Hong-Gyu Song

Degradation of 2,4,6-trinitrotoluene by Klebsiella sp. isolated from activated sludge

Klebsiella sp. strain C1 isolated from activated sludge metabolized 2,4,6-trinitrotoluene (TNT) by two different pathways. The typical metabolites in the nitro group reduction pathway of TNT, such as hydroxylamino-dinitrotoluenes and amino-dinitrotoluenes, were detected. Dinitrotoluenes and nitrite were also detected, possibly produced by a denitration pathway. After incubation of [U-14C]TNT for 28 and 77 d, 2.4 and 6.24%, respectively, were released as 14CO2. This mineralization rate was higher than those reported by any other TNT degrading bacteria and might be due to the dual pathways of degradation in this bacterium.

Comparison of 2,4,6-trinitrotoluene degradation by seven strains of white rot fungi.

The degradation of 2,4,6-trinitrotoluene (TNT) by seven strains of white rot fungi was examined in two different media containing 50 mg L−1 of TNT. When TNT was added into a nutrient-rich YMG medium at the beginning of the incubation, four of the fungal strains completely removed TNT during several days of incubation and showed higher removal rates than those of Phanerochaete chrysosporium. TNT added into YMG medium after a 5-day preincubation period completely disappeared within 12 hours, and the removal rates were higher than those in N-limited minimal medium. Isomers of hydroxylamino-dinitrotoluene were identified as the first detectable metabolites of TNT. These were transformed to amino-dinitrotoluenes, which also disappeared during further incubation from cultures of Irpex lacteus. During the initial phase of TNT degradation by I. lacteus, dinitrotoluenes were also detected after the transient formation of a hydride-Meisenheimer complex, indicating that I. lacteus used two different pathways of TNT degradation simultaneously.

Enhancement of growth and yield of tomato by Rhodopseudomonas sp. under greenhouse conditions

A greenhouse test was carried out to examine the effects on tomato growth of application of purple non-sulfur bacterium Rhodopseudomonas sp. which had enhanced germination and growth of tomato seed under axenic conditions. The shoot length of tomato plant inoculated by Rhodopseudomonas sp. KL9 increased by 34.6% compared to that of control in 8 weeks of cultivation. During the same period, this strain increased 120.6 and 78.6% of dry weight of shoot and root of tomato plants, respectively. The formation ratio of tomato fruit from flower was also raised by inoculation of KL9. In addition, Rhodopseudomonas sp. KL9 treatment enhanced the fresh weight and lycopene content in the harvested tomato fruits by 98.3 and 48.3%, respectively compared to those of the uninoculated control. When the effect on the indigenous bacterial community and fate of the inoculated Rhodopseudomonas sp. KL9 were monitored by denaturing gradient gel electrophoresis analysis, its application did not affect the native bacterial community in tomato rhizosphere soil, but should be repeated to maintain its population size. This bacterial capability may be applied as an environment-friendly biofertilizer to cultivation of high quality tomato and other crops including lycopene-containing vegetables and fruits.

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor

A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57∼97 % homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.

Growth promotion of Xanthium italicum by application of rhizobacterial isolates of Bacillus aryabhattai in microcosm soil

This study was conducted using rhizobacteria, which are able to exert beneficial effects upon plant growth in the infertile soil collected from barren lakeside areas. Four strains of plant growth promoting bacteria were isolated from the rhizosphere of a common wild plant, Erigeron canadensis. Isolated strains LS9, LS11, LS12, and LS15 were identified as Bacillus aryabhattai by 16S rDNA sequence analysis. B. aryabhattai LS9, LS11, LS12, and LS15 could solubilize 577.9, 676.8, 623.6, and 581.3 mg/L of 0.5% insoluble calcium phosphate within 2 days of incubation. Production of indole acetic acid, a typical growth promoting phytohormone auxin, by strain LS15 was 471.3 mg/L in 2 days with the addition of auxin precursor L-tryptophan. All the strains also produced other phytohormones such as indole butyric acid, gibberellins, and abscisic acid, and strain LS15 showed the highest production rate of gibberellin (GA3), 119.0 μg/mg protein. Isolated bacteria were used in a microcosm test for growth of wild plant Xanthium italicum, which can be utilized as a pioneer plant in barren lands. Seed germination was facilitated, and the lengths of roots, and shoots and the dry weights of germinated seedlings after 16 days were higher than those of the uninoculated control plants. Root lengths of seedlings of X. italicum increased by 121.1% in LS11-treated samples after 16 days. This plant growth-promoting capability of B. aryabhattai strains may be utilized as an environmentally friendly means of revegetating barren lands, especially sensitive areas such as lakeside lands.

