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Exploring the role and characterization of Burkholderia cepacia CD2: a promising eco-friendly microbial fertilizer isolated from long-term chemical fertilizer-free soil

  • HyunWoo Son;Justina Klingaite;Sihyun Park;Jae-Ho Shin
    • Journal of Applied Biological Chemistry
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    • v.66
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    • pp.394-403
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    • 2023
  • In the pursuit of sustainable and environmentally-friendly agricultural practices, we conducted an extensive study on the rhizosphere bacteria inhabiting soils that have been devoid of chemical fertilizers for an extended period exceeding 40 years. Through this investigation, we isolated a total of 80 species of plant growth-promoting rhizosphere bacteria and assessed their potential to enhance plant growth. Among these isolates, Burkholderia cepacia CD2 displayed remarkable plant growth-promoting activity, making it an optimal candidate for further analysis. Burkholderia cepacia CD2 exhibited a range of beneficial characteristics conducive to plant growth, including phosphate solubilization, siderophore production, denitrification, nitrate utilization, and urease activity. These attributes are well-known to positively influence the growth and development of plants. To validate the taxonomic classification of the strain, 16S rRNA gene sequencing confirmed its placement within the Burkholderia genus, providing further insights into its phylogenetic relationship. To delve deeper into the potential mechanisms underlying its plant growth-promoting properties, we sought to confirm the presence of specific genes associated with plant growth promotion in CD2. To achieve this, whole genome sequencing (WGS) was performed by Plasmidsaurus Inc. (USA) utilizing Oxford Nanopore technology (Abingdon, UK). The WGS analysis of the genome of CD2 revealed the existence of a subsystem function, which is thought to be a pivotal factor contributing to improved plant growth. Based on these findings, it can be concluded that Burkholderia cepacia CD2 has the potential to serve as a microbial fertilizer, offering a sustainable alternative to chemical fertilizers.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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Identification of Streptomyces scopuliridis KR-001 and Its Herbicidal Characteristics (Streptomyces scopuliridis KR-001의 분리 동정 및 잡초 방제효과)

  • Lee, Boyoung;Kim, Jae Deok;Kim, Young Sook;Ko, Young Kwan;Yon, Gyu Hwan;Kim, Chang-Jin;Koo, Suk Jin;Choi, Jung Sup
    • Weed & Turfgrass Science
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    • v.2 no.1
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    • pp.38-46
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    • 2013
  • With increasing environmental issues from synthetic chemical herbicides, microbe-originated herbicides could be a fascinating alternative in current agriculture. We isolated Streptomyces strains that produced herbicidal active metabolite(s) against a grass weed Digitaria sanguinalis. According to the result from 16S rDNA sequence comparison with the close strains, the best isolate (Code name MS-80673) was identified as Streptomyces scopuliridis KR-001. The closest type strain was Streptomyces scopuliridis RB72 which was previously reported as a bacteriocin producer. The optimal culture condition of S. scopuliridis KR-001 was $28^{\circ}C$, pH 7.0 and culture period 4 to7 days. Both of soil and foliar application of the crude culture broth concentrate was effective on several troublesome or noxious weed species such as a Sciyos angulatus in a greenhouse and field condition. Phytotoxic symptoms of the culture broth concentrate of S. scopuliridis KR-001 by foliar application were wilting and burndown of leaves, and stems followed by discoloration and finally plant death. In crops such as rice, wheat, barley, hot pepper and tomato, growth inhibition was observed. These results suggest that the new S. scopuliridis KR-001 strain producing herbicidal metabolites may be a new bio-herbicide candidate and/or may provide a new lead molecule for a more efficient herbicide.

Effect of Some Herbal Extracts on Entomopathogenic Nematodes, Silkworm and Ground Beetles (몇 가지 한약재 추출물이 곤충병원성선충과 누에 및 먼지벌레에 미치는 영향)

