Earticle

Among 31 water-born microbial strains isolated from various sites in Korea, strain DJ-4 was selected as a test organism for toxicity measurements in that its growth was completely inhibited by the presence of 668.4 mg/L of chloroform and 297.5 mg/L of toluene in the liquid LB medium whereas others did not. It was observed that lag periods and specific growth rates of DJ-4 batch vial cultures were prolonged and decreased, respectively, by phenol, benzene, toluene, ethylbenzene, p-xylene, perchloroethylene, trichloroethylene, and chloroform at the concentrations between 3.6 and 417.8 mg/L. There changes were found to be linear with respect to the concentrations of the toxic compounds. From the first-order regression equations, 50% effective concentrations (EC50 for concentrations of toxic compounds causing 50% decrease of specific growth rates and EC50lag for 50% increase of length of lag periods) were calculated for each compounds. By comparing DJ-4 EC50 values with Daphnia LC50's from a literature for benzene, ethylbenzene, toluene, and trichloroethlyene, it was concluded that microbial specific growth could be a new, fast, and reliable parameter for toxicity tests.

A biosensor with PZT piezoelectric ceramic crystal was developed for the detection of formaldehyde gas. Poled PZT piezoelectric ceramic disk was made from ZrO2, TiO2 and Nb2O5, together with the addition of PbO and polyvinyl alcohol, through various processes of mixing, calcination drying, crushing, forming, sintering, polishing, ion coating and poling. Oscillator circuit of sensor was made of operational amplifier(AD811AN). Formaldehyde dehydrogenase was immobilized onto a piezoelectic ceramic crystal, together with the cofactors, reduced glutathione and nicotinamide adenine dinucleotide. The effect of flow rate on the sensitivity was determined by varing the flow rate of carrier gas from 24.7mL/min to 111.7mL/min through detector cell. The results indicated that as the flow rate was increased, the recovery rate was increased. And a significant increase in the sensitivity was observed in enhanced flow rate of carrier gas. Frequency difference(ΔF) of immobilized PZT piezoelectic disk increased proportionally to the concentration gas and reproduced to repeated exposures of formaldehyde gas(28ppm, Δ68Hz).

Two unidentified methanotrophic strains (MM-white and MM-red) secreting soluble methane monooxygenase (sMMO) involved in thrichloroethylene biodegradation have been isolated from mixed methanotrophic consortium (MM) around Taejon area. Subsequently four methanotrophic strains were isolated from MM and named according to their color; white (MS-white), yellow (MS-yellow), pink (MS-pink) and reddish brown (MS-rbrown). All strains except MS-yellow which can take glucose as well as methane, metabolized methane as a sole carbon source. They all showed symbiotic behavior when methane was used as the sole carbon source. Optimum conditions of cell growth for MM were pH of 6.8 - 7.2, temperature of 29 - 32℃, and gas flow rate of 6 (for methane), 40 (for air), and 4 ml/min (for carbon dioxide). The sMMO activity was expressed as naphthalene oxidation rate (umol/ mg protein/ hr). The sMMO activity for MM grown in flask culture with 1 M of CuSO4 was 36, while it was 61 without copper. The activity for MM grown in the fermentor without CuSO4 was 1077, but is was 197 after reaction with 5 ppm of TCE. The methanotrophs showed significantly high sMMO activity despite the presence of 1uM of CuSO4, although most of other strains already known could not express sMMO activity under this condition.

The production of fibrinolytic enzyme from Bacillus subtilis BK-17 was studied in the shake flask cultures. The important medium components studied include nitrogen source, carbon source and inorganic salts. The environmental conditions include initial pH, temperature, shaking speed and working volume. Among various N-sources, C-sources and inorganic salts tested, soybean flour, D-glucose and Na2HPO4 gave the best results, and their optimal concentrations were 1.5%, 0.5% and 0.05%, respectively. The optimal pH and temperature were 9.0 and 37℃. With decreasing working volume in the range of 25∼100ml in the 250ml flask or increasing shaking speed in the range of 100∼300rpm, the enzyme production was greatly enhanced. The enzyme activity under the optimal conditions was about 1400I.U./ml with urokinase as a standard.

