Soil Microbiomes and their Arsenic Functional Genes in Chronically High-Arsenic Contaminated Soils.

Microbial arsenic transformations play essential roles in controlling pollution and ameliorating risk. This study combined high-throughput sequencing and PCR-based approaches targeting both the 16 S rRNA and arsenic functional genes to investigate the temporal and spatial dynamics of the soil microbiomes impacted by high arsenic contamination (9.13 to 911.88 mg/kg) and to investigate the diversity and abundance of arsenic functional genes in soils influenced by an arsenic gradient. The results showed that the soil microbiomes were relatively consistent and mainly composed of Actinobacteria (uncultured Gaiellales and an unknown_67 - 14 bacterium), Proteobacteria, Firmicutes (particularly, Bacillus), Chloroflexi, and Acidobacteria (unknown_Subgroup_6). Although a range of arsenic functional genes (e.g., arsM, arsC, arrA, and aioA) were identified by shotgun metagenomics, only the arsM gene was detected by the PCR-based method. The relative abundance of the arsM gene accounted for 0.20%-1.57% of the total microbial abundance. Combining all analyses, arsenic methylation mediated by the arsM gene was proposed to be a key process involved in the arsenic biogeochemical cycle and mitigation of arsenic toxicity. This study advances our knowledge about arsenic mechanisms over the long-term in highly contaminated soils.

Copper and chromium removal from industrial sludge by a biosurfactant-based washing agent and subsequent recovery by iron oxide nanoparticles.

Industrial wastewater treatment generates sludge with high concentrations of metals and coagulants, which can cause environmental problems. This study developed a sequential sludge washing and metal recovery process for industrial sludge containing > 4500 mg/kg Cu and > 5000 mg/kg Cr. The washing agent was formulated by mixing glycolipid, lipopeptide, and phospholipid biosurfactants from Weissella cibaria PN3 and Brevibacterium casei NK8 with a chelating agent, ethylenediaminetetraacetic acid (EDTA). These biosurfactants contained various functional groups for capturing metals. The optimized formulation by the central composite design had low surface tension and contained relatively small micelles. Comparable Cu and Cr removal efficiencies of 37.8% and 38.4%, respectively, were obtained after washing the sludge by shaking with a sonication process at a 1:4 solid-to-liquid ratio. The zeta potential analysis indicated the bonding of metal ions on the surface of biosurfactant micelles. When 100 g/L iron oxide nanoparticles were applied to the washing agent without pH adjustment, 83% Cu and 100% Cr were recovered. In addition, X-ray diffraction and X-ray absorption spectroscopy of the nanoparticles showed the oxidation of nanoparticles, the reduction of Cr(V) to the less toxic Cr(III), and the absorption of Cu. The recovered metals could be further recycled, which will be beneficial for the circular economy.

Development of a plastic waste treatment process by combining deep eutectic solvent (DES) pretreatment and bioaugmentation with a plastic-degrading bacterial consortium.

Polyethylene terephthalate (PET), a petroleum-based plastic, and polylactic acid (PLA), a biobased plastic, have a similar visual appearance thus they usually end up in municipal waste treatment facilities. The objective of this project was to develop an effective PET and PLA waste treatment process that involves pretreatment with deep eutectic solvent (DES) followed by biodegradation with a plastic-degrading bacterial consortium in a composting system. The DES used was a mixture of choline chloride and glycerol, while the bacterial strains (Chitinophaga jiangningensis EA02, Nocardioides zeae EA12, Stenotrophomonas pavanii EA33, Gordonia desulfuricans EA63, Achromobacter xylosoxidans A9 and Mycolicibacterium parafortuitum J101) used to prepare the bacterial consortium were selected based on their ability to biodegrade PET, PLA, and plasticizer. The plastic samples (a PET bottle, PLA cup, and PLA film) were pretreated with DES through a dip-coating method. The DES-coated plastic samples exhibited higher surface wettability and biofilm formation, indicating that DES increases the hydrophilicity of the plastic and facilitates bacterial attachment to the plastic surface. The combined action of DES pretreatment and bioaugmentation with a plastic-degrading bacterial consortium led to improved degradation of PET and PLA samples in various environments, including aqueous media at ambient temperature, lab-scale traditional composting, and pilot-scale composting.

