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.     

Degradation of CL-20 by white-rot fungi

In previous studies, we found that the emerging energetic chemical, CL-20 (C6H6N12O12, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane), can be degraded following its initial denitration using both aerobic and anaerobic bacteria. The C and N mass balances were not determined due to the absence of labeled starting compounds. The present study describes the degradation of the emerging contaminant by Phanerochaete chrysosporium using ring-labeled [15N]-CL-20 and [14C]-CL-20. Ligninolytic cultures degraded CL-20 with the release of nitrous oxide (N2O) in amounts corresponding to 45% of the nitrogen content of CL-20. When ring-labeled [15N]-CL-20 was used, both 14N14NO and 15N14NO were observed, likely produced from -NO2 and N-NO2, respectively. The incubation of uniformly labeled [14C]-CL-20 with fungi led to the production of 14CO2 (> 80%). Another ligninolytic fungus, Irpex lacteus, was also able to degrade CL-20, but as for P. chrysosporium, no early intermediates were observed. When CL-20 was incubated with manganese peroxidase (MnP), we detected an intermediate with a [M-H]- mass ion at 345 Da (or 351 and 349 Da when using ring-labeled and nitro-labeled [15N]-CL-20, respectively) matching a molecular formula of C6H6N10O8. The intermediate was thus tentatively identified as a doubly denitrated CL-20 product. The concomitant release of nitrite ions (NO2-) with CL-20 degradation by MnP also supported the occurrence of an initial denitration prior to cleavage and decomposition.     

Biodegradation of PCBs by ligninolytic fungi and characterization of the degradation products

The aim of the present study was to compare the degrading capabilities of eight ligninolytic fungal representatives towards a technical mixture of polychlorinated biphenyls (Delor 103). Axenic cultures of the fungi, either in complex or N-limited liquid media, were spiked with the technical mixture of Delor 103. All of the fungal strains were able to degrade the pollutant significantly after 6weeks of incubation in both media. Outstanding results were achieved by the treatment with Pleurotus ostreatus, which removed 98.4% and 99.6% of the PCB mixture in complex and mineral media, respectively. This fungus was the only one capable of breaking down penta- and hexachlorinated biphenyls in the complex medium. Ecotoxicological assays performed with the luminescent bacterium Vibrio fischeri demonstrated that all of the fungal strains employed in this study were able to remove the toxicity only temporarily (e.g., after 28d of incubation), while P. ostreatus was capable of suppressing the toxicity associated to PCBs along the whole incubation period in both media. We also performed an extensive set of qualitative GC/MS analyses and chlorinated derivatives of hydroxy- and methoxy-biphenyls were detected along with monoaromatic structures, i.e. chlorobenzoic acids, chlorobenzaldehydes and chlorobenzyl alcohols. This results indicate that both intracellular (cytochrome P-450 monooxigenase, aryl-alcohol dehydrogenase and aryl-aldehyde dehydrogenase) and extracellular (ligninolytic enzymes) enzymatic systems could be involved in the biotransformation of PCB by ligninolytic fungi. The data from this work also document that the fungi are able to degrade further the main metabolites on the PCB pathway (i.e. chlorobenzoic acids) simultaneously with PCBs.     

碱法麦草浆漂白废水脱色白腐菌的筛选及脱色研究

从腐木上采集,经多次分离纯化筛选出2株具有较高漂白废水脱色能力的菌株F7、F9,与Phanerochaete Chrysosporium在相同的条件下处理碱法麦草浆CE段混合漂白废水,培养4d脱色率分别为74．44％,46．67％和62．22％。延长培养时间至第8d,F7菌脱色效果不明显,而F9菌脱色可增至67．58％。     

Enantioselective recognition of mono-demethylated methoxychlor metabolites by the estrogen receptor

Metabolites of methoxychlor such as 2-(p-hydroxyphenyl)-2-(p-methoxyphenyl)-1,1,1-trichloroethane (mono-OH-MXC) and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (bis-OH-MXC), have estrogenic activity. Mono-OH-MXC is a chiral compound in which the carbon atom bridging two benzene rings is the chiral centre. In previous studies the estrogenic activity of racemic mono-OH-MXC has been measured, and the activity of each enantiomer of this compound has not yet been elucidated. In this study, we evaluated the estrogen receptor-binding activity of each enantiomer of mono-OH-MXC to clarify the enantioselective recognition by the estrogen receptor. (S)-mono-OH-MXC showed 3-fold higher binding activity than that of the (R) enantiomer. The activity of bis-OH-MXC was only 1.7-fold higher than that of (S)-mono-OH-MXC. This result suggests that the one hydroxy group and the orientation of the CCl3 group of mono- and bis-OH-MXCs are important for the interaction with the estrogen receptor. The result also points out the estrogenic activity of methoxychlor after metabolic activation in vivo, which predominantly produces the (S)-mono-OH-MXC, may be higher than estimated from the in vitro activity of racemic mixtures.     