A fungus with high phosphate solubilizing activites was isolated from soil using potato dextrose agar-calcium phosphate medium and identified to Penicillium sp. PS-113, based on the morphological characteristics of conidiophore and conidia; flask shape of phialide, simple branching type of conidiophore, and columnar shape of conidial head, in malt extract agar and potato dextrose agar media. The optimum temperature ad initial pH to solubilize rock phosphate in potato dextrose broth-rock phosphate medium were 30℃ and pH 8.0, respectively. In these conditions phosphate solubilizing activities of Penicillium sp. PS-113 against four types of insoluble phosphate like tricalcium-phosphate, aluminium phosphate, hydroxyapatite and rock phosphate were quantitatively determined. As results, this fungus highly produced free phosphates to the culture broth with the concentrations of 1,283 ppm against tricalcium-phosphate, 585 ppm against rock phosphate, 528 ppm against aluminium phosphate, and 242 ppm against hydroxyapatite, respectively.

Stabilization of antibody, which is specific to Salmonella typhimurium antigens, present in dry states on membranes was accomplished, and its shelf-life, i.e., duration for maintaining minimum 90% of the initial activity, under optimal conditions was determined. To prepare two major components of an immuno-strip, the antibody was not only immobilized on nitrocellulose membrane surfaces but also placed within the pores of glass fiber membrane after conjugating it with old colloids as signal generator. Among potential stabilizers of the immuno-components, a disaccharide, trehalose, showed a significant protection effect of immunoglobulin structure from thermal energy. Optimal concentrations of trehalose for the respective component were significantly different (8-fold higher for the antibody-gold conjugate than for the immobilized antibody), which probably resulted from distinct densities and configurations of antibody present on the membranes. An additional requirement for the gold conjugate was freeze-drying of this substance such that the conjugate can be readily resolubilized upon contact with aqueous medium. By using the components prepared under optimal conditions, immuno-strips were constructed and exposed to thermal energy. Signals with less than 10% decrease in the intensity were maintained for approximately 21 days at 60℃. Compared to previous reports, this result represented a 2-year shelf-life at room temperature. it was, however, two times longer if determined from thermal acceleration tests based on the theory of inactivation rate of protein. Such discrepancy between the two estimates could be mainly attributed to errors in accurately controlling temperatures and also to changes in the physical properties of membranes due to a high thermal energy.

Temperature and pH conditions were studied for an effective biosurfactant production by Pseudomonas aeruginosa YPJ-80. Efficient methods of biosurfactant separation were also investigated. pH-uncontrolled experiments at 35 and an initial pH of 8 resulted in the best cell growth (3.6 g/L) and biosurfactant production (0.073 g biosurfactant/g cell). Biosurfactant separation was most efficient using solvent extraction with chloroform/methanol (2:1 vol%) followed by acidification using 1N HCl.

A large quantity of ethylene glycol(EG) is remained in the effluent after pretreating polyester weight-loss wastewater physicochemically in the fist stage and must be treated biologically in the second stage. Therefore, an excellent EG-utilizing bacteria strain was isolated from the natural system and the optimal culture conditions of the strain were investigated. The optimal culture conditions of temperature, pH, and nitrogen source were found to be 35℃, 7.5 and ammonium chloride, respectively, when CODCr removal efficiency was more than 90%. The growth of stains and EG removal efficiency was slightly improved by adding elements such as niacin and biotin. With increasing inoculation size in a batch culture, the removal efficiency of EG was conspicuously increased. Growth rate was inhibited when the initial concentration of EG was more then 30g/L. The strain was identified as Pseudomonas sp. based on morphological and biological characteristics and named as Pseudomonas sp. EG1.

An agarase was partially purified from the culture broth of marine bacterium Bacillus cereus ASK202. Optimal pH and temperature of this agarase were found to be 7.0 and 40℃, respectively. The maximum productivity of agarooligosaccharides was obtained from 0.3 %(w/v) agar by using of 1 unit agarase. As the results of TLC and HPLC analysis, these oilgosaccharides consisted of neoagarobiose, neoagarotetraose and neoagarohexaose. Under the optimal reaction conditions, 77.5 %(w/v) neoagarobiose and 6.2 %(w/v) neoagarotetraose were produced from agar and the conversion yield of total agarooligosaccharides was 83.7 %(w/v) after for 2 h reaction at 40℃.

The addition of the ceramic powder as state of bare in culture medium has stimulated the growth of Achyranthes japonica in both the disorganized cell and the plantlet. The grwoth rate of Hyoscyamus niger adventitious root and Pylatycodon grandiflorum hairy root was enhanced up to 100 and 250%, respectively, even though Scopolia parviflora hairy root and Hyoscyamus albus adventitious root were not. The ceramic powder has enhanced the growth of H. niger adventitious root even in a test tube immersed into its culture medium to irradiate alone without any direct contact. The ceramic powder seems to have a significant role on both the growth and the physiological action of some plants.