Bioaugmentation with a defined bacterial consortium: A key to degrade high molecular weight polylactic acid during traditional composting.

Polylactic acid (PLA) is commercialized as a compostable bio-thermoplastic. PLA degrades under industrial composting conditions where elevated temperatures are maintained for a long timeframe. However, these conditions cannot be achieved in a non-industrial compost pile. Therefore, this study aims to degrade high molecular weight PLA films by adding a PLA-degrading bacterial consortium (EAc) comprised of Nocardioides zeae EA12, Stenotrophomonas pavanii EA33, Gordonia desulfuricans EA63, and Chitinophaga jiangningensis EA02 during traditional composting. With EAc-bioaugmentation, PLA films (5-30% w/w) had complete disintegration (35 d), 77-82% molecular weight reduction (16 d), and higher CO2 liberation and mineralization than non-bioaugmented composting. Bacterial community analyses showed that EAc-bioaugmentation increased the relative abundance of Schlegelella, a known polymer degrader, and interacted positively with beneficial indigenous microbes like Bacillus, Schlegelella and Thermopolyspora. The bioaugmentation also decreased compost phytotoxicity. Hence, consortium EAc shows potential in PLA-waste treatment applications, such as backyard and small-scale composting.

Formulation of a glycolipid:lipopeptide mixture as biosurfactant-based dispersant and development of a low-cost glycolipid production process.

Biosurfactant-based dispersants were formulated by mixing glycolipids from Weissella cibaria PN3 and lipopeptides from Bacillus subtilis GY19 to enhance the synergistic effect and thereby achieve hydrophilic-lipophilic balance. The proportions of each biosurfactant and dispersant-to-oil ratios (DORs) were varied to obtain the appropriated formulations. The most efficient glycolipid:lipopeptide mixtures (F1 and F2) had oil displacement activities of 81-88% for fuel and crude oils. The baffled flask test of these formulations showed 77-79% dispersion effectiveness at a DOR of 1:25. To reduce the cost of the dispersant, this study optimized the glycolipid production process by using immobilized cells in a stirred tank fermenter. Semicontinuous glycolipid production was carried out conveniently for 3 cycles. Moreover, food wastes, including waste coconut water and waste frying oil, were found to promote glycolipid production. Glycolipids from the optimized process and substrates had similar characteristics but 20-50% lower cost than those produced from basal medium with soybean oil in shaking flasks. The lowest cost dispersant formulation (F2*) contained 10 g/L waste-derived cell-bound glycolipid and 10 g/L lipopeptide and showed high dispersion efficiency with various oils. Therefore, this biosurfactant-based dispersant could be produced on a larger scale for further application.

Bio-based dispersants for fuel oil spill remediation based on the Hydrophilic-Lipophilic Deviation (HLD) concept and Box-Behnken design.

The high density and viscosity of fuel oil leads to its prolonged persistence in the environment and causes widespread contamination. Dispersants with a low environmental impact are necessary for fuel oil spill remediation. This study aimed to formulate bio-based dispersants by mixing anionic biosurfactant (lipopeptides from Bacillus subtilis GY19) with nonionic oleochemical surfactant (Dehydol LS7TH). The synergistic effect of the anionic-nonionic surfactant mixture produced a Winsor Type III microemulsion, which promoted petroleum mobilization. The hydrophilic-lipophilic deviation (HLD) equations for ionic and nonionic surfactant mixtures were compared, and it was found that the ionic equation was applicable for the calculation of lipopeptides and Dehydol LS7TH concentrations. The best formula contained 6.6% w/v lipopeptides and 11.9% w/v Dehydol LS7TH in seawater, and its dispersion effectiveness for bunker fuels A and C was 92% and 78%, respectively. The application of bio-based dispersants in water sources was optimized by Box-Behnken design. The efficiency of the bio-based dispersant was affected by the dispersant-to-oil ratios (DORs) but not by the water salinity. A suitable range of DORs for different oil contamination levels could be identified from the response surface plot. The dispersed fuel oil was further degraded by adding an oil-degrading bacterial consortium to the chemically enhanced water accommodated fractions (CEWAFs). After 7 days of incubation, the concentration of fuel oil was reduced from 3692 mg/L to 356 mg/L (88% removal efficiency). On the other hand, the abiotic control removed less than 40% fuel oil from the CEWAFs. This bio-based dispersant had an efficiency comparable to that of a commercial dispersant. The process of dispersant formulation and optimization could be applied to other surfactant mixtures.

