Enzymatic degradation of anthracene, dibenzothiophene and pyrene by manganese peroxidase in media containing acetone

The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) greatly hamper their degradation in liquid media. The use of an organic solvent can assist the degradative action of ligninolytic enzymes from white rot fungi. The enzymatic action of the enzyme manganese peroxidase (MnP) in media containing a miscible organic solvent, acetone (36% v/v), was evaluated as a feasible system for the in vitro degradation of three PAHs: anthracene, dibenzothiophene and pyrene. These compounds were degraded to a large extent after a short period of time (7, 24 and 24h, respectively), at conditions maximizing the MnP-oxidative system. The initial amount of enzyme present in the reaction medium was determinant for the kinetics of the process. The order of degradability, in terms of degradation rates was as follows: anthracene>dibenzothiophene>pyrene. The intermediate compounds were determined using gas chromatography-mass spectrometry and the degradation mechanisms were proposed. Anthracene was degraded to phthalic acid. A ring cleavage product of the oxidation of dibenzothiophene, 4-methoxybenzoic acid, was also observed.     

Biphenyl degradation kinetics by Burkholderia xenovorans LB400 in two-phase partitioning bioreactors

Biphenyl could be successfully degraded by Burkholderia xenovorans LB400, initially described as Pseudomonas sp. LB400, in two-phase partitioning bioreactors (TPPBs). TPPBs are comprised of an aqueous, cell containing phase, and an immiscible, biocompatible organic phase that partitions toxic and/or poorly soluble substrates (in this case biphenyl) based on maintaining a thermodynamic equilibrium. The critical LogKO/W of the organism was found to be approximately 5.5, indicating that solvents with a LogKO/W larger than 5.5 are suitable as delivery phases for B. xenovorans LB400. Two solvents selected for the TPPB system were octadecene and bis(2-ethylhexyl)sebacate (BES). In one experiment a total of 6.6 g biphenyl per l aqueous-phase-equivalent (biphenyl delivered in solvent, at an aqueous phase to solvent ratio of 10) could be degraded in 25 h during batch operation with octadecene. The specific growth rate and the half saturation constant of the Monod model were estimated to be mumax=0.25 h-1 and KS=0.0001 g l-1, and the yield coefficient was YX/S=0.48 g biomass per g biphenyl. These parameter estimates were used to predict the time course of biphenyl degradation at different initial substrate concentrations and with biphenyl delivered from the two solvents with different partitioning behaviour for biphenyl. The predictions were validated by experimental data, confirming the microbial kinetics as well as the expected partitioning effects.     

Response of a solid-liquid two-phase partitioning bioreactor to transient BTEX loadings

A two-phase partitioning bioreactor (TPPB) consisting of an aqueous phase containing a bacterial consortium and a polymeric phase of silicone rubber pellets (solid volume fraction 0.1) was used to treat a gaseous waste stream containing benzene, toluene, ethylbenzene and o-xylene (BTEX). The function of the solid polymer phase was to absorb/desorb the gaseous volatile organic compounds providing a buffering effect to protect the cells from high transient loadings and to sequester the BTEX for subsequent degradation. The TPPB was subjected to high and fluctuating inlet loadings of BTEX in the form of 4h step changes of 2, 4, 6 and 10 times the nominal inlet loading of 60 gm(-3) h(-1) total BTEX in approximately equal amounts, and removal efficiencies and elimination capacities were determined. It was found that overall removal efficiencies of greater than 95% can be achieved while obtaining overall elimination capacities of up to 282 gm(-3) h(-1) during transient operation and TPPB operation succumbs to toxic substrate levels between step changes of 6 and 10 times the nominal loading value (360-600 gm(-3) h(-1)). BTEX concentrations in the aqueous phase and the polymer phase of the TPPB were monitored throughout the imposed step changes to determine the extent to which the sequestering phase can buffer the aqueous phase from BTEX. With the polymer phase comprising only 10% of the total working volume of the reactor, the polymer beads accounted for up to 93%, 91% and 70% of the total BTEX present in the working volume for step changes of 2, 4 and 6 times the nominal loading, respectively.     

