• Title/Summary/Keyword: $CaCrO_4$

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The Treatment of Night Soil using Bacillus sp. (Bacillus sp.를 이용한 분뇨처리)

  • 염혜경;이은숙;이병헌;이민규;정일호;김중균
    • Journal of Life Science
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    • v.12 no.6
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    • pp.700-707
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    • 2002
  • To study the characteristics of organic and nutrient removal by Bacillus species at high COD concentration of influent, three lab-scale batch reactors(R1, R2, R3), each of which has different substrate composition, were operated. More than 95% of $NH_4^+$-N and $COD_{cr}$, concentrations were removed under an aerobic condition, and their removal efficiencies were found to be 22.6 and 90.5%(R1), 23.9 and 65.8%(R2), 30.2 and 86.4%(R3), respectively. The removal efficiency of $NH_4^+$-N was high when an enough amount of $NO_3^{-}$-N was supplied, and that of $COD_{cr}$. was low when a high concentration of initial $NO_2^{-}$-N was added. The amount of carbon utilized in denitrification was a little. In all reactors,$NO_3^{-}$-N was removed under an anoxic condition, but in the R3 reactor, 10% of $NO_3^{-}$-N could be removed even undo, an aerobic condition. The removal efficiencies of TN and TP were 41.8 and 49.5%(R1), 40.1 and 35.8%(R2), 47.0 and 57.6%(R3), respectively. Alkalinities destructed under an aerobic condition for each reactor were 4.96, 5.41 and 3.93 mg/L (as $CaCO_3$) per each gram of $NH_4^+$-N oxidized, respectively, while 3.06, 3.17 and 2.60 mg/L (as $CaCO_3$) of alkalinities were produced for each gram of ,$NO_3^{-}$-N reduced to $N_2$. The SOUR were found to be 38.5, 52.7 and 42.0 mg $O_2$/g MLSS/hr, which indicated that Bacillus sp. had a higher cell activity than activated sludge. The OLR and sludge production were estimated to be 0.69 and 0.28(Rl), 0.77 and 0.20(R2), 0.61 kg COD/$m^3$/day and 0.25 kg MLSS/kg COD(R3), respectively. From the N-balance, the highest percentage(40.9%) of nitrogen lost to $N_2$ was obtained in the R3 reactor. From all the results, the possibility of aerobic denitrification Bacillus sp. has been shown and the B3 process seemed to have two advantages: a little amount of carbon was required in denitrification and not much amount of alkalinity was destructed under an aerobic condition.

Composting of Compostable Wet Household Wastes Using Waste Newspapers as Humidity Amendment (수분개선제로 폐신문지를 이용한 가정쓰레기의 퇴비화)

  • Yun, Eun-Jin;Park, Ju-Won;Seo, Jeoung-Yoon
    • Korean Journal of Environmental Agriculture
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    • v.19 no.1
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    • pp.32-37
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    • 2000
  • Because the household garbage had high water contents, it is difficult to compost it without an additive. Therefore, this study was performed to investigate possibility of using the waste newspapers as a humidity conditioner for the household garbage composting. The maximum temperature was $66.0^{\circ}C$ in spring, $69.2^{\circ}C$ in summer, $60.9^{\circ}C$ in fall and $56.0^{\circ}C$ in winter for composting periods. The seasonal pH value reached around 8.5 after 1 week and then repeated fluctuation at the narrow range in spring and fall, while it was stabilized at the range of $8{\sim}9$ after increasing to 8.5 after 1 week in winter. The water content was reduced little in winter, while decreased significantly in the other seasons. The water content after 8 weeks was 22.2% in spring, 47.6% in summer, 25.5% in fall and 72.5% in winter. The mass was reduced rapidly during the first week of each season, but it did not show much decrease. The volume reduced after 8 weeks to 59%, 32%, 27%, and 34% in spring, summer, fall and winter respectively. Organic matter content decreased over the four seasons. Nitrogen contents were in the range of 0.7% to 2.2% during the four seasons. The contents of inorganic compounds based on dry matyter were in the range of $0.94{\sim}2.59%\;P_2O_5$, $1.23{\sim}1.87%\;CaO$, $0.37{\sim}0.46%\;MgO$, $0.55{\sim}1.98%\;K_2O$. Concentration of heavy metals(Hg, Cd, Pb, Cu, Cr, Zn, As) based on dry matter were less than the limiting value of the by-product compost.