Arsenic speciation, the abundance of arsenite-oxidizing bacteria and microbial community structures in groundwater, surface water, and soil from a gold mine.

The arsenic speciation, the abundance of arsenite-oxidizing bacteria, and microbial community structures in the groundwater, surface water, and soil from a gold mining area were explored using the PHREEQC model, cloning-ddPCR of the aioA gene, and high-throughput sequencing of the 16S rRNA gene, respectively. The analysis of the aioA gene showed that arsenite-oxidizing bacteria retrieved from groundwater, surface water, and soil were associated with Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. In groundwaters from the mining area, there were relatively high ratios of aioA/total 16S rRNA gene copies and the dominance of As5+, which suggested the presence and activity of arsenite-oxidizing bacteria. Metagenomic analysis revealed that the majority of the soil and surface water microbiomes were Proteobacteria, Actinobacteria, Bacteroidetes, and Chloroflexi, whereas the groundwater microbiomes were dominated exclusively by Betaproteobacteria and Alphaproteobacteria. Geochemical factors influencing the microbial structure in the groundwater were As, residence time, and groundwater flowrate, while those showing a positive correlation to the microbial structure in the surface water were TOC, ORP, and DO. This study provides insights into the groundwater, surface water, and soil microbiomes from a gold mine and expands the current understanding of the diversity and abundance of arsenite-oxidizing bacteria, playing a vital role in global As cycling.

Enrichment and characterization of bacterial consortia for degrading 2-mercaptobenzothiazole in rubber industrial wastewater.

Benzothiazoles especially 2-mercaptobenzothiazole (2-MBT) in rubber industrial wastewater can be released into the environment. They can cause adverse health impacts. This study aimed to obtain efficient 2-MBT-degrading bacteria for wastewater application. The bacterial consortia were enriched by incubating rubber wastewater sludge in a medium containing 2-MBT for 28 days. Stepwise acclimatization was conducted with increasing 2-MBT concentrations from 50 to 200 mg L-1 in nitrogen-containing medium for 76 days. The process significantly increased the bacterial number and changed the dominant populations. Among these consortia, the EN consortium from benzothiazole-containing sludge had the highest specific 2-MBT biodegradation rate of 5.2 ± 0.5 mg L-1 day-1 mg protein-1 and could degrade up to 300 mg L-1 2-MBT. From 16S rRNA gene analysis, Pseudomonas was the dominant genus at approximately 70 % of the total population. Stenotrophomonas was the second most abundant populations and have never been reported for 2-MBT biodegradation. The EN consortium removed 65-79 % and 90-93 % of 112 mg L-1 2-MBT and ∼4000 mg L-1 COD in rubber wastewater, respectively, which were significantly higher than the values of natural attenuation. Therefore, the EN consortium could be an ideal inoculum for the post-treatment of benzothiazoles in rubber industrial wastewater.

Formulation of Bio-Based Washing Agent and Its Application for Removal of Petroleum Hydrocarbons From Drill Cuttings Before Bioremediation.