Detoxification of hexachlorobenzene by dechlorination with potassium-sodium alloy

Dechlorination of hexachlorobenzene (HCB) was achieved by a liquid potassium–sodium (K–Na)-alloy. HCB in a cyclohexane/benzene solution (22 mmol/l, 4.67 g/l as chlorine) was dechlorinated by almost 100% after a 30-min reaction, indicating high reactivity of K–Na alloy and high proton donating power of cyclohexane. Decreasing orders of chlorobenzenes identified after a 15-min reaction, by amount were 1,2,3,4- > 1,2,3,5- > 1,2,4,5- for tetrachlorobenzenes, 1,2,4- > 1,2,3- > 1,3,5- for trichlorobenzenes, and 1,4- > 1,3- > 1,2- for dichlorobenzenes. It was hypothesized that once one chlorine atom in HCB was replaced with a proton, the adjacent chlorine atom to the proton tended to be replaced with another hydrogen atom. A total of 63 PCBs formed via the Wurtz–Fittig reaction were identified as by-products in the sample after a 15-min reaction. Among PCBs found, 2,3^′,4^′,5-tetrachlorobiphenyl, which was a product from 1,2,4-trichlorobenzene formed via the Wurtz–Fittig reaction, was detected in relatively high concentration (48.9 nmol/ml). The sample obtained from a reaction mixture after 30 min contained only 14 PCBs in trace amounts, indicating that the PCBs formed were also further dechlorinated by K–Na alloy. Non-chlorinated compounds––such as methylbenzene, dimethylbenzene, dimer of tetrahydrofuran, and dicyclohexyl (dimer of cyclohexane)––were also identified in the samples. A method using K–Na alloy developed in the present study dechlorinated satisfactorily HCB at room temperature.     

Abiotic reductive dechlorination of chlorinated ethylenes by soil

Abiotic reductive dechlorination of chlorinated ethylenes by soil in anaerobic environments was characterized to improve knowledge of the behavior of chlorinated ethylenes in natural systems, including systems modified to promote attenuation of contaminants. Target organics in the soil suspension reached sorption equilibrium in 2 days and the sorption isotherm of target organics was properly described by the linear sorption model. A modified Langmuir-Hinshelwood model was developed to describe the kinetics of reductive dechlorination of target organics by soil. The rate constants for the reductive dechlorination of chlorinated ethylenes at the reactive surfaces of reduced soils were found in the range between 0.055 (+/- 8.9%) and 2.60 (+/- 3.2%) day(-1). The main transformation products in reduced soil suspensions were C2 hydrocarbons. No chlorinated intermediates were observed at concentrations above detection limits. Five cycles of reduction of the soil followed by oxidation of the soil with trichloroethylene (TCE) did not affect the removal of TCE. The removal was affected by the reductants used and increased in the order: Fe(II) < dithionite < Fe(II) + dithionite.     

Oxidative ring cleavage of low chlorinated biphenyl derivatives by fungi leads to the formation of chlorinated lactone derivatives