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Changes in Chemical Properties of Paddy Field Soils as Influenced by Regional Topography in Jeonbuk Province (지형특성에 따른 전북지역 논토양 화학성 변화)

  • Ahn, Byung-Koo;Lee, Jin-Ho;Kim, Kab-Cheol;Kim, Hyung-Gook;Jeong, Seong-Soo;Jeon, Hye-Won;Zhang, Yong-Sun
    • Korean Journal of Soil Science and Fertilizer
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    • v.45 no.3
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    • pp.393-398
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    • 2012
  • We investigated the changes in chemical properties of paddy field soils at 300 different sampling sites containing 4 topography in Jeonbuk province, Korea. The soil samples were collected 43.0% from local valley and fans, 39.3% from fluvio-marine deposits, 15.0% from alluvial plains, and 2.7% from diluvium sites. The optimal values of soil properties in the total soil samples were as follows: 65.3% of total samples in soil pH value, 48.3% of total samples in cation exchange capacity (CEC) value, and 22.3% of total samples in available phosphorus content, whereas the deficient values of soil properties were 63.3% of total samples in soil organic matter (SOM) content, 75.7% of total samples in available silicate content, and 61.3%, 51.0%, and 59.3% of total samples in exchangeable $K^+$, $Ca^{2+}$, and $Mg^{2+}$ concentrations, respectively. There were different soil types in the paddy fields: that is, 34.4% immature paddy and 33.6% sandy paddy in the local valley and fans, 57.8% sandy paddy in the alluvial plains, 47.4% normal paddy in the fluvio-marine deposits, and 75.7% immature paddy in the diluvium. Soil textures were also different: 53.5% loam in the local valley and fans, 37.8% sandy loam in the alluvial plains, and 55.1% silty loam in the fluvio-marine deposits. Soil pH and SOM contents were not different among the different topographical sampling sites. However, the mean value of available phosphorus content, 224 mg $kg^{-1}$, was exceeded optimal values in the diluvium. The contents of exchangeable cations were optimal in all the sites, except exchangeable $Ca^{2+}$ contents in the local valley and fans. The contents of available silicate ranged between 112 and 127 mg $kg^{-1}$ in all the sites, which were lower than optimal value. In addition, soil pH values were proportionally correlated to the order of available silicate, exchangeable $Ca^{2+}$, $Mg^{2+}$, $Na^+$, CEC, and exchangeable $K^+$. The contents of SOM were proportionally correlated to the order of CEC, available $P_2O_5$, exchangeable $Ca^{2+}$, and available silicate. The contents of heavy metals, Cd, Cr, Cu, Ni, Pb, and Zn, were only 10% of the threshold levels of the metals, and As content was about 20 to 30% of the threshold level.

Influence of Toxic Heavy Metals on Germination of Rice Seeds and Growth of Rice Seedling (수도생육(水稻生育)에 대한 유해(有害) 중금속(重金屬)의 영향(影響) - 발아 및 묘대기(苗垈期) 생육(生育)에 대하여 -)