The monellin, a sweet-taste protein, was expressed and secreted in Saccharomyces cerevisiae. The secreted menellin was concentrated using an ultrafiltration membrane with a nominal molecular weight cut off of 3,000 or by ammonium sulfate precipitation. The monellin was purified by G-25 gel filtration chromatography, followed by CM-Sepharose ion exchange chromatography. The purified monellin was characterized by SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis) and PHLC. The molecular weight of monellin was found to be 10,700 dalton, and its purity was over 95%.

For the purpose of screening the functionality in aloe, the mucilage from Aloe vera Linne was prepared by extraction of mucilaginous parenchyma with ethanol followed homogenation, filtration and centrifigation. Flocculability of this mucilage from Aloe vera L. was investigated and the identification fro flocculating active materials were carried out. The mucilage exhibited an excellent flocculability for 1% bentonite and 5,000 ppm kaolin suspensions. The good correlations between mucilage volume(concentration) and flocculability for 1% bentonite suspension were obtained. The flocculated volume or flocculation rate was affected depending upon the degrees of processing such as a heat treatment and purification. A major component showing the flocculaing activity was considered as a highly polymerized polysaccharide having flocculating activity was about 1,000,000 daltons.

For the efficient production of a new exo-polysaccharide from Ganoderma lucidum ASI 7004, the optimum conditions and methods in submerged cultivation were investigated with an airlift fermenter system. The optimum aeration rate was 2.5 Wm at the initial pH 5.0 and 28℃. The increase of dissolved oxygen concentration by pure oxygen supply during cultivation did not improved the exo-polysaccaride production and the mycelial growth. The maximum exo-polysaccharide production and the mycelial growth under the optimum culture condition were obtained in media of glucose 60g/L, yeast extract 6g/L, (NH4)2HPO4 1g/L and KH2PO4 0.5g/L. Under these optimum medium and culture conditions, about 7.15g/L of exo-polysaccharide and 13.9g/L of mycelial growth were producted, respectively.

Biopolymer from alkaline-tolerant Bacillus so. was purified, and its physico-chemical and structural properties were investigated. Crude biopolymer, precipitated by acetone from culture broth was fractionated into two fractions by gel chromatography on Sephadex G-200. Among two fractions, one fraction(PS I), which an acidic biopolymer precipitated by the CPC(cetylpyridinium chloride) treatment was studied further. PS I fraction had carboxyl groups and was positive at color reaction of sugar. PS I fraction also showed UV absorbance at 190-225nm. The purified acidic biopolymer was composed of 4% glucose, 8% glucosamine and 88% glutamic acid. Sugar components of the purified acidic biopolymer seemed to be linked to PGA(polyglutamic acid) which existed in the from of r-peptide bond. By the results of Smith degradation of sugar components, glucose and glucosamine was bound by 1,3 glocosidic linkage. Therefore, this biopolymer was a glycopeptide, oligosaccaride r-PGA. We concluded that the equivalent weight and the molecular weight of this biopolymer were estimated as about 171 and 5x105 dalton, respectively.

Two cometabolic trichloroethylene (TC) degraders, Pseudomonas putida F1 and Burkholderia (Pseudomonas) cepacia G4, were found to catabolize phenol, benzene, toluene, and ethylbenzene as carbon and energy sources. Resting cells of P. putida F1 and B. cepacia G4 grown in the presence of toluene and phenol, respectively, were able to degrade not only benzene, toluene and ethylenzene but also TCE and p-xylene. However, these two strains grown in the absence of toluene or phenol did not degrade TCE and p-xylene. Therefore, it was tentatively concluded that cometabolic degradation of TC and p-xylene was mediated by toluene dioxygenase (P. putida F1) or toluene-2-monooxygenase (B. cepacia G4). Maximal degradation rates of BTEX and TCE by toluene- and phenol-induced resting cells of P. putida F1 and B. cepacia G4 were appeared to be 4-530 nmol/(minmg cell protein) when a single compound was solely served as a target substrate. In case of double substrates, the benzene degradation rate by P. putida F1 in the presence of toluene was decreased up to one seventh of that for the single substrate. TCE degradation rate was also linearly decreased as toluene concentration increased. On the other hand, toluene degradation rate was enhanced by benzene and TCE. For B. cepacia G4, degradation rates of TCE and toluene increased 4 times in the presence of 50 M phenol. From these results, it was concluded that a degradation rate of a compound in the presence of another cosubstrate(s) could not be predicted by simply generalizing antagonistic or synergistic interactions between substrates.

