Yosuke Iimura

Biodegradation of polycyclic aromatic hydrocarbons by Sphingomonas sp. enhanced by water-extractable organic matter from manure compost

The effectiveness of in-situ bioremediation of polycyclic aromatic hydrocarbons (PAHs) may be inhibited by their low aqueous solubility and strong absorption to soil constituents. The aim of this research was to evaluate the effect of water-extractable organic matter (WEOM) from manure compost on the biodegradation of various PAHs. The aqueous solubilities of PAHs including phenanthrene, pyrene and benzo[a]pyrene under different concentrations of WEOM from cow manure compost were initially evaluated. The contribution of WEOM on the degradation of PAHs by Sphingomonas sp. was then investigated. Dissolution results confirmed the ability of WEOM to increase the apparent solubility of the 3PAHs. Time course of biodegradation also revealed its positive contribution to their removal. For example, the degradation of pyrene was 118% higher in the presence of 1000 mg-C L(-1) WEOM as compared to the mineral salt medium (MSM) alone after 48 h incubation. In addition, degradation was 12% higher with WEOM than with Glucose-Ammonium nitrate despite the more than 6 times higher cell concentration in the latter. WEOM from other manure composts such as chicken and pig were found to have the same effect. Finally, additional tests confirmed that high molecular weight WEOM (>1000 Da) contributed mainly to solubility and biodegradation enhancements. On the basis of these results, the increase in apparent solubility of PAHs in WEOM solutions may have a significant impact on their biodegradation. It is postulated that the application of WEOM-rich manure composts may be extended in the in-situ bioremediation of PAHs-polluted soil.

Application of aqueous saponin on the remediation of polycyclic aromatic hydrocarbons-contaminated soil

The aim of this research was to evaluate the feasibility of aqueous saponin for the removal and biodegradation of polycyclic aromatic hydrocarbons (PAHs) from contaminated soil. Dissolution test confirmed the ability of saponin to increase the apparent solubility of the tested 3–5 rings PAH above the critical micelle concentration (approximately 1000 mg/L). Microbial test with pure culture of Sphingomonas sp. showed that saponin significantly enhanced the degradation of pyrene. For example, the percent degradation was 2.1 times higher in the presence of 2500 mg/L saponin than that of control without saponin after 60 hours incubation at around 108 CFU/mL initial cell loading. These results suggest that the binding of pyrene with saponin does not pose a serious constraint to bacterial uptake. Contrary to pyrene, saponin was chemically stable against the PAHs degrader. It is also not toxic to the cell at least up to 2500 mg/L. Finally, using a spiked soil sample, extraction tests with 10,000 mg/L of saponin showed that around 52.7% and 0.3% of pyrene was removed from low and high organic spiked soils, respectively. The results from this study indicate that aqueous saponin is appropriate as a washing agent as well as biodegradation enhancer for the detoxification of PAHs-contaminated low organic carbon soil.

Isolation of mRNAs induced by a hazardous chemical in white‐rot fungus, Coriolus versicolor, by differential display

White‐rot fungus Coriolus versicolor, a ligninolytic basidiomycete, has been studied because of its ability to degrade hazardous chemicals. In this study, we searched for genes that are induced by a hazardous chemical using the mRNA differential‐display technique and C. versicolor IFO30340 that has been exposed to pentachlorophenol (PCP). Five cDNA fragments were cloned and the DNA sequences of two fragments were analyzed in further detail. The clones corresponded to novel genes that have not previously been identified in C. versicolor. One of the cDNAs exhibited strong sequence homology to the gene for an enolase and the other exhibited homology to a heat shock protein. The expression of the two genes was up‐regulated in PCP‐treated C. versicolor.

Treatment of PAHs in contaminated soil by extraction with aqueous DNA followed by biodegradation with a pure culture of Sphingomonas sp

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) in aqueous deoxyribonucleic acid (DNA) solution from contaminated soil washing was investigated. Initial data with a model effluent consisting of anthracene, phenanthrene, pyrene and benzo[a]pyrene that were individually dissolved in 1% aqueous DNA solution confirmed their positive degradation by Sphingomonas sp. at around 10(8)CFU mL(-1) initial cell loading. For anthracene and phenanthrene, complete removal was achieved within 1h treatment. Degradation of pyrene and benzo[a]pyrene took a relatively longer time of a few days and weeks, respectively. DNA-dissolved PAHs were also degraded relatively faster than PAH crystals in aqueous medium to suggest that the binding of the PAHs in the polymer does not pose serious constraint to bacterial uptake. The DNA was stable against the PAH-degrading bacteria. Parallel experiments with actual DNA solutions obtained during pyrene extraction from an artificially spiked soil also showed similar results. Close to 100% pyrene degradation was achieved after 1d treatment. With its chemical stability, the cell-treated DNA was re-used up to four cycles without a considerable decline in extraction performance.