  • Kim, B.J.;Ha, Y.L.;Kim, J.O.;Han, K.H.
    • Korean Journal of Soil Science and Fertilizer
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    • v.11 no.2
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    • pp.119-126
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    • 1979
  • Rice seeds, suweon 264, were germinated under 5 levels of toxic heavy metals, Cd(0, 0.05, 0.5, 5, 20ppm), Cu(0, 0.05, 0.5, 5, 20, ppm), Cr(0, 0.5, 1, 5, 10ppm), Ni(0, 0.5, 1, 5, 10ppm), Co(0, 0.5, 1, 5, 10ppm), Zn(0, 0.5, 5, 20, 40, ppm), Pb(0, 0.5, 5, 20, 40ppm) and Mn(0, 1, 10, 25, 50, ppm) in culture solution, and then grown with supplying culture solution contained respective concentrations. Germination and growth response to the toxic heavy metals were studied. Results obtained are as follows : 1) The germination injury of rice seeds by excess concentration of toxic heave metal in culture solution occured in Cd and Cu; below 0.05 ppm, Ni; below 0.5 ppm, Mn; below 1.0 ppm, Co and Cr; 0.5-1.0 ppm, and 0.5-5 ppm, Zn and Pb. Thereby, in the order of degrees of the elements toxicity to germination, they were arranged as follows : Cd>Cu>Ni>Co>Cr>Mn>Zn$$\geq_-$$Pb. 2) Toxic heavy metal concentrations in culture solution, which result in decreasing dry weight due to the injury of excess concentration of treated elements, were Cd: below 0.05 ppm, Ni, Cr and Co; below 0.5 ppm, Cu and Zn; 0.5-5 ppm, Pb; 5-20 ppm and Mn; 10-25 ppm. The order was Ni>Cd>Cr>Co>Cu$$\geq_-$$Zn>Pb>Mn. 3) The critical contents of Cd, Ni, Pb, Cu, Zn, Mn, and Co in dry matter, Which result in decreasing dry weight, were considered to be 0.05-15.5, 1.50-25.0, 24.0-28.0, 26.5-62.5, 470-645.0, 231.0-500.0 and below 15.0 ppm, respectively. 4) The contents of Cr, Co, Cd, Pb, Ni, Cu and Zn in dry matter by 0.5 ppm treatment concentration of each heavy metals was trace, 15.0, 17.5, 24.0, 25.0, 84.5 and 470.0 ppm, respectively. Thereby, in the order of each element to uptaked by rice seedlings, they were arranged as follow; Zn>Cu>Ni>Pb>Cd>Co>Cr. 5) The hazardous concentrations of root activity by toxic heavy metals in culture solution were Cd; below 0.05, Cu; 0.05-0.5, Cr; below 0.5, Ni; 0.5-1.0, Co; 0.5-1.0, Zn; above 0.5, Pb; 0.5-5.0 and Mn; 1.0-10.0 ppm. The hazardous degree of root activity by toxic heave metals was in the order of Cd>Cu>Cr>Zn>Ni>Co>Pb>Mn.

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White Mica and Chemical Composition of Samdeok Mo Deposit, Republic of Korea (삼덕 Mo 광상에서 산출되는 백색운모 및 화학조성)

  • Yoo, Bong Chul
    • Journal of the Mineralogical Society of Korea
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    • v.32 no.3
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    • pp.223-234
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    • 2019
  • The geology of the Samdeok Mo deposit consists of Paleozoic Hwajeonri formation, Kowoonri formation, Suchangri formation, Iwonri formation, Hwanggangri formation, Cretaceous, leucocratic porphyritic granite and granitic porphyry. This deposit consists of three quartz veins that filled NS oriented fractured zones in Suchangri formation. Quartz veins vary from 0.05 m to 0.3 m in thickness and extend to about 400 m in strike length. Quartz veins occur as massive, breccia, and cavity textures. Wallrock alteration has silicification, sericitization, argillitization and chloritization. The mineralogy of the quartz veins consists of quartz, fluorite, white mica, biotite, apatite, monazite, rutile, ilmenite, molybdenite, chalcopyrite, Fe-Mg-Mn oxide and Fe oxide. White mica from Samdeok Mo deposit occurs as fine or coarse grains in quartz vein and hostrock and has four mineral assemblages (I type: quartz, molybdenite, Fe oxide and Fe-Mg-Mn oxide, II type: quartz, Fe oxide and Fe-Mg-Mn oxide, III type: quartz and biotite, and IV type: quartz). The structural formular of white mica from quartz vein is $(K_{0.89-0.60}Na_{0.05-0.00}Ca_{0.01-0.00}Sr_{0.02-0.00})_{0.94-0.62}(Al_{1.54-1.12}Mg_{0.36-0.18}Fe_{0.26-0.09}Mn_{0.04-0.00}Ti_{0.02-0.00}Cr_{0.02-0.00}Zn_{0.01-0.00})_{1.91-1.72}(Si_{3.40-3.11}Al_{0.92-0.60})_{4.00}O_{10}(OH_{1.68-1.42}F_{0.58-0.32})_{2.00}$, but white mica of I type has higher FeO content, and lower $SiO_2$ and MgO contents than white micas of other types. Also, compositional variations in white mica from the Samdeok Mo deposit are caused by phengitic or Tschermark substitution ($(Al^{3+})^{VI}+(Al^{3+})^{IV}{\leftrightarrow}(Fe^{2+}{\text{ or }}Mg^{2+})^{VI}+(Si^{4+})^{IV}$) and direct $(Fe^{3+})^{VI}{\leftrightarrow}(Al^{3+})^{VI}$ substitution.