Ratio of the concentration of anthraquinone to anthracene in coastal marine sediments

The ratio of the concentration of the oxidation product anthraquinone to that of its parent polycyclic aromatic hydrocarbon anthracene is reported for several coastal marine sediments. The ratio ranges from 0.317 in a highly contaminated industrialized harbor to 2.81 in a remote, less contaminated site. We hypothesize that differences in this ratio result from the input source of PAHs, with input from atmospheric deposition at remote sites resulting in a predominance of anthraquinone (ratio > 1), and direct discharge to highly contaminated industrialized harbors resulting in a predominance of anthracene (ratio < 1). To support this hypothesis, the fate of anthracene in the marine environment was investigated with respect to conversion to its oxidation product, anthraquinone. Once associated with sediments, anthracene is believed to be relatively persistent; however, it can potentially be subjected to oxidation via biological (microbial degradation) and chemical (chemical oxidation and photooxidation) processes. An assessment of the extent of oxidation of anthracene associated with sediments was conducted both under conditions simulating those found in the marine environment and under rigorous conditions by exposure to UV radiation. Results of this study show that while anthracene associated with marine sediments does not readily undergo oxidation to anthraquinone under conditions normally encountered in the marine environment, under extreme conditions anthracene is photooxidized by exposure to UV radiation. The extent of oxidation is influenced by sediment characteristics such as percent organic carbon, humic acid content and sediment surface area. The relative stability of anthracene under normal conditions may help to validate the use of the anthraquinone to anthracene ratio in marine sediments as an environmental marker of contaminant source.     

Combined UV-biological degradation of PAHs

The UV-photolysis of PAHs was tested in silicone oil and tetradecane. In most cases, the degradation of a pollutant provided within a mixture was lower than when provided alone due to competitive effects. With the exception of anthracene, the larger pollutants (4- and 5-rings) were always degraded first, proving that UV-treatment preferentially acts on large PAHs and thereby provides a good complement to microbial degradation. UV-photolysis was also found to be suitable for treatment of soil extract from contaminated soils. The feasibility of UV-biological treatment was demonstrated for the removal of a mixture of phenanthrene and pyrene in silicone oil. UV-irradiation of the silicone oil led to 83% pyrene removal but no phenanthrene photodegradation. Subsequent treatment of the oil in a two-phases partitioning bioreactor (TPPB) system inoculated with Pseudomonas sp. was followed by complete phenanthrene biodegradation but no further pyrene removal. Totally, the combined process allowed 92% removal of the PAH mixture. Further work should focus on characterizing the photoproducts formed and studying the influence of the solvent on the photodegradation process.     

Sequential UV-biological degradation of polycyclic aromatic hydrocarbons in two-phases partitioning bioreactors

A method based on UV-irradiation in organic solvent followed by transfer of the remaining pollutants into silicone oil for subsequent biodegradation in a biphasic system inoculated with a phenanthrene degrading Pseudomonas sp. was tested for the treatment of various mixtures of PAHs. Acetone was first selected as the most suitable solvent compared to methanol, acetonitrile and silicone oil for the removal of pyrene and phenanthrene. The sequential treatment was then applied to the treatment of a mixture of fluorene, phenanthrene, anthracene, fluoranthrene, pyrene, benzo(a)anthracene and benzo(a)pyrene in acetone. These compounds were photodegraded in the following order of initial removal rates (mg l(-1) d(-1)): benzo(a)pyrene (7.8) > anthracene (5.0) > benzo(a)anthracene (2.5) > fluoranthrene (1.8) > pyrene (1.5) > phenanthrene (1.2) > fluorene (0.2). UV-treatment allowed complete removal of, anthracene, benzo(a)anthracene and benzo(a)pyrene and removals of 63% of pyrene and 37% of fluorene after 434 h or irradiation. The subsequent biological treatment removed the remaining phenanthrene and fluorene by 100% and 90%, respectively, after 790 h of cultivation. Although less efficient due to the presence of interfering compounds, the UV-biological treatment of a soil extract allowed a 63% removal of the seven PAHs named above. Microbial growth did not occur when the pollutants were directly supplied to the microorganism showing that biphasic systems reduced the toxicity effects cause by mixtures of PAHs at high concentrations. This study demonstrates the potential of selective UV treatment of high molecular weight PAHs followed by biological treatment of the low molecular weight species in biphasic systems.     

Hydrolytic and ligninolytic enzyme activities in the Pb contaminated soil inoculated with litter-decomposing fungi

The impact of Pb contamination was tested to five hydrolytic (beta-glucosidase, beta-xylosidase, beta-cellobiosidase, alpha-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40 g Pb of kg ww soil(-1)) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at H?lv?l? (37 g Pb of kg ww soil(-1)) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40 g Pb of kg ww soil(-1)) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities beta-cellobiosidase and beta-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40 g Pb of kg ww soil(-1)) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil.     