Effect of fungal pellet morphology on enzyme activities involved in phthalate degradation

Pellet size of white rot fungus, Pleurotus ostreatus may affect the secretion of its degradative enzymes and accompanying biodegrading capability, but could be controlled by several physical culture conditions in liquid culture. The pellet size of P. ostreatus was affected by the volume of inoculum, flask, and medium, but the agitation speed was the most important control factor. At the lower agitation speed of 100 rpm, the large pellets were formed and the laccase activity was higher than that of small pelleted culture at 150 rpm, which might be due to loose intrapellet structure. However, the biodegradation rates of benzylbutylphthalate and dimethylphthalate were higher in the small pelleted culture, which indicated the involvement of other degradative enzyme rather than laccase. The activity of esterase which catalyzes the nonphenolic compounds before the reaction of ligninolytic enzymes was higher in the small pelleted culture, and coincided with the degradation pattern of phthalates. This study suggests the optimization of pellet morphology and subsequent secretion of degradative enzymes is necessary for the efficient removal of recalcitrants by white rot fungi.

Comparison of pyrene biodegradation by white rot fungi

Six strains of white rot fungi, isolated from soil in Korea, were evaluated as to their ability to biodegrade the 4-ring polycyclic aromatic hydrocarbon pyrene. While growing in a complex fungal medium, Irpex lacteus, Trametes versicolor KR11W, and Phanerochaete chrysosporium mineralized 15.6, 12.7 and 7.0% of the added 0.84 nmol of radioactive pyrene, respectively. In these cultures, 33–46% of the added pyrene was converted to water-soluble polar metabolites, and 22–40% was incorporated into fungal biomass. Pleurotus ostreatus mineralized only 2.5% of the added pyrene, while T. versicolor KR65W and Microporus vernicipes failed to evolve 14CO2 from pyrene. The information obtained aids in strain selection for clean-up of polycyclic aromatic hydrocarbon contamination.

Quantitative improvement of 16S rDNA DGGE analysis for soil bacterial community using real-time PCR

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments has been frequently used to profile a structure of the bacterial community in a given soil. However, this procedure has various types of intrinsic error and bias, thus often misleads the relative abundance of bacterial populations. In order to establish a reliability for the current 16S rDNA DGGE method, we investigated various parameters and potential sources of errors in the DGGE procedures, such as primer mismatch, dNTP concentration, DNA polymerase, PCR cycles, uneven amplification of templates, secondary structure of PCR product, melting domain profiles, and acrylamide/bis concentration. Our result showed that the relative band intensities of the corresponding 16S rDNA templates were closely correlated with the differences of the melting temperature between the higher and lower melting domains of the PCR products. In addition, application of i) real-time PCR, ii) combination of PCR primers and iii) optimization of both dNTP and acrylamide/bis concentrations significantly improved the quantitative representation of bacterial 16S rDNA levels in the mixed samples. Especially, identification of the inflection points of DNA samples through the real-time PCR was crucial for the accurate representation of soil bacterial populations. Beyond these points DNA templates can be over-amplified to a saturated level independently of their initial amounts. Therefore for the accurate analysis of soil bacterial community, a quantitative 16S rDNA DGGE analysis needs to be performed in combination with a real-time PCR.

Nitroreductase II involved in 2,4,6-trinitrotoluene degradation: Purification and characterization from Klebsiella sp. Cl

Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. CI have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygen-insensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreductase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-amino-dinitrotoluene).

Degradation of humus-bound metabolites generated from toluene and o-xylene in soil

Abstract We investigated the fate of 14 C -labeled toluene and o-xylene in soil. Some of the metabolites of toluene and o-xylene were bound to the humic matrix rather than mineralized or incorporated into biomass. The distribution of the bound radioactive metabolites from toluene and o-xylene were respectively, 33.4% and 32.1% of original radioactivity. Most of the humus-bound metabolites (83.5–85.4%) from toluene and o-xylene were found in the humin fraction and less than 10% were incorporated to fulvic and humic acid, respectively. The bound metabolites from radioactive toluene and o-xylene were not extracted with various solvents, and they showed slow biodegradation. The mineralization rates of bound metabolites generated from toluene and o-xylene did not differ significantly, and the turnover times ranged between 3.9 and 4.6 years.