Assessing microbial stability and predicting biogas production in full-scale thermophilic dry methane fermentation of municipal solid waste.

Compared to typical anaerobic digestion processes, little is known about both sludge microbial compositions and biogas production models for full-scale dry methane fermentation treating municipal solid waste (MSW). The anaerobic sludge composed of one major hydrogenotrophic methanogen (Methanoculleus) and syntrophic acetate oxidizing bacteria (e.g., Caldicoprobacter), besides enrichment of MSW degraders such as Clostridia. The core population remained phylogenetically unchanged during the fermentation process, regardless of amounts of MSW supplied (∼35 ton/d) or biogas produced (∼12000 Nm3/d). Based on the correlations observed between feed amounts of MSW from 6 days in advance to the current day and biogas output (the strongest correlation: r = 0.77), the best multiple linear regression (MLR) model incorporating the temperature factor was developed with a good prediction for validation data (R2 = 0.975). The proposed simple MLR method with only data on the feedstock amounts will help decision-making processes to prevent low-efficient biogas production.

Bacterial Community Coexisting with White-Rot Fungi in Decayed Wood in Nature.

Lignin-decomposing ability of several bacteria and the degradation mechanism have been revealed in vitro. However, the abundance of such bacteria in decayed wood in nature remains unknown at genus and species levels. This study was aimed at identifying bacterial communities in the decayed wood coexisting with white-rot fungi, which play a potential role in lignin degradation, and predicting the functional profile of bacterial lignin degradation in wood via bacterial community analyses. The bacterial flora of forest soil and four decayed wood samples showed marked differences; particularly, in addition to Methylobacterium and Acidibrevibacterium, sphingomonads, which degrade the major skeleton of lignin in vitro, were more abundant in the decayed wood than in forest soil, suggesting that multiple bacteria were involved in lignin degradation. The bacterial community in the decayed wood was more influenced by wood type and lignin structure than the fungal species observed in the decayed wood.

Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida.

Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

Heterologous expression of Trametes versicolor laccase in Saccharomyces cerevisiae.

Laccase is used in various industrial fields, and it has been the subject of numerous studies. Trametes versicolor laccase has one of the highest redox potentials among the various forms of this enzyme. In this study, we optimized the expression of laccase in Saccharomyces cerevisiae. Optimizing the culture conditions resulted in an improvement in the expression level, and approximately 45 U/L of laccase was functionally secreted in the culture. The recombinant laccase was found to be a heavily hypermannosylated glycoprotein, and the molecular weight of the carbohydrate chain was approximately 60 kDa. These hypermannosylated glycans lowered the substrate affinity, but the optimum pH and thermo-stability were not changed by these hypermannosylated glycans. This functional expression system described here will aid in molecular evolutionary studies conducted to generate new variants of laccase.

Expression of a fungal laccase fused with a bacterial cellulose-binding module improves the enzymatic saccharification efficiency of lignocellulose biomass in transgenic Arabidopsis thaliana.

Delignification is effective for improving the saccharification efficiency of lignocellulosic biomass materials. We previously identified that the expression of a fungal laccase (Lac) fused with a bacterial cellulose-binding module domain (CBD) improved the enzymatic saccharification efficiency of rice plants. In this work, to evaluate the ability of the Lac-CBD fused chimeric enzyme to improve saccharification efficiency in a dicot plant, we introduced the chimeric gene into a dicot model plant, Arabidopsis thaliana. Transgenic plants expressing the Lac-CBD chimeric gene showed normal morphology and growth, and showed a significant increase of enzymatic saccharification efficiency compared to control plants. The transgenic plants with the largest improvement of enzymatic saccharification efficiency also showed an increase of crystalline cellulose in their cell wall fractions. These results indicated that expression of the Lac-CBD chimeric protein in dicotyledonous plants improved the enzymatic saccharification of plant biomass by increasing the crystallinity of cellulose in the cell wall.

In vitro regeneration and Agrobacterium-mediated transformation of male- sterile marigold (Tagetes erecta L.).