The yeast Trichosporon mucoides and the filamentous fungus Paecilomyces lilacinus as biphenyl oxidizing organisms are able to oxidize chlorinated biphenyl derivatives. Initial oxidation of derivatives chlorinated at C4 position started at the non-halogenated ring and went on up to ring cleavage. The products formed were mono- and dihydroxylated 4-chlorobiphenyls, muconic acid derivatives 2-hydroxy-4-(4-chlorophenyl)-muconic acid and 2-hydroxy-5-(4-chlorophenyl)-muconic acid as well as the corresponding lactones 4-(4-chlorophenyl)-2-pyrone-6-carboxylic acid and 3-(4-chlorophenyl)-2-pyrone-6-carboxylic acid. Altogether T. mucoides formed 12 products and P. lilacinus accumulated five products. Whereas the rate of the first oxidation step at 4-chlorobiphenyl seems to be diminished by the decreased bioavailability of the compound, no considerable differences were observed between the degradation of 4-chloro-4'-hydroxybiphenyl and 4-hydroxybiphenyl. Twofold chlorinated biphenyl derivatives did not serve as substrates for oxidation by either organism with the exception of 2,2'-dichlorobiphenyl, transformed by the yeast Trichosporon mucoides to two monohydroxylated derivatives. The results show, that soil fungi may contribute to the aerobic degradation of low chlorinated biphenyls accumulating from anaerobic dehalogenation of PCB by bacteria.     

The fate of methoxychlor in soils and transformation by soil microorganisms

Abstract

Methoxychlor was found to be sufficiently persistant in soil and its residues were present even 18 months after the soil treatment. Saprophytes, fungi and actinomyces were unaffected by varying concentrations of methoxychlor, azotobacter however was susceptable. Soil strains isolated did not utilize methoxychlor as a sole carbon source except for 9 cultures belonging to the genera Bacillus, Acineto‐bacter and Rhodococcus which carried out the complete dechlorination, demethylation and splitting of one of methoxychlor aromatic rings. Anaerobic conditions were more favorable for methoxychlor biodegradation by soil and pure microbial cultures.     

Photodechlorination of methoxychlor induced by hydroquinone: Rearrangement and conjugate formation

Reductive degradation of the pesticide methoxychlor by photolysis in the presence of hydroquinone in dilute solutions of acetonitrile or 1:1 aqueous t-butanol leads to processes other than simple dechlorination. Direct radical coupling with hydroquinone forms a markedly estrogenic conjugate. A pH dependant molecular rearrangement gives pro-estrogenic 4,4′-dimethoxy-α,α′-dichlorostilbenes, and condensation with hydroquinone yields the chlorine free polycyclic aromatic, 12-hydroxy-3,6-dimethoxybenzo[b]phenanthro[9,10-d]furan.     

The fate of methoxychlor in soils and transformation by soil microorganisms

Methoxychlor was found to be sufficiently persistent in soil and its residues were present even 18 months after the soil treatment. Saprophytes, fungi and actinomyces were unaffected by varying concentrations of methoxychlor, azotobacter however was susceptable. Soil strains isolated did not utilize methoxychlor as a sole carbon source except for 9 cultures belonging to the genera Bacillus, Acinetobacter and Rhodococcus which carried out the complete dechlorination, demethylation and splitting of one of methoxychlor aromatic rings. Anaerobic conditions were more favorable for methoxychlor biodegradation by soil and pure microbial cultures.     

Biodegradation of crosslinked acrylic polymers by a white-rot fungus

Two synthetic superabsorbent crosslinked acrylic polymers were mineralized by the white-rot fungusPhanerochaete chrysosporium. The amount of polymer converted to CO₂ increased as the amount of polymer added to the cultures increased. In the presence of sufficiently large amounts of the superabsorbents, such that all of the culture fluid was absorbed and a gelatinous matrix was formed, the fungus still grew and mineralization was observed. Neither the polymers, nor their degradation products were toxic to the fungus. While the rates of mineralization were low, all of the polymers incubated in the liquid fungal cultures were completely depolymerized to water soluble products within 15–18 days. The depolymerization of the polymers was observed only in nitrogen limited cultures of the fungus which secrete the lignin degradation system, however, the water soluble products of depolymerization were mineralized in both nutrient limited and sufficient cultures of the fungus. The rate of mineralization of the depolymerized metabolites was more than two times greater in nutrient sufficient cultures. Following longer incubation periods, most (> 80 %) of the radioactivity was recovered in the fungal mycelial mat suggesting that carbon of the polymer had been converted to fungal metabolites.     