Drill cuttings from petroleum exploration and production sites can cause diverse environmental problems. Total petroleum hydrocarbons (TPHs) are a major pollutant from the use of polyolefin-based mud. As an alternative to incineration, this study investigated the application of surfactant-enhanced washing technology prior to bioremediation. The washing step was necessary because the initial TPH concentrations were quite high at approximately 15% (w/w). Washing agents were formulated by varying the concentration of lipopeptide biosurfactant (in foamate or cell-free broth), Dehydol LS7TH (fatty alcohol ethoxylate 7EO, oleochemical surfactant) and butanol (as a lipophilic linker) at different salinities. The most efficient formula produced a Winsor Type I microemulsion (oil-in-water microemulsion) with polyolefin and contained only 20% (v/v) foamate and 2% (v/v) Dehydol LS7TH in water. Due to the synergistic behavior between the anionic lipopeptides and non-ionic Dehydol LS7TH, the formula efficiently removed 92% of the TPHs from the drill cuttings when applied in a jar test. To reduce the cost, the concentrations of each surfactant should be reduced; thus, the formula was optimized by the simplex lattice mixture design. In addition, cell-free broth, at a pH of 10, containing 3.0 g/L lipopeptides was applied instead of foamate because it was easy to prepare. The optimized formula removed 81.2% of the TPHs and contained 72.0% cell-free broth and 1.4% Dehydol LS7TH in water. A 20-kg soil washing system was later tested where the petroleum removal efficiency decreased to 70.7% due to polyolefin redeposition during separation of the washing solution. The remaining TPHs (4.5%) in the washed drilled cuttings were further degraded by a mixture of Marinobacter salsuginis RK5, Microbacterium saccharophilum RK15 and Gordonia amicalis JC11. To promote TPH biodegradation, biochar and fertilizer were applied along with bacterial consortia in a microcosm experiment. After 49-day incubation, the TPHs were reduced to 0.9% by both physical and biological mechanisms, while the TPHs in the unamended samples remained unaffected. With the use of the formulated bio-based washing agent and bioremediation approach, the on-site treatment of drill cuttings could be conducted with an acceptable cost and low environmental impacts.

Reuse of Immobilized Weissella cibaria PN3 for Long-Term Production of Both Extracellular and Cell-Bound Glycolipid Biosurfactants.

Lactic acid bacteria (LABs) are generally recognized as safe (GRAS), and therefore, LAB biosurfactants are beneficial with negligible negative impacts. This study aims to maintain the biosurfactant producing activity of an LAB strain, Weissella cibaria PN3, by immobilizing the bacterial cells on a commercial porous carrier. For biosurfactant production, 2% soybean oil was used as the carbon source. After 72 h, immobilized cells were reused by replacing production medium. The extracellular and cell-bound biosurfactants were extracted from the resulting cell-free broth and cell pellets, respectively. SEM images of used immobilizing carriers showed increased surface roughness and clogged pores over time. Thus, the immobilizing carriers were washed in PBS buffer (pH 8.0) before reuse. To maintain biosurfactant production activity, immobilized cells were reactivated every three production cycles by incubating the washed immobilizing carriers in LB medium for 48 h. The maximum yields of purified extracellular (1.46 g/L) and cell-bound biosurfactants (1.99 g/L) were achieved in the 4th production cycle. The repeated biosurfactant production of nine cycles were completed within 1 month, while only 2 g of immobilized cells/L were applied. The extracellular and cell-bound biosurfactants had comparable surface tensions (31 - 33 mN/m); however, their CMC values were different (1.6 and 3.2 g/L, respectively). Both biosurfactants had moderate oil displacement efficiency with crude oil samples but formed emulsions well with gasoline, diesel, and lavender, lemongrass and coconut oils. The results suggested that the biosurfactants were relatively hydrophilic. In addition, the mixing of both biosurfactants showed a synergistic effect, as seen from the increased emulsifying activity with palm, soybean and crude oils. The biosurfactants at 10 - 16 mg/mL showed antimicrobial activity toward some bacteria and yeast but not filamentous fungi. The molecular structures of these biosurfactants were characterized by FTIR as different glycolipid congeners. The biosurfactant production process by immobilized Weissella cibaria PN3 cells was relatively cheap given that two types of biosurfactants were simultaneously produced and no new inoculum was required. The acquired glycolipid biosurfactants have high potential to be used separately or as mixed biosurfactants in various products, such as cleaning agents, food-grade emulsifiers and cosmetic products.

Inhibitory effect of phenol on wastewater ammonification.

