Degradation of pyrene by Mycobacterium flavescens

 A strain of Mycobacterium flavescens was isolated from polluted sediments. It was capable of utilizing pyrene as a sole source of carbon and energy. When pyrene was supplied as a suspension at 50 μg/ml, the generation time was 9.6 h and the rate of pyrene utilization was 0.56 μg ml^-1 day^-1. In addition to pyrene, the strain could mineralize phenanthrene (17.7%) and fluoranthene (17.9%), but failed to mineralize naphthalene, chrysene, anthracene, fluorene, acenaphthene and benzo[a]pyrene, as determined by recovery of radiolabeled CO₂ in incubations conducted for 2 weeks under growth conditions. Metabolites produced during growth on pyrene were detected and characterized by HPLC and GC-MS. The product of initial ring oxidation, 4,5-dihydroxy-4,5-dihydropyrene was identified, as well as ring-fission products including 4-phenanthroic acid, phthalic acid, and 4,5-phenanthrenedioic acid. Received: 3 October 1995/Received last revision: 1 April 1996/Accepted: 15 April 1996     

Degradation of pyrene byRhodococcus sp. UW1

Summary A Rhodococcus species, designated strain UW1, was isolated from contaminated soil using conventional enrichment and isolation techniques. The isolate was able to use pyrene as sole source of carbon and energy; it mineralized 72% of the pyrene within 2 weeks. During growth a metabolite was detected in the culture fluid and further characterized by UV- and mass spectrometry. There is evidence that this metabolite resulted from a recyclization of the direct meta-ring-fission product of pyrene after dihydroxylation in either the 1,2- or 4,5-position. At pH 7.0 and 30°C Rhodococcus sp. UW1 showed a maximum degradation rate of 0.08 mg pyrene/ml per day, while growing with a doubling time of 30 h. The activity of the initial dioxygenase system was characterized by measuring the oxygen-consumption rates of pyrene-induced resting cells, the maxima of which occurred at pH 7.2 and 45°C. Rhodococcus sp. UW1 could also use phenathrene, anthracene, fluoranthene and chrysene as sole sources of carbon and energy, whereas naphthalene, dibenzofuran, fluorene and dibenzothiophene were only co-metabolized. Offprint requests to: U. Walter     

The microbiological fate of polycyclic aromatic hydrocarbons: carbon and oxygen balances for bacterial degradation of model compounds

A series of pure bacterial strains belonging mainly to theRhodococcus andPseudomonas genera were grown on one of the following polycyclic aromatic hydrocarbons (PAH) supplied as sole carbon and energy source: naphthalene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene. In each case, a quantitative evaluation of the carbon repartition of the PAH degraded into CO₂, biomass and water-soluble metabolites was carried out. In addition, the kinetics of oxygen consumption and of water-soluble metabolite accumulation during PAH biodegradation was followed with respirometric equipment. Satisfactory carbon balances were obtained and the data correlated well with oxygen consumption values. The results show that growth on PAH presents high mineralization yields (from 56% to 77% of carbon) and sizeable production of biomass (from 16% to 35% of carbon) and limited but significant accumulation of metabolites (from 5% to 23% of carbon). The mineralization yields were higher and biomass yields lower in the case of higher PAH. Some differences between strains were also observed.     

Utilization of solubilized and crystalline mixtures of polycyclic aromatic hydrocarbons by a Mycobacterium sp.

A strain of Mycobacterium, that is able to degrade fluorene, phenanthrene, fluoranthene and pyrene was grown on various mixtures of these substrates. The polycyclic aromatic hydrocarbons (PAH) were provided either as crystals or solubilized by a surfactant. Mixed PAH were degraded simultaneously, but not in parallel, indicating that the degradation pathways were not incompatible. Certain interactions of the substrates were observed. For example, the degradation of solubilized pyrene was delayed in the presence of fluorene and enhanced in the presence of phenanthrene. Fluorene was degraded cometabolically with the other PAH serving as growth substrates, but not as the only source of carbon. The utilization of phenanthrene occurred at the fastest rate and was not affected by the presence of fluorene, pyrene or fluoranthene.     

Degradation of polycyclic aromatic hydrocarbons by an immobilized mixed bacterial culture

The mixed bacterial culture MK1 was capable of degrading a wide spectrum of aromatic compounds both as free and as immobilized cells. By offering anthracene oil or a defined mixture of phenol, naphthalene, phenanthrene, anthracene and pyrene (in concentrations of 0.1–0.2 mm, respectively) as sources of carbon and energy, a specific degradation pattern correlating with the condensation degree was observed. Regarding the defined mixture of aromatic hydrocarbons, complete metabolism was reached for phenol (0.1 mm) after 1 day, for naphthalene (0.1 mm) after 2 days and for phenanthrene (0.1 mm) after 15 days of cultivation. The conversion of anthracene (0.1 mm) and pyrene (0.1 mm) resulted in minimal residual concentrations, analogous to fluoranthene and pyrene of the anthracene oil (0.1%). Maximal total degradation for the tricyclic compounds dibenzofurane, fluorene, dibenzothiophene, phenanthrene and anthracene of the anthracene oil (0.1%) occurred after 5 days. In general, a significant metabolisation of the tetracyclic aromatic hydrocarbons fluoranthene and pyrene was observed after the degradation of phenol, naphthalene and most of the tricyclic compounds. Doubling the start concentrations of the polycyclic aromatic hydrocarbons effected higher degradation rates. Cell growth occurred simultaneously with the conversion of phenol, naphthalene and the tricyclic compounds. The immobilized cells showed stable growth and, compared to freely suspended cells, the same degradation sequence as well as an equivalent degradation potential — even in a model soil system. Correspondence to: I. Wiesel     

