufficiently could be useful for the microbiological indicator.

• Korean Journal of Microbiology
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• v.50 no.3
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• pp.191-200
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• 2014

Gas hydrates play a significant role in the global carbon cycle and climate change because methane, a greenhouse gas, can be released from the dissociation of gas hydrate. Anaerobic oxidation of methane (AOM) is an important process that consumes more than 90% of the methane released into the hydrosphere and atmosphere. In this study, the microbial community associated with the methane gas hydrate sediment in the Ulleung basin, East Sea of Korea (UBGH) was analyzed by phylogenetic analysis of the mcrA and 16S rRNA gene libraries. A vertical stratification of the dominating anaerobic methane oxidizer (ANME)-1 group was observed at the surface and the sulfate methane transition zone (SMTZ). The ANME-2c group was found to be dominant in the high methane layer. The archaea of marine benthic group B, which is commonly observed in the AOM region, accounted for more than 50% of the identifications in all sediments. Nitrate reducing bacteria were predominant at SMTZ (Halomonas: 56.5%) and high methane layer (Achromobacter: 52.6%), while sulfate reducing bacteria were not found in UBGH sediments. These results suggest that the AOM process may be carried out by a syntrophic consortium of ANME and nitrate reducing bacteria in the gas hydrates of the Ulleung Basin of the East Sea.