  • Lee, Dong-Woon;Choi, Hyeon-Cheol;Kim, Tae-Su;Park, Jong-Kyun;Park, Jung-Chan;Yu, Hwang-Bin;Lee, Sang-Myoung;Choo, Ho-Yul
    • Korean journal of applied entomology
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    • v.48 no.3
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    • pp.335-345
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    • 2009
  • Effect of four nematicidal herbal extracts (Daphne genkwa, Eugenia caryophyllata, Quisqualis indica and Zingiber officinale) and 3 acricidal herbal extracts (Pharbitis nil, Xanthium strumarium, and Desmodium caudatum) on entomopathobenic nematodes [Steinernema carpocapsae Pocheon strain (ScP) and Heterorhabditis sp. Gyeongsan strain (HG)], silkworm (Bombyx mori), and ground beetles (Synuchus sp.) were investigated in the laboratory and field. D. genkwa was highly toxic to SCP and HG (100% mortality) at the concentration of 5,000 ppm in X-plate. All the infective juveniles of HG were dead after 3 days by E. caryophyllata and Q. indica. The mortality of ScP and HG was below 10% by D. genkwa, D. caudatum, E. caryophyllata, Q. indica and Z. officinale at the concentration of 1,000 ppm two days after treatment while mortality of HG was 62.8% by D. genkwa at the concentration of 1,000 ppm in X-plate. However, 1,000 ppm had not effect on nematode survival and pathogenicity of ScP in sand column. On the contrary, E. caryophyllata had effect on pathogenicity of HG. Mean number of dead Galleria mellonella larva of HG was 0.5 in E. caryophyllata treatment. Q. indica did not effect silkworm reared on mulberry leaves at the treatment of 1,000 ppm in 10 days after treatment. However, there were 20.0 and 100% mortalities in the treatment of D. genkwa 3 and 10 days after treatment, respectively. The weight of silkworm was low in D. genkwa and did not pupate. The weight of pupa and cocoon were not different in E. caryophyllata, P. nil, Q. indica, X. strumarium and Z. officinale. D. genkwa, E. caryophyllata, P. nil, Q. indica and Z. officinale had no effect on ground beetles, Synuchus sp. in forest soil.

Studies on Identification and Drug Resistance of Atypical Mycobacteria isolated from Patients with Pulmonary Tuberculosis (폐결핵환자에서 비정형항산균의 분리, 동정 및 약제감수성에 관하여)

  • Chung, Dong-Hyun;Kim, Sung-Kwang;Kim, Joo-Deuk
    • Journal of Yeungnam Medical Science
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    • v.1 no.1
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    • pp.49-58
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    • 1984
  • The differential diagnosis of atypical mycobacteriosis caused by atypical mycobacteria (with the exception of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium leprae) which are widly distributed in soil and water, from pulmonary tuberculosis is possible only when atypical mycobacteria are isolated and identified. In this investigation, attempts were made to isolate atypical mycobacteria from persons registered as tuberculosis patients in the Anyang Health Center in Anyang City, Kyungki province, Korea. Biological and biochemical tests were performed for the atypical mycobacteria isolated from these patients, also retrospective analysis of clinical and X-ray findings of the patients with bacteriologically confirmed atypical mycobacteriosis were done. The results can be summarized as follows: 1. 103 strains of mycobacteria were isolated among 334 sputum samples from patients. 2. Among the isolated mycobacteria, 10 strains (9.7%) were found to be a atypical mycobacteria and 93 strains (90.3%) were tubercle bacilli of human type. 3. On the basis of Runyon's grouping of atypical mycobacteria, there were 3 strains (30.0 %) of scotochromogen and nonphotochromogen respectively, 4 strains (40.0%) of rapid grower, and no photochromogen. 4. By biochemical tests, 3 strains of scotochromogen were identified as Mycobacterium scrofulaceum (2 strains) and Mycobacterium szulgai (1 strain) 3 strains of nonphotochromogen were Mycobacterium avium-complex (2 strains) and Mycobacterium terriae (1 strain), and 4 strains of rapid grower were Mycobacterium fortuitum (3 strains) and Mycobacterium chelonei. 5. In drug sensitivity tests, all 10 strains isolated atypical mycobacteria showed resistance to various concentration of INH and SM and low concentration (10mcg, 40mcg and 50mcg) of EB, TH, and CS, and were sensitive to only high concentration (20mcg and 100mcg) of EB, TH, CS, and RFP. 6. In analysis of clical findings by the patients with bacteriologically confirmed atypical mycobacteriosis, it was found that clinical symptoms of these patients appeared not to be mild than those of patients with pulmonary tuberculosis. The patients with atypical mycobacteriosis had been treated for pulmonary tuberculosis for a long time and they showed no improvement.