This study aimed to elucidate inhibitory effect of phenol on ammonification of dissolved organic nitrogen (DON) in wastewater. Laboratory incubation experiments were conducted using primary and secondary effluent samples spiked with phenol (100-1000 mg/L) and inoculated with mixed cultures, pure strains of phenol-degrading bacteria (Acinetobacter sp. and Pseudomonas putida F1), and/or an ammonia oxidizing bacterium (Nitrosomonas europaea). DON concentration was monitored with incubation time. Phenol suppressed the ammonification rate of DON up to 62.9%. No or minimal ammonification inhibition was observed at 100 mg/L of phenol while the inhibition increased with increasing phenol concentration from 250 to 1000 mg/L. The inhibition was curtailed by the presence of the phenol-degrading bacteria. DON was ammonified in the samples inoculated with only N. europaea and the ammonification was also inhibited by phenol. The findings suggest that high phenol in wastewater could result in low ammonification and high DON in the effluent.

Bioaugmentation coupled with phytoremediation for the removal of phenolic compounds and color from treated palm oil mill effluent.

The potential for coupling bioaugmentation with phytoremediation to simultaneously treat and utilize treated palm oil mill effluent (TPOME) in animal feed production was determined from a reduction in phenolic compounds and color in soil leachates, as well as from an increased yield of pasture grass. Two phenol-degrading bacteria-Methylobacterium sp. NP3 and Acinetobacter sp. PK1-were inoculated into the Brachiaria humidicola rhizosphere before the application of TPOME. A pot study showed that the soil with both grass and inoculated bacteria had the highest dephenolization and decolorization efficiencies, with a maximum capability of removing 70% from 587 mg total phenolic compounds added and 73% from 4438 color units during ten TPOME application cycles. The results corresponded to increases in the number of phenol-degrading bacteria and the grass yield. In a field study, this treatment was able to remove 46% from 21,453 mg total phenolic compounds added, with a maximum color removal efficiency of 52% from 5105 color units, while the uninoculated plots removed about 24-39% and 29-46% of phenolic compounds and color, respectively. The lower treatment performance was probably due to the increased TPOME concentrations. Based on the amounts of phenolic compounds, protein, and crude fiber in the grass biomass, the inoculated TPOME-treated grass had a satisfactory nutritional quality and digestibility for use as animal feed.

Potential microbial consortium involved in the biodegradation of diesel, hexadecane and phenanthrene in mangrove sediment explored by metagenomics analysis.

Hydrocarbon contamination is a serious problem that degrades the quality of mangrove ecosystems, and bioremediation using autochthonous bacteria is a promising technology to recover an impacted environment. This research investigates the biodegradation rates of diesel, hexadecane and phenanthrene, by conducting a microcosm study and survey of the autochthonous microbial community in contaminated mangrove sediment, using an Illumina MiSeq platform. The biodegradation rates of diesel, hexadecane and phenanthrene were 82, 86 and 8 mg kg-1 sediment day-1, respectively. The removal efficiencies of hexadecane and phenanthrene were >99%, whereas the removal efficiency of diesel was 88%. A 16S rRNA gene amplicon sequence analysis revealed that the major bacterial assemblages detected were Gammaproteobacteria, Deltaproteobacteria, Alphaproteobacteria. The bacterial compositions were relatively constant, while reductions of the supplemented hydrocarbons were observed. The results imply that the autochthonous microorganisms in the mangrove sediment were responsible for the degradation of the respective hydrocarbons. Diesel-, hexadecane- and phenanthrene-degrading bacteria, namely Bacillus sp., Pseudomonas sp., Acinetobacter sp. and Staphylococcus sp., were also isolated from the mangrove sediment. The mangrove sediment provides a potential resource of effective hydrocarbon-degrading bacteria that can be used as an inoculum or further developed as a ready-to-use microbial consortium for the purpose of bioremediation.

Formulation of crude oil spill dispersants based on the HLD concept and using a lipopeptide biosurfactant.