Assessment of Soil Contamination and Hydrogeochemistry for Drinking Water Sites in Korea (국내 먹는샘물 개발지역의 토양 오염 평가 및 수리지구화학적 특성)

  • 이두호;전효택
    • Journal of the Korean Society of Groundwater Environment
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    • v.4 no.1
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    • pp.41-53
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    • 1997
  • Geochemical data of soil and water samples were presented in order to assess the environmental impart for drinking water sites. Microscopic observation of rock samples and physical and chemical analysis of soil and water samples were undertaken. The geology of study areas are classified into three groups such as granitic rocks, meta-sedimentary rocks and sedimentary rocks. Enrichment of heavy metals derived from those rocks is not found in this study areas. Soils were analyzed for Cu, Pb, Zn, Cd and Cr using AAS extracted by HNO$_3$+HClO$_4$ and 0.1 N HCl. Heavy metal concentrations in soils are within the range of those in uncontaminated soils. In comparison of metal contents extracted by 0.1 N HCl and HNO$_3$+HC1O$_4$, less than 10% of the heavy metals are present in the exchangeable fraction. In particular, an pollution index has been proposed to assess the degree of soil contamination. Pollution index in soils are between 0.03 and 0.47 therefore, soils are not polluted with heavy metals. Deep groundwaters within granitic rocks have been evolved into Na$\^$+/-HCO$_3$$\^$-/ type, whereas other deep groundwaters evolved into Ca$\^$2+/-HCO$_3$$\^$-/ type. The predominance of Na$\^$+/ over Ca$\^$2+/ in deep groundwaters within granitic rocks is a result of dissolution of plagioclase, but for sedimentary and meta-sedimentary rocks, dissolution of calcite is a dominant factor for their hydrogeochemistry. The pH, conductivity and contents of the most dissolved ions in the water increase with depth. Shallow groundwaters, however, are highly susceptible to pollution owing to agricultural activities, considering the fact that high contents of nitrate, chloride and potassium, and high K/Na ratio are observed in some shallow groundwaters. In a thermodynamic approach, most natural water samples are plotted within the stability fields of kaolinite and smectite. Therefore, microcline and other feldspars will alter to form clay minerals, such as kaolinite and smectite. From the modelling for water-rock interactions based on mass balance equation, models accord well with behavior of the ions and results of thermodynamic studies are derived.

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Geochemical Origin, Behavior and Enrichment of Environmental Toxic Elements in Coaly Metapelite from the Deokpyeong Area, Korea (덕평지역의 탄질 변성니질암에 관한 환경적 독성원소의 지구화학적 기원, 거동 및 부화)

  • Lee, Hyun Koo;Lee, Chan Hee
    • Economic and Environmental Geology
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    • v.30 no.6
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    • pp.553-566
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    • 1997
  • Origin, behavior and enrichment of environmental toxic elements from the Deokpyeong area were investigated on the basis of major, trace and rare earth element geochemistry. Coaly metapelites of the Deokpyeong area are subdivided into grey phyllite, dark grey phyllite, coaly slate and black slate, which are interbedded along the Ogcheon Supergroup. The coaly slate had been mined for coal, but mining is closed. The coaly and black slates are lower contents of $SiO_2$ and $Al_2O_3$, and higher contents of LOI, CaO, $Na_2O$ and BaO as compared with the phyllitic rocks. Rare earth elements are highly enriched in the coaly and black slate. Average compositions (ppm) of minor and/or environmental toxic elements in the coaly and black slate are revealed as As=127, Ba=30,163, Cd=18, Cr=740, Cu=84, Mo=378, Pb=43, Sb=12, Se=44, U=144, V=8,147 and Zn=292, which are extremely high concentrations than those in the NASC compositions. Major elements (average enrichment index; 5.34) in the coaly metapelites are mostly depleted, excepting $P_2O_5$ and BaO, normalized by NASC. Rare earth elements (average enrichment index; 1.48) are enriched in the coaly slate. On the basis of NASC, minor and/or environmental toxic elements in the coaly metapelites were strongly enriched of all the elements with the exception of Co, Cs, Ni and Sr. Average enrichment index of trace elements in coaly metapelite is 31.51 (coaly slate; 51.94 and black slate; 15.46). Especially, enrichment index of potentially toxic elements (As, Ba, Cr, Cu, Mo, Ni, Sb, Se, U, V and Zn) of the rock is 46.10 (grey phyllite; 7.15, dark grey phyllite; 4.77, coaly slate; 88.96 and black slate; 22.11). These coal formations were deposited in basin of boundary between terrestrial and marine environments deduced to carbon, sulfur (C/S=2.2 to 275.7), trace and rare earth elements characteristics. Irregular behavior and dispersion between major, minor and rare earth elements of those metapelites indicates a variable source materials, incomplete mixing of differential source and/or reequilibrium of diagenesis and metamorphism.