The effects of carbohydrates, hormones and elicitors on both cell growth and betaine production were investigated in the cell cultures of Lycium chinense Mill. The maximum effect of glucose and sucrose was observed in cells cultured in the presence of 3% and 7% for cell growth and betaine production, respectively. The effect of hormones on cell growth and betaine production was prominent in the presence of 10 M 2, 4-D, 10 M NAA and 2.5 M IAA, whereas cell growth and betaine production were excellent at 2.5 M BA and 10 M BA, respectively. Abiotic elicitors such as KCI, MnCl2 and NaCl exhibited an inhibitory role on cell growth in all treatment groups. Betaine production was increased according to increase of concentration of abiotic elicitors. methanol-soluble and insoluble components as biotic elicitor remarkably inhibited cell growth from 2 mg and 6 mg, respectively. Betaine production was increased maximally at 2 mg of biotic elicitors. When growth medium was switched to production medium at two stage culture, it resulted that cell fresh weight and dry weight decreased but betaine content increased about 2.2-fold.

A sensitive membrane strip assay for plasma lipoprotein cholesterol that can be performed without handling reagents has been investigated. We previously developed an assay system with immobilized enzymes (cholesterol esterase and cholesterol oxidase) on the surfaces of nitrocellulose membrane(1). In such a case, the amount of enzymes present on the membrane was limited by its surface area and, thus, the detection capability was relatively poor (> 50 mg/dL cholesterol). To overcome this problem, we devised a new system with non-immobilized enzymes by placing them within interstitial spaces of a celullose membrane pad in a dry state. Upon contact with sample medium, the enzymes were immediately dissolved and participated in the reactions with cholesterol in a liquid phase. We constructed a user-friendly system consisting of four membrane pads fro sample application, cholesterol decomposition, color development as signal, and medium absorption to invoke a continuous flow (sequential location from the bottom). A sample containing lipoproteins was added into the application pad by capillary action and transferred to the next pad for decomposition. The decomposition pad (namely, enzyme pad) contained a detergent (sodium cholate) for the destruction of lipoprotein particles, the two enzymes for cholesterol decomposition, and a chromogen (3,3'-diaminobenzidine). As a consequence of the enzyme reactions, hydrogen peroxide was produced, and then reacted in the presence of the chromogen with horseradish peroxidase immobilized on the signal generation pad. Finally, a colorimetric signal directly proportional to the cholesterol concentration was produced. The detection limit determined from this system under optimal conditions was at least 2 times lower than of the enzyme-immobilized system.

The effect of intracellular Ca2+ level on the hybridoma cell growth and monoclonal antibody(MAb) production was examined. For the manipulation of intracellular Ca2+ concentration, the cells were treated with A23187, ryanodine, and thapsigargin at about 1x106 cells/mL. The treated cells were recultivated by using the Iscove's Modified Dulbecco's Medium(MDM) containing 1.49mM CaCl2. The ryanodine-treated cells showed better cell growth, MAb concentration, and specific MAb productivity than others. In comparison with control, the maximum cell concentration, MAb concentration, and specific MAb productivity were increased by 40.6%, 48.1% and 83.3%, respectively. Confocal microscopic images of Fura-2/AM loaded cells indicate that the increase in intracellular Ca2+ level can enhance the MAb productivity by allowing the calcium influx into the endoplasmic reticulumn.

Biological deinking process of used papers was studied by the polymer modified cellulase. Cellulase was modified with copolymers which consist of polyoxyethylene derivative and maleic anhydride(MA). The MA functional groups of copolymer can react with amino acids groups of the cellulase without much loss of activity. Modified degree of amino acids was controlled by the added copolymer. The maximum modified degree was about 60% and it was obtained when the weight ratio of copolymer and cellulase was 4. The remained activity of the maximum modified cellulase(MMC) was higher than 80% of native cellulase. The MMC's concentration was 0.05-2.0 wt% relative to the dry paper. In mechanical pulping process, cellulase enhanced the detachment of the ink particle from the used paper by partial hydrolysis of the fiber. The polyoxyethylene of modified cellulase produced the forms which can float the separated ink particle. Compared to the convention deinking method with NaOH or organic chemicals, the new biological deinnking process improved the physical properties such as freeness, tearing strength and whiteness.