Mechanistics of trichloroethylene mineralization by the white-rot fungus Trametes versicolor

The white-rot fungus Trametes versicolor degraded trichloroethylene (TCE), a highly oxidized chloroethene, and produced 2,2,2-trichloroethanol and carbon dioxide as the main products of degradation, based on the results obtained using [13C]-TCE as the substrate. For a range of concentrations of TCE between 2 and 20 mg l(-1), 53% of the theoretical maximum chloride expected from complete degradation of TCE was observed. Laccase was shown to be induced by TCE, but did not appear to play a role in TCE degradation. Cytochrome P-450 appears to be involved in TCE degradation, as evidenced by marked inhibition of degradation of TCE in the presence of 1-aminobenzotriazole, a known inhibitor of cytochrome P-450. Our results suggested that chloral (trichloroacetaldehyde) was an intermediate of the TCE degradation pathway. The results indicate that the TCE degradation pathway in T. versicolor appears to be similar to that previously reported in mammals and is mechanistically quite different from bacterial TCE degradation.     

The dechlorination of cyclodiene pesticides by methanogenic granular sludge

Cyclodiene pesticides: aldrin, isodrin, dieldrin and endrin were dechlorinated by methanogenic granular sludge in spiked batch tests. Initial pesticides concentration was 7 or 9 mgl(-1). Two monodechlorinated analogues were formed during the conversions of aldrin and isodrin. Dieldrin was transformed into two monodechlorinated products as well as into aldrin and two monodechlorinated derivatives of aldrin. In respect of endrin three monodechlorinated and three didechlorinated products were found. The dechlorination of endrin was the most rapid, and was almost complete within 28 days. The dechlorinations of aldrin, isodrin and dieldrin were much slower: over a period of 3 months only 59%, 70% and 88% was transformed, respectively. The amounts of released chloride corresponded 0.54 +/- 0.23, 1.05 +/- 0.25, 1.10 +/- 0.12 of theoretical value for suggested reactions, for aldrin, isodrin and dieldrin respectively. For endrin it was much higher. These transformations did not occur in control samples containing autoclaved granules or in control blank samples without sludge. However, in aldrin spiked blanks, a conversion into a six-chlorinated analogue was found.     

Abiotic reductive dechlorination of chlorinated ethylenes by iron-bearing phyllosilicates

Abiotic reductive dechlorination of chlorinated ethylenes (tetrachloroethylene (PCE), trichloroethylene (TCE), cis-dichloroethylene (c-DCE), and vinylchloride (VC)) by iron-bearing phyllosilicates (biotite, vermiculite, and montmorillonite) was characterized to obtain better understanding of the behavior of these contaminants in systems undergoing remediation by natural attenuation and redox manipulation. Batch experiments were conducted to evaluate dechlorination kinetics and some experiments were conducted with addition of Fe(II) to simulate impact of microbial iron reduction. A modified Langmuir-Hinshelwood kinetic model adequately described reductive dechlorination kinetics of target organics by the iron-bearing phyllosilicates. The rate constants stayed between 0.08 (+/-10.4%) and 0.401 (+/-8.1%) day(-1) and the specific initial reductive capacity of iron-bearing phyllosilicates for chlorinated ethylenes stayed between 0.177 (+/-6.1%) and 1.06 (+/-7.1%) microM g(-1). The rate constants for the reductive dechlorination of TCE at reactive biotite surface increased as pH (5.5-8.5) and concentration of sorbed Fe(II) (0-0.15 mM g(-1)) increased. The appropriateness of the model is supported by the fact that the rate constants were independent of solid concentration (0.0085-0.17 g g(-1)) and initial TCE concentration (0.15-0.60 mM). Biotite had the greatest rate constant among the phyllosilicates both with and without Fe(II) addition. The rate constants were increased by a factor of 1.4-2.5 by Fe(II) addition. Between 1.8% and 36% of chlorinated ethylenes removed were partitioned to the phyllosilicates. Chloride was produced as a product of degradation and no chlorinated intermediates were observed throughout the experiment.     

Regiospecific dechlorination of spiked tetra- and trichlorodibenzo-p-dioxins by anaerobic bacteria from PCDD/F-contaminated Spittelwasser sediments

Samples were taken from sediment of the River Spittelwasser (district Bitterfeld, Germany), which is highly polluted with PCDD/Fs and other chloroorganic compounds. The sediment cores were separated into 10-20 cm thick layers, spiked with 50 microM of 1,2,3,4-tetrachlorodibenzo-p-dioxin and incubated for 8 months under anaerobic conditions in the presence of cosubstrates. Reductive dechlorination of the tetrachlorinated congener and formation of tri- and dichlorinated products were observed in all biologically active incubations. Analysis of subcultures spiked with 1,2,3- and 1,2,4-trichlorodibenzo-p-dioxin, respectively, revealed two different dechlorination pathways within the sediment cores. Pathway M was characterized by the simultaneous dechlorination of peri- and lateralchlorine atoms, whereas sequence SP was restricted to the dechlorination at positions flanked by chlorine atoms on both sides.     