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Oxide perovskite crystals type ABCO4:application and growth

  • Pajaczkowska, A.
    • Proceedings of the Korea Association of Crystal Growth Conference
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    • 1996.06a
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    • pp.258-292
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    • 1996
  • In the last year great interest appears to YBCO thin films preparation on different substrate materials. Preparation of epitaxial film is a very difficult problem. There are many requirements to substrate materials that must be fullfilled. Main problems are lattice mismatch (misfit) and similarity of structure. From paper [1] or follows that difference in interatomic distances and angles of substrate and film is mire important problem than similarity of structure. In this work we present interatomic distances and angle relations between substrate materials belonging to ABCO4 group (where A-Sr or Ca, B-rare earth element, C-Al or Ga) of different orientations and YBCO thin films. There are many materials used as substrates for HTsC thin films. ABCO4 group of compounds is characterized by small dielectric constants (it is necessary for microwave applications of HTsC films), absence of twins and small misfit [2]. There most interesting compounds CaNdAlO4, SrLaAlO4 and SrLaGaO4 were investigated. All these compounds are of pseudo-perovskite structure with space group 14/mmm. This structure is very similar to structure of YBCO. SLG substrate has the lowest misfit (0.3%) and dielectric constant. For preparation of then films of substrates of this group of compound plane of <100> orientation are mainly used. Good quality films of <001> orientations are obtained [3]. In this case not only a-a misfit play role, but c-3b misfit is very important too. Sometimes, for preparation of thin films substrates of <001> and <110> orientations were manufactured [3]. Different misfits for different YBCO faces have been analyzed. It has been found that the mismatching factor for (100) face is very similar to that for (001) face so there is possibility of preparation of thin films on both orientations. SrLaAlO4(SLA) and SrLaGaO4(SLG) crystals of general formula ABCO4 have been grown by the Czochralski method. The quality of SLA and SLG crystals strongly depends on axial gradient of temperature and growth and rotation rates. High quality crystals were obtained at axial gradient of temperature near crystal-melt interface lower than 50℃/cm, growth rate 1-3 mm/h and the rotation rate changing from 10-20pm[4]. Strong anisotropy in morphology of SLA and SLG single crystals grown by the Czochralski method is clearly visible. On the basics of our considerations for ABCO4 type of the tetragonal crystals there can appear {001}, {101}, and {110} faces for ionic type model [5]. Morphology of these crystals depend on ionic-covalent character of bonding and crystal growth parameters. Point defects are observed in crystals and they are reflected in color changes (colorless, yellow, green). Point defects are detected in directions perpendicular to oxide planes and are connected with instability of oxygen position in lattice. To investigate facets formations crystals were doped with Cr3+, Er3+, Pr3+, Ba2+. Chromium greater size ion which is substituted for Al3+ clearly induces faceting. There appear easy {110} faces and SLA crystals crack even then the amount of Cr is below 0.3at.% SLG single crystals are not so sensitive to the content of chromium ions. It was also found that if {110} face appears at the beginning of growth process the crystal changes its color on the plane {110} but it happens only on the shoulder part. The projection of {110} face has a great amount of oxygen positions which can be easy defected. Pure and doped SLA and SLG crystals measured by EPR in the<110> direction show more intensive lines than in other directions which allows to suggest that the amount of oxygen defects on the {110} plane is higher. In order to find the origin of colors and their relation with the crystal stability, a set of SLA and SLG crystals were investigated using optical spectroscopy. The colored samples exhibit an absorption band stretching from the UV absorption edge of the crystal, from about 240 nm to about 550 m. In the case of colorless sample, the absorption spectrum consists of a relatively weak band in the UV region. The spectral position and intensities of absorption bands of SLA are typical for imperfection similar to color centers which may be created in most of oxide crystals by UV and X-radiation. It is pointed out that crystal growth process of polycomponent oxide crystals by Czochralski method depends on the preparation of melt and its stoichiometry, orientation of seed, gradient of temperature at crystal-melt interface, parameters of growth (rotation and pulling rate) and control of red-ox atmosphere during seeding and growth (rotation and pulling rate) and control of red-ox atmosphere during seeding and growth. Growth parameters have an influence on the morphology of crystal-melt interface, type and concentration of defects.