两相分配式生物反应器处理高盐废水中的苯酚

采用新型两相分配式生物反应器(TPPB)和前期研究得到的高效苯酚降解菌对高盐废水中苯酚的降解进行研究,研究中确定煤油为反应系统的最佳有机溶剂,并考察了废水苯酚含量、废水盐度以及搅拌器搅拌速度对苯酚降解的影响。结果表明,反应系统能正常降解苯酚含量为1 000～2 500 mg/L的高盐苯酚废水;反应系统在含盐量为100 g NaCl/L、搅拌速度为50 r/min的运行工况条件下,降解时间缩短为52 h,总酚去除率为20.58 mg/(L.h)。     

Degradation of tetraphenylphosphonium bromide at high pH and its effect on radionuclide solubility

Recently, tetraphenylphosphonium bromide (TPPB) has been used to remove technetium from some radioactive waste streams. However, before TPPB could be approved for use it was necessary to show that TPPB and its degradation products would not have a significant detrimental effect on post-closure performance of a radioactive waste repository. TPPB is known to be stable at neutral pH, however, under alkaline conditions it degrades by an alkaline hydrolysis mechanism to triphenylphosphonium oxide (TPPO). Degradation can also occur by radiolysis to produce triphenylphosphine (TPP). The kinetics of the alkaline hydrolysis degradation of TPPB is described and the solubility of europium, iodine, nickel, technetium(VII) and uranium(VI) in aqueous solutions of TPPB and its degradation products is reported. These results were used to support the use of TPPB in removing technetium from some waste streams.     

Manganese peroxidase-catalyzed oxidative degradation of vanillylacetone

When 4-(4-hydroxy-3-methoxy-phenyl)-2-butanone (vanillylacetone) was tested for manganese peroxidase (MnP)-catalyzed oxidation, it was found to be degraded with the cleavage of an aromatic ring. Among numerous products of vanillylacetone oxidation, four major ones were purified by thin-layer chromatography and identified using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analysis. Three of them maintained the aromatic ring structure and were identified as 4-[6,2'-dihydroxy-5,3'-dimethoxy-5'-(3-oxo-butyl)-biphenyl]-butan-2-one, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and 4-[6,2'-dihydroxy-5,3'-dimethoxy-5'-(3-oxo-butyl)-biphenyl]-3-buten-2-one. Even though the fourth product could not be purified to a single compound, data from infrared spectroscopy showed that it did not have a benzene ring. From MS and NMR analysis, 3-(3-oxo-butyl)-hexa-2,4-dienedioic acid-1-methyl ester was tentatively suggested as the dominant species. The reaction mechanism was suggested on the basis of the structural information of these products. To our knowledge, this paper is the first report on aromatic ring cleavage of the phenolic compound by MnP.     

Degradation of the antifouling compound Irgarol 1051 by manganese peroxidase from the white rot fungus Phanerochaete chrysosporium

Irgarol 1051 (2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine), a derivative of s-triazine herbicide, is an antifouling compound used as an alternative to organotins. The compound is highly persistent and is known to be biodegraded only by the white rot fungus, Phanerochaete chrysosporium. We used partially purified manganese peroxidase (MnP) prepared from P. chrysosporium to evaluate its capacity to degrade Irgarol 1051. MnP degraded Irgarol 1051 to two major products, one identified as M1 (identical to GS26575, 2-methylthio-4-tert-butylamino-6-amino-s-triazine) and the other not identified but with same mass spectrum as M1 and a different ultraviolet spectrum. This report clearly demonstrates that this ligninolytic enzyme is involved in the degradation of Irgarol 1051.     

Dissipation of polycyclic aromatic hydrocarbons from soil added with manure or vermicompost

The dissipation of three PAHs, i.e., 500 mg phenanthrene kg(-1) soil, 350 mg anthracene kg(-1) soil and 150 mg benzo(a)pyrene kg(-1) soil, was investigated in soil from Acolman (México) added with cow manure or vermicompost while production of CO(2) and inorganic N was monitored. At day 0, recovery of added phenanthrene was 95%, anthracene 96% and benzo(a)pyrene 100% in sterilized soil and concentrations did not change significantly in sterilized soil over time. Application of organic material did not affect the concentration of phenanthrene and anthracene, which decreased sharply in the unsterilized soil in the first weeks of the incubation. Less than 3% of the added phenanthrene was detected after 100 days and less than 8.5% of the added anthracene (mean of the two experiments). The decrease in concentration of benzo(a)pyrene (BaP) was not fast as that of phenathrene and anthracene, and 22% was extractable from soil still after 100days. It was concluded that addition of farm yard manure (FYM) and vermicompost only had an effect on the initial dissipation of phenanthrene, anthracene and benzo(a)pyrene in soil of Acolman.     