Influence of compost amendment on pyrene availability from artificially spiked soil to two subspecies of Cucurbita pepo

The dissolved organic matter (DOM) fraction of soil organic matter (SOM) may positively contribute to polycyclic aromatic hydrocarbons (PAHs) bioavailability. This work investigated the effects of DOM-rich and PAHs-free compost amendment on the plant uptake of pyrene. Two subspecies of Cucurbita pepo (ssp. pepo cv. Raven and ssp. texana cv. Sunray) were grown for three weeks in a spiked soil containing 83.9 mg kg(-1) pyrene under four different treatments; inorganic fertilizer (IF) alone, 15% (v/v) mixed gardening compost with IF (MX15%+IF), MX30% alone, and no fertilization (NF). Equilibrium pyrene desorptions from a spiked soil (104 mg kg(-1)) under different concentrations (35-590 mg-C L(-1)) of DOM extracts derived from two types of composts including MX and cow manure were also conducted. After harvest, the decrease in the pyrene concentration of the soil ranged from 46-65% for the different treatments. The total dry biomass for both plants was highest under MX15%+IF. The bioconcentration factors of pyrene for both also tended to decrease with increasing MX dose from 15% to 30%. However, the total uptakes of pyrene with IF and MX15%+IF were not statistically different (36.7 and 33.7 microg for Raven, and 5.20 and 7.90 microg for Sunray, respectively). These values were around 100% higher than that with NF (17.4 microg for Raven and 2.0 microg for Sunray). The pyrene desorption data confirmed the ability of DOM to associate with pyrene as indicated by its increase in apparent water solubility. On the basis of these results, MX application at 15% (v/v) does not significantly reduce the phytoextraction of pyrene due to the enhancement of plant growth as well as the possible contribution of DOM fractions to pyrene bioavailability. The application of compost may not pose serious concerns regarding the efficiency of phytoremediation of PAHs-polluted soil.

Effective Removal of Bisphenol a from Contaminated Areas by Recombinant Plant Producing Lignin Peroxidase

We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. Lignin peroxidase is a well-known enzyme used for the degradation of some environmental pollutants. In the present study, we introduced an extracellular fungal enzyme, the lignin peroxidase of Trametes versicolor , into tobacco plants. Six transgenic plant, designated FLP-1, 2, 3, 4, 5 and 8, produced lignin peroxidase in a crude extract of the root. The FLP-1, FLP-2 and FLP-8 were able to remove 10μmol of bisphenol A g -1 dry weight from hydroponic culture. The efficiency of this removal was approximately 4-fold greater than that of control lines. Our results should stimulate efforts to develop plant-based technologies for the removal of environmental pollutants from contaminated environments.

Hybrid aspen with a transgene for fungal manganese peroxidase is a potential contributor to phytoremediation of the environment contaminated with bisphenol A

To assess the possible utility of a fungal gene for manganese-dependent peroxidase (MnP) produced by a transgenic plant in phytoremediation, we transformed hybrid aspen with a chimeric gene for MnP. Our gene construct allowed expression of the gene for MnP in plants and relatively high MnP activity was detected in the hydroponic medium in which roots of plants that expressed the transgene had been cultured. Some of our transgenic plants were able to remove bisphenol A from the medium more efficiently than wild-type plants. Our results demonstrate that, without any modification of the coding sequence, a chimeric gene for fungal MnP can be expressed in a woody plant, with secretion of active MnP from roots into the rhizosphere. Our strategy suggests new options using woody plants for phytoremediation.

Molecular cloning of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene that contributes to the construction of a new transformation system in Coriolus versicolor

The white-rot basidiomycete Coriolus versicolor secretes several enzymes that participate in the degradation of lignin and various persistent organic pollutants. In this study, we attempted to establish a genetic transformation system with a homogenous promoter sequence for driving the gene for antibiotic resistance. We succeeded in cloning the promoter sequence of the gene for glyceraldehyde-3-phosphate dehydrogenase (gpd), which is expressed at high levels in C. versicolor. The expression vector pT7GPTHPT was constructed, which included a gene for resistance to hygromycin B under control of the gpd promoter. The successful selection of transformants on medium that contained hygromycin B indicated that the system should be useful not only for the genetic transformation of C. versicolor, but also for the overproduction of useful fungal enzymes such as laccase and peroxidase.

Specific degradation of β-aryl ether linkage in synthetic lignin (dehydrogenative polymerizate) by bacterial enzymes of Sphingomonas paucimobilis SYK-6 produced in recombinant Escherichia coli

Sphingomonas paucimobilis SYK-6 produces unique and specific enzymes, such asβ-etherases,O-demethylases, and ring fission dioxygenases, for lignin degradation. Cleavage of arylglycerol-β-aryl ether linkage is the most important process in the lignin metabolic pathway ofS. paucimobilis SYK-6. We reported the genes (ligD, ligE, ligF) for enzymes that cleavedβ-aryl ether linkage of dimeric compounds in previous studies. In this study we synthesized the fluorescent high-molecular-weight lignin (UBE-DHP) by dehydrogenative polymerization. We investigated theβ-aryl ether cleavage ability of these enzymes produced in recombinantEscherichia coli. When UBE-DHP was incubated with LigF, 4-methylumbeliferone was released as a result ofβ-aryl ether cleavage of αO-methylumbelliferyl-β-hydroxypropiovanillone (compound III) incorporated in UBE-DHP. Here, we report thatβ-etherase ofS. paucimobilis SYK-6 can be expressed inE. coli and is able to cleave theβ-aryl ether linkage in synthetic high-molecular-weight lignin.

Application of fungal laccase fused with cellulose-binding domain to develop low-lignin rice plants.

We observed a reduction of lignin content linked to the expression of fungal laccase in rice plants. The lignin content of L-4, which showed the highest LAC activity among transgenic lines produced, was lower than that of the control line. However, this change was not reflected to the saccharification efficiency.