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Petrochemistry of the Granitic Rocks in the Chungju, Wolaksan and Jecheon Granite Batholiths (충주(忠州)-월악산(月岳山)-제천(提川) 화강암류(花崗岩類)의 암석화학적(岩石化學的) 연구(硏究))

  • Kim, Kyu Han;Shin, Yun Soo
    • Economic and Environmental Geology
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    • v.23 no.2
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    • pp.245-259
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    • 1990
  • Petrochemical analyses of granitic rocks including trace element, REE and oxygen isotope were carried out to understand petrogenesis of plutonic rocks from the Chungju, Wolaksan and Jecheon granite batholiths, which might be related with tungsten-base metal-fluorite mineralization in the Hwanggangri metallogenic province. Different geochemical characteristics such as major and trace elements were found between Jurassic Daebo granitic rocks (Chungju, Jecheon, Wonju, and Boeun granitic rocks) and Cretaceous Bulgugsa granitic rocks (Wolaksan, Muamsa and Sokrisan granitic rocks). Cretaceous granitoids are characterized by high $SiO_2$and $K_2O$ contents and low $TiO_2$, $Al_2O_3$, MgO and CaO contents. They also have relatively high contents of trace elements(Zn, V, Co, Cr, Sr, and Ba) in comparison with the Jurassic granitoids. (Eu)/($Eu^*$) and $(La/Lu)_{CN}$ ratios of Jurassic plutons vary from 0.78 to 1.13 and from 26.02 to 30.5, respectively, while the ratios of Cretaceous ones range from 0.22 to 0.28 and from 4.42 to 14.2, respectively. The REE patterns of the Cretaceous and Jurassic granitic rocks have quite different Eu anomalies: large negative Eu anomaly in the former, and mild or absent Eu anomaly in the latter. The large Eu negative of Cretaceous granitic rocks are interpreted as a differentiated product of fractional crystallization of granitic magma deduced by Rayleigh fractionation model(Tsusue et al., 1987). Oxygen isotopic compositions of quartz for Daebo and Bulgugsa granitic rocks range from 9.98 to 10.51‰ and from 8.26 to 9.56‰, respectively. The Daebo granitic rocks enriched in $^{18}0$ suggest that the magma be undergone different partial melting processes from the Bulgugsa ones. Of the Bulgugsa granitoids, Wolaksan and Sokrisan mass have different contents of trace elements and ${\delta}\;^{18}0$ values of the silicate minerals, which indicate that they are not from the identical source of magma. Many mineral deposits are distributed in and/or near the Wolaksan and Muamsa granitic rocks, but a few mineral deposits are found in and near the Chungju and Jecheon granite batholiths. It might be depend on geochemisty of the related igneous rocks which have low contents of Ba, Sr, Co, V, Cr, Ni, Zn and high contents of Nb and Y, and on lithology of country rocks such as cabonate and noncarbonate rocks.

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Plant Regeneration of B.juncea Through Plant Tissue and Protoplast Culture

  • Lian, Yu-Ji;Lim, Hak-Tae
    • Journal of Plant Biotechnology
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    • v.3 no.1
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    • pp.27-31
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    • 2001
  • New types of cytoplasmic male sterility in Brassica species would be very useful for the production of F$_1$, hybrid seeds. Leaves and stems of rapid cycling stock of B.juncea (CrGC4-3) containing Anand CMS were used as experimental materials for plant regeneration from protoplast culture. Very high plant regeneration rate (85%) was found in the Kao & Michayluk medium supplemented with 2 mg/L zeatin, 0.5 mg/L BAP, and 1 mg/L NAA when only leaf, not stem, segments were cultured. Protoplasts were isolated from leaves using mixtures of enzymes (1% Cellulycin, 0.5% Macerozyme) in 0.4 M mannitol and 50 mM $CaCl_2$.$2H_2$O. Mcrocalli induced from protoplasts were transferred to the shoot regeneration medium containing 2 mg/L BAP, 2 mg/L zeatin, and 0.5 mg/L NAA. After 60 days of initial protoplast culture, regenerated plantlets were obtained, acclimatized, transplanted into the pots, and grown up to the flowering stage.

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