Solvent-free dispersants for crude oil spills were formulated based on the hydrophilic-lipophilic deviation (HLD) concept and using lipopeptides from Bacillus sp. GY19. The lipopeptides were recovered and concentrated from cell-free broth by foam fractionation and freeze-drying. They had good surface activity under varying temperatures, pH and NaCl levels. Moreover, the lipopeptides had low toxicity to copepods (LC50 1174mg/L) and whiteleg shrimp (LC50 1050mg/L). The characteristic curvature (Cc) of the lipopeptides showed that they were more hydrophobic (Cc 4.93) than sodium dihexyl sulfosuccinate (SDHS, Cc -0.92). The HLD equation was used to calculate the lipopeptide and the SDHS fractions in the dispersant formulations according to the equivalent alkane carbon number (EACN) of hydrocarbons and seawater salinity. The molar fraction of lipopeptides increased with increasing EACN. The lipopeptide-SDHS mixtures formed microemulsion Type III with specific hydrocarbons and crude oils. Oil displacement and baffled flask tests showed that the formulations reduced the interfacial tension and solubilized crude oil in the water column at higher efficiency than commercial dispersants or lipopeptides alone. In summary, the effectiveness of the lipopeptide-based dispersant corresponded to the optimal HLD.

Airlift bioreactor containing chitosan-immobilized Sphingobium sp. P2 for treatment of lubricants in wastewater.

An internal loop airlift bioreactor containing chitosan-immobilized Sphingobium sp. P2 was applied for the removal of automotive lubricants from emulsified wastewater. The chitosan-immobilized bacteria had higher lubricant removal efficiency than free and killed-immobilized cells because they were able to sorp and degrade the lubricants simultaneously. In a semi-continuous batch experiment, the immobilized bacteria were able to remove 80-90% of the 200 mg L(-1) total petroleum hydrocarbons (TPH) from both synthetic and carwash wastewater. The internal loop airlift bioreactor, containing 4 g L(-1) immobilized bacteria, was later designed and operated at 2.0 h HRT (hydraulic retention time) for over 70 days. At a steady state, the reactor continuously removed 85±5% TPH and 73±11% chemical oxygen demand (COD) from the carwash wastewater with 25-200 mg L(-1) amended lubricant. The internal loop airlift reactor's simple operation and high stability demonstrate its high potential for use in treating lubricants in emulsified wastewater from carwashes and other industries.

Isolation and application of Gordonia sp. JC11 for removal of boat lubricants.

Boat lubricants are continuously released into the marine environment and thereby cause chronic oil pollution. This study aims to isolate lubricant-degrading microorganisms from Thai coastal areas as well as to apply a selected strain for removal of boat lubricants. Ten microorganisms in the genera of Gordonia, Microbacterium, Acinetobacter, Pseudomonas, Brucella, Enterococcus and Candida were initially isolated by crude oil enrichment culture techniques. The lubricant-removal activity of these isolates was investigated with mineral-based lubricants that had been manufactured for the 4-stroke diesel engines of fishing boats. Gordonia sp. JC11, the most effective strain was able to degrade 25-55% of 1,000 mg L(-1) total hydrocarbons in six tested lubricants, while only 0-15% of the lubricants was abiotically removed. The bacterium had many characteristics that promoted lubricant degradation such as hydrocarbon utilization ability, emulsification activity and cell surface hydrophobicity. For bioaugmentation treatment of lubricant contaminated seawater, the inoculum of Gordonia sp. JC11 was prepared by immobilizing the bacterium on polyurethane foam (PUF). PUF-immobilized Gordonia sp. JC11 was able to remove 42-56% of 100-1,000 mg L(-1) waste lubricant No. 2 within 5 days. This lubricant removal efficiency was higher than those of free cells and PUF without bacterial cells. The bioaugmentation treatment significantly increased the number of lubricant-degrading microorganisms in the fishery port seawater microcosm and resulted in rapid removal of waste lubricant No. 2.

Cometabolic degradation of trichloroethene by Rhodococcus sp. strain L4 immobilized on plant materials rich in essential oils.

The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 muM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE.

Diversity and activity of PAH-degrading bacteria in the phyllosphere of ornamental plants.