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Biodegradation of Phenol by Comamonas testosteroni DWB-1-8 Isolated from the Activated Sludge of Textile Wastewater (섬유 폐수 활성 슬러지에서 분리한 Comamonas testosteroni의 생물학적 페놀 분해)

  • Kwon, Hae Jun;Choi, Doo Ho;Kim, Mi Gyeong;Kim, Dong-Hyun;Kim, Young Guk;Yoon, Hyeokjun;Kim, Jong-Guk
    • Journal of Life Science
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    • v.30 no.2
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    • pp.156-161
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    • 2020
  • Since industrialization, the production and utilization of various chemicals has contributed to improving the quality of our lives, but the subsequent discharge of massive waste is inevitable, and environmental pollution is becoming more serious every day. Exposure to chemicals as a result of environmental pollution is having a negative effect on human health and the ecosystem, and cleaning up the polluted environment that can affect our lives is a very important issue. Toxic aromatic compounds have been detected frequently in soil, groundwater, and wastewater because of the extensive use of oil products, and phenol, which is used to produce synthetic resins, textiles, and dyes, is one of the major pollutants, along with insecticides and preservatives. Phenol can cause dyspnea, headache, vomiting, mutation, and carcinogenesis. Phenol-degrading bacterium DWB-1-8 was isolated from the activated sludge of textile wastewater; this strain was identified as Comamonas testosteroni by 16S rRNA gene sequencing. The optimal culture conditions for the cell growth and degradation of phenol were 0.7% K2HPO4, 0.6% NaH2PO4, 0.1% NH4NO3, 0.015% MgSO4·7H2O, 0.001% FeSO4·7H2O, an initial pH of 7, and a temperature of 30℃. The strain was also able to grow by using other toxic compounds, such as benzene, toluene, or xylene (BTX), as the sole source of carbon.

Identification of Antagonistic Bacteria, Pseudomonas aurantiaca YC4963 to Colletotri­chum orbiculare Causing Anthracnose of Cucumber and Production of the Antibiotic Phenazine-l-carboxylic acid (Colletotrichum orbiculare에 대한 길항세균 Pseudomonas aurantiaca YC4963의 분리 동정 및 항균물질 Phenazine-1-carboxylic acid의 생산)

  • Chae Hee-Jung;Kim Rumi;Moon Surk-Sik;Ahn Jong-Woong;Chung Young-Ryun
    • Korean Journal of Microbiology
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    • v.40 no.4
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    • pp.342-347
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    • 2004
  • A bacterial strain YC4963 with antifungal activity against Colletotrichum orbiculare, a causal organism of cucumber anthracnose was isolated from the rhizosphere soil of Siegesbeckia pubescens Makino in Korea. Based on physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bac­terial strain was identified as Pseudomonas aurantiaca. The bacteria also inhibited mycelial growth of several plant fungal pathogens such as Botrytis cinerea, Fusarium oxysporum and Rhizoctonia solani on PDA and 0.1 TSA media. The antifungal activity was found from the culture filtrate of this isolate and the active compound was quantitatively bound to XAD adsorption resin. The antibiotic compound was purified and identified as phenazine-l-carboxylic acid on the basis of combined spectral and chemical analyses data. This is the first report on the production of phenazine-l-carboxylic acid by Pseudomonas aurantiaca.

A Study on the Behaviour of Prebored and Precast Steel Pipe Piles from Full-Scale Field Tests and Class-A and C1 Type Numerical Analyses (현장시험과 Class-A 및 C1 type 수치해석을 통한 강관매입말뚝의 거동에 대한 연구)

  • Kim, Sung-Hee;Jung, Gyoung-Ja;Jeong, Sang-Seom;Jeon, Young-Jin;Kim, Jeong-Sub;Lee, Cheol-Ju
    • Journal of the Korean GEO-environmental Society
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    • v.18 no.7
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    • pp.37-47
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    • 2017
  • In this study, a series of full-scale field tests on prebored and precast steel pipe piles and the corresponding numerical analysis have been conducted in order to study the characteristics of pile load-settlement relations and shear stress transfer at the pile-soil interface. Dynamic pile load tests (EOID and restrike) have been performed on the piles and the estimated design pile loads from EOID and restrike tests were analysed. Class-A type numerical analyses conducted prior to the pile loading tests were 56~105%, 65~121% and 38~142% respectively of those obtained from static load tests. In addition, design loads estimated from the restrike tests indicate increases of 12~60% compared to those estimated in the EOID tests. The EOID tests show large end bearing capacity while the restrike tests demonstrate increased skin friction. When impact energy is insufficient during the restrike tests, the end bearing capacity may be underestimated. It has been found that total pile capacity would be reasonably estimated if skin friction from the restrike tests and end bearing capacity from the EOID are combined. The load-settlement relation measured from the static pile load tests and estimated from the numerical modelling is in general agreement until yielding occurs, after which results from the numerical analyses substantially deviated away from those obtained from the static load tests. The measured pile behaviour from the static load tests shows somewhat similar behaviour of perfectly-elastic plastic materials after yielding with a small increase in the pile load, while the numerical analyses demonstrates a gradual increase in the pile load associated with strain hardening approaching ultimate pile load. It has been discussed that the load-settlement relation mainly depends upon the stiffness of the ground, whilst the shear transfer mechanism depends on shear strength parameters.