Decolorization of RBBR by plant cells and correlation with the transformation of PCBs

An extracellular H2O2-requiring Remazol Brilliant Blue R (RBBR) decolorizing enzyme activity was detected after cultivation of cells of various plant species both in liquid medium and when growing on agar plates containing RBBR. Level of the enzyme activity was compared with the ability to metabolize polychlorinated biphenyls (PCBs). The ability to decolorize RBBR was tested in the presence and absence of PCBs. The cultures with high PCB-transforming activity proved to exhibit RBBR oxidase much more resistant towards the influence of PCBs. In addition low activities of lignin peroxidase (LiP) and manganese dependent peroxidase (MnP) were detected in medium and in plant cells. No correlation of MnP and LiP activities with PCB degradation could be found. The RBBR decolorization could be used as a rough screening method for plant cultures able to metabolize PCBs.     

Removal of estrogenic activity of natural steroidal hormone estrone by ligninolytic enzymes from white rot fungi

Natural steroidal hormone estrone (E1) was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. E1 decreased by 98% after 5 d of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, which suggested that the disappearance of E1 is related to ligninolytic enzymes produced extracellularly by white rot fungus. Therefore, E1 was treated with MnP and laccase prepared from the culture of white rot fungi. HPLC analysis demonstrated that E1 disappeared completely in the reaction mixture after 1 h of treatment with either MnP or laccase. Using the yeast two-hybrid assay system, it was also confirmed that both enzymatic treatments completely removed the estrogenic activity of E1 after 2 h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of E1.     

Synthesis of zirconia-immobilized copper chelates for catalytic decomposition of hydrogen peroxide and the oxidation of polycyclic aromatic hydrocarbons

Chelating sorbents with diethylenetriaminepenta(methylene-phosphonic acid) (DTPMPA) and ethylenediaminetetraacetic acid ligands immobilized on zirconia matrix were prepared and subsequently saturated with Cu(II). All the Cu chelates catalyzed decomposition of H(2)O(2) yielding highly reactive hydroxyl radicals. All of them were also able to catalyze degradation of polycyclic aromatic hydrocarbons (anthracene, benzo[a]pyrene and benzo[b]fluoranthene). The most effective DTPMPA-based catalysts G-32 and G-35 (10 mg ml(-1) with 100 mmol H(2)O(2)) caused almost complete decomposition of 15 ppm anthracene and benzo[a]pyrene during a five day catalytic cycle at 30 degrees C. Anthracene-1,4-dione was the main product of anthracene oxidation by all catalysts. The catalysts were active in several cycles without regeneration.     

Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes

Bisphenol A (BPA) and nonylphenol (NP) were treated with manganese peroxidase (MnP) and laccase prepared from the culture of lignin-degrading fungi. Laccase in the presence of 1-hydroxybenzotriazole (HBT), the so-called laccase-mediator system, was also applied to remove the estrogenic activity. Both chemicals disappeared in the reaction mixture within a 1-h treatment with MnP but the estrogenic activities of BPA and NP still remained 40% and 60% in the reaction mixtures after a 1-h and a 3-h treatment, respectively. Extension of the treatment time to 12 h completed the removal of estrogenic activities of BPA and NP. The laccase has less ability to remove these activities than MnP, but the laccase-HBT system was able to remove the activities in 6 h. A gel permeation chromatography (GPC) analysis revealed that main reaction products of BPA and NP may be oligomers formed by the action of enzymes. Enzymatic treatments extended to 48 h did not regenerate the estrogenic activities, suggesting that the ligninolytic enzymes are effective for the removal of the estrogenic activities of BPA and NP.     

Evaluation of the stability of arsenic immobilized by microbial sulfate reduction using TCLP extractions and long-term leaching techniques