Soybean peroxidase was extracted from soybean hulls and purified by ammonium sulfate precipitations (25% and 75% saturation), pl fractionation, and anionic exchange and gel filtration chromatographies (DEAE-Sephadex A-50 and Superose 12). Modlecular weight and pl value were estimated to be ca. 45 kD and 4.2, respectively. Purified soybean peroxidase had an RZ value of 0.43. Compared with horseradish peroxidase, it showed superior thermal and pH stability. Assuming the first-order kinetics, the thermal deactivation rate constant of soybean peroxidase at 80℃ was about 8 times lower than that of horseradish peroxidase. Deactivation energy was calculated to be 69.3 kcal/mol. Soybean peroxidase showed about 10% higher H2O2 degradation capacity than horseradish peroxidase. Exploiting these advantages, the soybean peroxidase purified from the domestic soybean hull is expected to replace horseradish peroxidase in various applications.

In this study, biodegradation rate of Arabian light crude oil by mixed cultures of selected petroleum-degraders was determined. Their modes of hydrocarbon uptake were then observed to determine whether there are differences in biodegradation rate by the mixed cultures. By the mixed cultures of petroleum-degraders having same modes of hydrocarbon uptake, such as strain US1 and K1 (using pseudo-solubilized hydrocarbons by a biosurfactants), K2-2 and P1(using hydrocarbons by direct contact), CL 180 and IC-10 (mixed type of uptake modes), the biodegradation rates of aliphatics in the crude oil were increased more than those by their pure cultures, about 40%, 25% and 20%, respectively. Biodegradation rate of strain KH3-2 (using only water- dissolved hydrocarbons) was increased by mixed cultures with strain K1, CL180 or IC-10 possessing high emulsifying activity. However, the biodegradation rate of the crude oil was decreased about 20%-40% by the mixed cultures of petroleum-degraders having different mode of hydrocarbon uptake, such as addition of strain US1 or K1 in the cultures of K2-2 or P1. Biosurfactants produced by US1 or K1 seems to enhance the emulsification of crude oil in aqueous phase but inhibit the attachment of K2-2 or P1 to crude oil. As same phenomena, the addition to Triton X-100 into the culture of strain US1, K1, CL180, IC-10 or KH3-2 increased the biodegradation rate, but the addition in the culture of strain K2-2 or P1 decreased the biodegradation rate. The mixed culture made of CL180, IC-10 and KH3-2 degraded 61.5% of aliphatics and 69% of aromatics in 3% (v/v) of Arabian light crude oil added.

The readily fermentable carbon sources in swine were acetic acid, propionic acid and butyric acid at the average concentrations of 7.2 g/L, 2.2 g/L and 2.7 g/L, respectively. The swine waste also contained excess nitrogen and other mineral sources. In shake flask experiments, the optimal range of cell growth for Azotobacter vinelandii UWD were 1.0∼3.5 g/L of acetic acid, 0.7∼2.0 g/L of propionic acid and 0.5∼2.0 g/L of butyric acid. A mixture of these three acids simulating two times diluted swine waste supported the best cell growth but the amount of carbon sources was limited. In shake flask and fermentor experiments, an addition of 30 g/L of glucose increased the final cell dry weight 8 times while the final poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) concentration increased 86 times compared with using acid mixture only. A. vinelandii UWD preferred organic acids in the sequence of acetic acid, propionic acid, butyric acid, and valeric acid.

The raw starch hydrolysis by amylase prepared from alkaline-tolerant Bacillus sp. were investigated. Degree of hydrolysis(%) of 5%(w/v) raw rice, corn and potato starch by this enzyme were about 40, 25 and 20%, respectively. The hydrolysis action on raw starch by change of blue value was similar to the action pattern of exo B-amylase. The hydrolysis products of rice starch were mainly glucose and maltose. Oligosaccarides were also detected. From the above results, this enzyme was considered as exo type a-amylase. This enzyme activity on the raw starch and the gelatinized starch were 28.40 and 86.60 IU/mg protein, respectively, and the ratio of raw starch-digesting activity to gelatinized starch-digesting activity (raw starch digestivity) was about 32%. The Km values for the raw and the gelatinized starch were 4.22 and 3.0mg/mL, respectively, and the VmaX values were 0.20 and 0.31mg/mL/min, respectively.

Ammonium limitation did not promote ply(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production of Azotobacter vinelandii UWD. In acid phase, ammonium limitation during utilization of propionic acid and butyric acid led to 35% decrease in product yield. In glucose phase, both biomass yield and polymer yield decreased about 22% under ammonium limitation. However, in nitrogen-fixing culture glucose was consumed 25% faster and the final PHBV wt% decreased slightly. Under nitrogen limitation a portion of the carbon sources was used fro nitrogen fixation rather than biomass and polymer formation, resulting in a decrease in biomass yield and polymer yield.