Phyllosphere bacteria on ornamental plants were characterized based on their diversity and activity towards the removal of polycyclic aromatic hydrocarbons (PAHs), the major air pollutants in urban area. The amounts of PAH-degrading bacteria were about 1-10% of the total heterotrophic phyllosphere populations and consisted of diverse bacterial species such as Acinetobacter, Pseudomonas, Pseudoxanthomonas, Mycobacterium, and uncultured bacteria. Bacterial community structures analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis from each plant species showed distinct band patterns. The uniqueness of these phyllosphere bacterial communities was partly due to the variation in leaf morphology and chemical properties of ornamental plants. The PAH degradation activity of these bacteria was monitored in gas-tight systems containing sterilized or unsterilized leaves. The results indicated that phyllosphere bacteria on unsterilized leaves were able to enhance the activity of leaves for phenanthrene removal. When compared between plant species, phenanthrene removal efficiency corresponded to the size of phenanthrene-degrading bacteria. In addition, phyllosphere bacteria on Wrightia religiosa were able to reduce other PAHs such as acenaphthylene, acenaphthene, and fluorine in 60-ml glass vials and in a 14-l glass chamber. Thus, phyllosphere bacteria on ornamental plants may play an important role in natural attenuation of airborne PAHs in urban areas.

Trichloroethylene cometabolic degradation by Rhodococcus sp. L4 induced with plant essential oils.

Cometabolic degradation of TCE by toluene-degrading bacteria has the potential for being a cost-effective bioremediation technology. However, the application of toluene may pose environmental problems. In this study, several plant essential oils and their components were examined as alternative inducer for TCE cometabolic degradation in a toluene-degrading bacterium, Rhodococcus sp. L4. Using the initial TCE concentration of 80 microM, lemon and lemongrass oil-grown cells were capable of 20 +/- 6% and 27 +/- 8% TCE degradation, which were lower than that of toluene-grown cells (57 +/- 5%). The ability of TCE degradation increased to 36 +/- 6% when the bacterium was induced with cumin oil. The induction of TCE-degrading enzymes was suggested to be due to the presence of citral, cumin aldehyde, cumene, and limonene in these essential oils. In particular, the efficiency of cumin aldehyde and cumene as inducers for TCE cometabolic degradation was similar to toluene. TCE transformation capacities (T (c)) for these induced cells were between 9.4 and 15.1 microg of TCE mg cells(-1), which were similar to the known toluene, phenol, propane or ammonia degraders. Since these plant essential oils are abundant and considered non-toxic to humans, they may be applied to stimulate TCE degradation in the environment.

Characterization of Anopheles minimus CYP6AA3 expressed in a recombinant baculovirus system.

Metabolism by cytochrome P450 monooxygenases is a major mechanism implicated in resistance of insects to insecticides, including pyrethroids. We previously isolated the cytochrome P450 CYP6AA3 from deltamethrin-selected resistant strain of Anopheles minimus mosquito, a major malaria vector in Thailand. In the present study, we further investigated the role of CYP6AA3 enzyme in deltamethrin metabolism in vitro. The CYP6AA3 was expressed in Spodoptera frugiperda (Sf9) insect cells via baculovirus-mediated expression system. The enzymatic activity of CYP6AA3 in deltamethrin metabolism was characterized after being reconstituted with An. minimus NADPH-cytochrome P450 reductase and a NADPH-regenerating system. The contribution of CYP6AA3 responsible for deltamethrin metabolism was determined by measurement of deltamethrin disappearance following the incubation period and deltamethrin-derived compounds were detected using combined gas chromatography mass spectrometry analysis. 3-Phenoxybenzaldehyde was a major product of CYP6AA3-mediated deltamethrin metabolism. Deltamethrin degradation and formation of metabolites were NADPH-dependent and inhibited by piperonyl butoxide. Deltamethrin was catalyzed by CYP6AA3 with an apparent K(m) of 80.0 +/- 2.0 and V(max) of 60.2 +/- 3.6 pmol/min/pmol P450. Furthermore, deltamethrin cytotoxicity assays by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue dye exclusion were examined in Sf9 insect cells, with and without expression of CYP6AA3. Results revealed that CYP6AA3 could play a role in detoxifying deltamethrin in the cells. Thus, the results of this study support the role of CYP6AA3 in deltamethrin metabolism.