In this study, we devised a method for the in vitro regeneration and subsequent genetic transformation of male sterile marigold. To our knowledge, this is the first report of generation of transgenic plants with a single genotype of marigold via Agrobacterium-mediated transformation. We obtained four transgenic lines from two independent experiments with 496 leaf explants, which were inoculated by an Agrobacterium strain LBA4404 harboring the plasmid, pIG121-Hm. Although the efficiency of the transformation in our system was low, stable expression of uidA gene in adventitious shoots and compound leaves could be detected in ß-glucuronidase histochemical analysis. This protocol contributes to the progress of genetic studies and molecular breeding of this species.

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.

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 10(8) 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.

Nature of bioavailability of DNA-intercalated polycyclic aromatic hydrocarbons to Sphingomonas sp.

The nature of bioavailability of DNA-intercalated PAHs in aqueous solution was investigated. The degradability of different PAHs including anthracene, phenanthrene and pyrene by Sphingomonas sp. was not inhibited even at a high DNA concentration of 2%. The DNA was stable against the PAH-degrader as indicated by the unchanged electrophoresis gel chromatograms after treatment. This shows that a structural change in the polymer is not necessary for the release of PAHs. Partitioning experiments using phenanthrene as a model PAH illustrated the presence of an initial passive uptake by autoclaved cells. Subsequent intracellular degradation became apparent from parallel data with live cells. Phenanthrene transfer from the DNA was diffusion-controlled and the exit of this molecule from their intercalation sites is favored in lieu of the presence of stronger hydrophobic binding sites in the cell membrane.

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.

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.

Removal of polycyclic aromatic hydrocarbons from contaminated soil by aqueous DNA solution.

An aqueous DNA solution was applied for the extraction of polycyclic aromatic hydrocarbons (PAHs) from a spiked soil. Solubilities of 0.56, 11.78, and 11.24 mg L(-1) for anthracene, phenanthrene, and pyrene, respectively, were achieved after 1 day equilibration in 1% DNA. Using a spiked soil that contained 72 mg kg(-1) anthracene, 102 mg kg(-1) phenanthrene, and 99 mg kg(-1) pyrene, extractions of close to 88, 78, and 94%, respectively, were attained with 5% DNA at a 1:50 soil/extractant ratio. Maximum PAH dissolution occurred after 4-6 h. Comparative tests showed the main advantage of DNA over methyl-beta- and gamma-cyclodextrins and Tween 80 for pyrene removal. An ionic strength of 0.1 M NaCl was found necessary for maximum PAH dissolution and extraction. The performance of hexane regenerated DNA also remained stable after three stages of recycling. These results show the huge potential of DNA as an aqueous washing agent for PAH-contaminated soil.

Transgenic tobacco expressing fungal laccase promotes the detoxification of environmental pollutants.

The phytoremediation of soils contaminated with organic pollutants offers a low-cost method for removal of such pollutants. We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. In the present study, we introduced an extracellular fungal enzyme, the laccase of Coriolus versicolor, into tobacco plants. One transgenic plant, designated FL4, produced laccase that was secreted into the rhizosphere. FL4 was able to remove 20 micromol bisphenol A or pentachlorophenol per gram dry weight. The efficiency of this removal was apparently 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.

Overproduction of recombinant laccase using a homologous expression system in Coriolus versicolor.

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants microg(-1) of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.

Structure of genes for Hsp30 from the white-rot fungus Coriolus versicolor and the increase of their expression by heat shock and exposure to a hazardous chemical.

We isolated and analysed two genomic DNAs that encode the heat-shock protein Hsp30 from Coriolus versicolor. The amino acid sequences substitute only three amino acid substitutions. The promoter regions contain the consensus heat-shock element, a xenobiotic-response element, a stress-response element, and a metal-response element. The levels of mRNAs for Hsp30 increased markedly after exposure of C. versicolor to pentachlorophenol and levels were higher than those after heat shock.