An investigation was conducted to evaluate the stability or leachability of arsenic immobilized by microbial sulfate reduction. Anoxic solid-phase samples taken from a bioreactor previously used to treat metal and As contaminated water using sulfate reducing bacteria (SRB) were subjected to the toxicity characteristic leaching procedure (TCLP) and long-term column leaching tests. The results from TCLP experiments showed that the concentration of As leached from solid-phase sulfide material (SSM) samples after an 18 h extraction time was <300 microgl(-1), which is below the current maximum Australian TCLP leachate value for As, and thus would not be characterized as a hazardous waste. In terms of percent total As leached, this was equivalent to <8.5% for SSM samples initially containing 61.3 mgkg(-1) As. The levels of As extracted by the TCLP was found to be significantly lowered or underestimated in the presence of dissolved oxygen, with As concentrations increasing with decreasing headspace-to-leachant volume ratios. The concentration of As was also consistently higher in nitrogen purged extractions compared to those performed in air. This was attributed to the dissolution of Fe-sulfide precipitates and subsequent oxidation of Fe(II) ions and precipitation of ferric(hydr)oxides, resulting in the adsorption of soluble As and corresponding decrease in As concentrations. According to the experimental data, it is recommended that TCLP tests for As leachability should be performed at least in zero-headspace vessels or preferably under nitrogen to minimize the oxidation of Fe(II) to ferric(hydr)oxides. In long-term leaching studies (approximately 68 days), it was found that the low solubility of the SSM ensured that rate of release of As was relatively slow, and the resulting leachate concentrations of As were below the current Australian guideline concentration for arsenic in drinking water.     

Cosolvent-enhanced chemical oxidation of perchloroethylene by potassium permanganate

A laboratory study was conducted to examine cosolvent-enhanced in-situ chemical oxidation (ISCO) of perchloroethylene (PCE) using potassium permanganate (KMnO4). The conceptual basis for this new technique is to enhance permanganate oxidation of dense non-aqueous phase liquids (DNAPLs) with the addition of a cosolvent, thereby increasing DNAPL solubility while avoiding mobilization. Among 17 cosolvent candidates screened, tertiary butyl alcohol (TBA) and acetone were the most stable in the presence of KMnO4, both of which increased PCE aqueous solubility significantly, and therefore are suitable to be used as cosolvent in this study. Batch experiments indicated that the second-order rate constant for PCE oxidation by potassium permanganate was 0.043+/-0.002 M(-1) s(-1) in the purely aqueous (no cosolvent) solution. In the presence of 20% cosolvent (volume fraction=fc=0.2), the rate constant decreased to 0.036+/-0.003 M(-1) s(-1) with TBA and to 0.031+/-0.002 M(-1) s(-1) with acetone. However, in the presence of free-phase PCE, chloride ion concentration from PCE oxidation in acetone/water solutions (fc=0.2) was about twice that in aqueous solutions, indicating that the increase in PCE solubility more than compensated for the decrease in reaction rate constant, such that the oxidation efficiency of PCE was increased with cosolvent. A complete chlorine mass balance was observed in the aqueous system, whereas approximately 70% was obtained in TBA/water or acetone/water (fc=0.2). In soil columns containing residual DNAPL and subjected to isocratic flushing with step-wise increases in f(c) cosolvent, TBA at fc=0.2 resulted in PCE mobilization, whereas acetone at fc相似文献   

Study of the degradation of dyes by MnP of Phanerochaete chrysosporium produced in a fixed-bed bioreactor

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium in a fixed-bed tubular bioreactor, filled with cubes of nylon sponge, operating in semi-solid-state conditions, was studied. Maximum individual manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1293 and 225 U/l were detected.The in vitro decolourisation of two structurally different dyes (Poly R-478, crystal violet) by the extracellular liquid obtained in the above-mentioned bioreactor was monitored in order to determine its degrading capability. The concentration of some compounds (sodium malonate, manganese sulphate) from the reaction mixture was optimised in order to maximise the decolourisation levels. A percentage of Poly R-478 decolourisation of 24% after 15 min of dye incubation was achieved.On the other hand, a methodology for a long treatment of these dyes based on the continuous addition of MnP enzyme and H(2)O(2) was developed. Moreover, this enzymatic treatment was compared with a photochemical decolourisation process. The former allowed to maintain the degradation rate almost constant for a long time, resulting in a decolourisation percentage of 70% and 30% for crystal violet and Poly R-478, respectively, after 2 h of treatment. As for the latter, it was not able to degrade Poly R-478, whereas crystal violet reached a degradation of 40% in 2 h.     

Prediction of Fenton oxidation positions in polycyclic aromatic hydrocarbons by Frontier electron density

Five recalcitrant polycyclic aromatic hydrocarbons (PAHs) in ethanol were subjected to Fenton oxidation, and following GC-MS identification of respective oxidation products, their oxidation positions were compared to those predicted by Frontier electron density. Quinone forms of oxidation products were identified in each PAH. With the exception of fluorene, oxidation positions of quinone forms of products of acenaphthylene, anthracene, benz(a)anthracene, and benzo(a)pyrene corresponded with predicted positions in which Frontier electron density was high. From these results, it appears that determining the Frontier electron density of a PAH is a promising method for predicting the Fenton oxidation position.