Biodegradation of heptachlor and heptachlor epoxide-contaminated soils by white-rot fungal inocula

The ability of certain white-rot fungi (WRF) inocula to transform heptachlor and heptachlor epoxide and its application in artificially contaminated soil were investigated. Fungal inoculum of Pleurotus ostreatus eliminated approximately 89 % of heptachlor after 28 days of incubation, and chlordene was detected as the primary metabolite. The fungal inoculum of Pleurotus ostreatus had the highest ability to degrade heptachlor epoxide; approximately 32 % were degraded after 28 days of incubation, and heptachlor diol was detected as the metabolite product. Because Pleurotus ostreatus transformed heptachlor into a less toxic metabolite and could also effectively degrade heptachlor epoxide, it was then selected to be applied to artificially contaminated soil. The spent mushroom waste (SMW) of Pleurotus ostreatus degraded heptachlor and heptachlor epoxide by approximately 91 and 26 %, respectively, over 28 days. This finding indicated that Pleurotus ostreatus SMW could be used to bioremediate heptachlor- and heptachlor epoxide-contaminated environments.     

纳米级铁粉脱氯降解四氯化碳

四氯化碳的生产和使用，给人类带来了较大危害。为此，采用纳米铁粉这一新方法对其进行脱氯处理。试验以纳米级铁粉对四氯化碳的脱氯率为考察指标，选用L25（5^6）正交试验方案，考察了降解介质的初始pH值、纳米铁粉的质量、降解温度、摇床转速和脱氯时间5个影响因素。结果表明，pH值这一因素有极显著影响；在得出的纳米铁粉对四氯化碳脱氯的最佳工艺条件下，获得了99．5％的脱氯率，为有机氯化物脱氯开辟了一条新途径。     

Reductive dechlorination of polychlorinated biphenyls as affected by sediment characteristics

The effect of sediment sources on the selection of polychlorinated biphenyl (PCB) dechlorinating competence was investigated using sediments from two different locations, the Grasse River and Owasco Lake. These two sediments had a similar organic carbon content but different particle size distribution. The two PCB-free sediments were spiked with Aroclor 1248 and inoculated with microorganisms from the Reynolds and General Motors sites in the St. Lawrence River, which exhibited different dechlorination patterns. When each inoculum was serially transferred into fresh sediments four times (every 8-10 weeks), they still maintained the initial dechlorination patterns regardless, the source of sediments and the number of transfers, and dechlorination patterns of the two inocula in the same sediments did not converge. In a parallel approach, when the acclimated microorganisms from the Reynolds site were inoculated into fresh sediments from both sources as well as sediments enriched with organic carbon (2%, w/v), the dechlorination pattern remained unchanged after a 40-week incubation. These results suggest that the sediment characteristics or organic carbon content did not play a role in the selection of dechlorinating populations.     

白腐菌S.commune降解微囊藻毒素-LR的研究

为研究真菌对微囊藻毒素的降解作用,以白腐菌S.commune为降解菌,微囊藻毒素-LR(MC-LR)为降解目标进行生物降解,考察了白腐菌预培养方式及降解过程中的培养方式、充氧方式、温度、初始pH以及MC-LR初始浓度对降解效果的影响.结果表明,白腐菌可有效降解MC-LR,经液体预培养白腐菌对MC-LR的降解效果好于固体预培养,白腐菌静置培养过程中每天充入纯氧1min有助于MC-LR的降解,白腐菌降解MC-LR的最佳初始pH为4.5,适宜温度为30～35℃.白腐菌对MC-LR的降解能力随MC-LR初始浓度的增加而降低.在最佳条件下,当MC-LR初始质量浓度为1 mg/L时,其完全降解需要2d;当MC-LR初始质量浓度为15 mg/L时,其完全降解需要7d.高浓度MC-LR(30 mg/L以上)会对白腐菌生长产生抑制作用.MC-LR降解中间产物的具体结构尚不清楚,有待未来深入分析研究.