A Study on the Production of Yeast Utilizing Ethanol as a Sole Carbon Source (Ethanol 이용 미생물에 의한 단세포 단백질 생산에 관한연구)

  • Lee, Ke-Ho;Ha, Jin-Hong
    • Applied Biological Chemistry
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    • v.16 no.1
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    • pp.1-11
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    • 1973
  • In order to obtain the basic informations on the production of single cell protein from ethanol, 145 yeast strains utilizing ethanol as a sole carbon source were isolated from 32 soil samples in Korea. A yeast strain showing the highest cell yield among the isolated strains was selected and identified. The optimum culture condition, utilization of other carbon sources and the cultural characteristics for the selected yeast, and the chemical analysis of the yeast cell composition, and utilization of ethanol by the selected yeast were investigated. All the culture was carried out in the shaking flasks. The results obtained were as follows: 1. The selected yeast strain was identified as Debaryomyces nicotianae-SNU 72. 2. The optimum composition of the medium for the selected yeast is : Ethanol 40 ml, Urea 0.5 g, Potassium phosphate (dibasic) 0.5 g, Ammoium phosphate (monobasic) 0.15 g, Magnesium sulfate 0.05 g, Calcium chloride 0.01g, Yeast extract 0.005 g, Tap water 1000 ml. 3. The optimum pH was 5.0-5.5, the optimum temperature $30-33^{\circ}C$ and the aerobic state was unimportant. 4. Utilization of methanol, n-propanol, iso-propanol, n-butanol, iso-butanol, tert-amyl alcohol and acetic acid by the selected yeast was very weak. So substitution of the subtrate was thought to be impossible. 5. Studies on the propagation of the yeast cells showed that the lag phase of the yeast cells lasted 16 hours, and the logarithmic growth phase extended 16 to 28 hours. The specific growth rate was about $0.19\;hr^{-1}$ and the doubling time was 3.6 hours during the logarithmic growth phase. 6. As the result of the chemical analysis of the dry yeast cells, the content rate of the crude protein was 55.19 %, the content of others was similar to the average content of the yeast component. 7. After 34 hours cultivation, under the optimum culture condition investigated, the dry cell yield against the amount of the added ethanol was 53.4 % (W/V%), the dry cell yield against the amount of the utilized ethanol was 73.6 % (W/V%), the evaporation rate of ethanol was about 19.1 %.

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A study on the field tests and development of quantitative two-dimensional numerical analysis method for evaluation of effects of umbrella arch method (UAM 효과 평가를 위한 현장실험 및 정량적 2차원 수치해석기법 개발에 관한 연구)

  • Kim, Dae-Young;Lee, Hong-Sung;Chun, Byung-Sik;Jung, Jong-Ju
    • Journal of Korean Tunnelling and Underground Space Association
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    • v.11 no.1
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    • pp.57-70
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    • 2009
  • Considerable advance has been made on research on effect of steel pipe Umbrella Arch Method (UAM) and mechanical reinforcement mechanism through numerical analyses and experiments. Due to long analysis time of three-dimensional analysis and its complexity, un-quantitative two-dimensional analysis is dominantly used in the design and application, where equivalent material properties of UAM reinforced area and ground are used, For this reason, development of reasonable, theoretical, quantitative and easy to use design and analysis method is required. In this study, both field UAM tests and laboratory tests were performed in the residual soil to highly weathered rock; field tests to observe the range of reinforcement, and laboratory tests to investigate the change of material properties between prior to and after UAM reinforcement. It has been observed that the increase in material property of neighboring ground is negligible, and that only stiffness of steel pipe and cement column formed inside the steel pipe and the gap between steel pipe and borehole contributes to ground reinforcement. Based on these results and concept of Convergence Confinement Method (CCM), two dimensional axisymmetric analyses have been performed to obtain the longitudinal displacement profile (LDP) corresponding to arching effect of tunnel face, UAM effect and effect of supports. In addition, modified load distribution method in two dimensional plane-strain analysis has been suggested, in which effect of UAM is transformed to internal pressure and modified load distribution ratios are suggested. Comparison between the modified method and conventional method shows that larger displacement occur in the conventional method than that in the modified method although it may be different depending on ground condition, depth and size of tunnel, types of steel pipe and initial stress state. Consequently, it can be concluded that the effect of UAM as a beam in a longitudinal direction is not considered properly in the conventional method.