A comparative study of one- and two-photon absorption properties of pyrene and perylene diimide derivatives

Two important classes of organic molecules, perylene diimide (PDI) and pyrene derivatives have been found to possess relatively excellent photophysical and photochemical properties and especially high two-photon absorption cross sections (δ ^T _max). Herein, one-photon absorption (OPA) and two-photon absorption (TPA) properties of some novel PDI and pyrene derivatives were comparatively investigated by the density functional theory (DFT) and Zerner’s intermediate neglect of differential overlap (ZINDO) methods. The calculated results indicate that with respect to PDI derivatives, the maximum TPA cross-sections for pyrene compounds increase obviously, the maximum peaks of OPA and TPA spectra are blue-shifted, the ΔE _H-L (energy gaps between the highest occupied orbital and the lowest unoccupied orbital) increase. The different π-conjugated bridges (fluorene and pyrene) and terminal groups have slight effect on the OPA properties. Nevertheless, the molecules bearing 1,6-disubstituted pyrene as the π-conjugated bridge display the largest δ ^T _max in both series of compounds 3 and 4. Moreover, the δ ^T _max values of molecules with benzothiazole-substituted terminal groups are larger than those of the molecules with diphenylamine, which is attributed to benzothiazole groups stabilizing the planarity of the branch parts, extending the conjugated length and increasing the π-electron delocalized extent. Furthermore, the molecular size has marked effect on OPA and TPA properties. It is worthy to mention that cruciform 8 displays the largest δ ^T _max among all the studied molecules in the range of 600–1100 nm. This research could provide a better understanding for the origin of the linear and nonlinear optical properties, and it would be helpful to gain more information about designing two-photon absorption materials with large δ ^T _max.     

Effect of surfactants on pyrene degradation by Pseudomonas fluorescens 29L

The effect of surfactants on pyrene degradation in Pseudomonas fluorescens 29L was investigated. This strain produced 30.1 μM of rhamnolipid equivalents (RE) of biosurfactants on 50 mg of pyrene per liter of medium. The production of biosurfactants was significantly correlated with the water solubility (S _w) of the substrate and the growth rate on it. When chrysene, with a S _w of 2.8 × 10⁻³ mg per liter of water, was the carbon source, 13.1 μM of RE of biosurfactants were produced compared to 10.3 μM of RE of biosurfactants on acenaphthene with a S _w of 1.9 mg per liter of water. No biosurfactants were produced on salicylic acid, catechol, and citrate. All of the strain 29L mutants which grew on pyrene produced biosurfactants while among the mutants which grew on naphthalene, only 88.4% produced biosurfactants. The rhamnolipid mixture, JBR425, inhibited the growth of Strain 29L wild type (WT) and all of its mutants on pyrene. However, these mutants were able to grow in the presence of pyrene when the growth medium was supplemented with 10⁻⁶ mg of emulsan per milliliter of medium. This study implies biosurfactants are produced by Strain 29L as a physiological response to the hydrophobicity of pyrene. The combined use of indigenous biosurfactants and the added biosurfactant, emulsan, is a biotechnology to enhance pyrene degradation by Pseudomonas fluorescens 29L.     

Molecular characteristics of xenobiotic-degrading sphingomonads

The genus Sphingomonas (sensu latu) belongs to the α-Proteobacteria and comprises strictly aerobic chemoheterotrophic bacteria that are widespread in various aquatic and terrestrial environments. The members of this genus are often isolated and studied because of their ability to degrade recalcitrant natural and anthropogenic compounds, such as (substituted) biphenyl(s) and naphthalene(s), fluorene, (substituted) phenanthrene(s), pyrene, (chlorinated) diphenylether(s), (chlorinated) furan(s), (chlorinated) dibenzo-p-dioxin(s), carbazole, estradiol, polyethylene glycols, chlorinated phenols, nonylphenols, and different herbicides and pesticides. The metabolic versatility of these organisms suggests that they have evolved mechanisms to adapt quicker and/or more efficiently to the degradation of novel compounds in the environment than members of other bacterial genera. Comparative analyses demonstrate that sphingomonads generally use similar degradative pathways as other groups of microorganisms but deviate from competing microorganisms by the existence of multiple hydroxylating oxygenases and the conservation of specific gene clusters. Furthermore, there is increasing evidence for the existence of plasmids that only can be disseminated among sphingomonads and which undergo after conjugative transfer pronounced rearrangements.     

Recombination in Polymer:Fullerene Solar Cells with Open‐Circuit Voltages Approaching and Exceeding 1.0 V

Polymer:fullerene solar cells are demonstrated with power conversion efficiencies over 7% with blends of PBDTTPD and PC₆₁BM. These devices achieve open‐circuit voltages (V_oc) of 0.945 V and internal quantum efficiencies of 88%, making them an ideal candidate for the large bandgap junction in tandem solar cells. V_oc’s above 1.0 V are obtained when the polymer is blended with multiadduct fullerenes; however, the photocurrent and fill factor are greatly reduced. In PBDTTPD blends with multiadduct fullerene ICBA, fullerene emission is observed in the photoluminescence and electroluminescence spectra, indicating that excitons are recombining on ICBA. Voltage‐dependent, steady state and time‐resolved photoluminescence measurements indicate that energy transfer occurs from PBDTTPD to ICBA and that back hole transfer from ICBA to PBDTTPD is inefficient. By analyzing the absorption and emission spectra from fullerene and charge transfer excitons, we estimate a driving free energy of –0.14 ± 0.06 eV is required for efficient hole transfer. These results suggest that the driving force for hole transfer may be too small for efficient current generation in polymer:fullerene solar cells with V_oc values above 1.0 V and that non‐fullerene acceptor materials with large optical gaps (>1.7 eV) may be required to achieve both near unity internal quantum efficiencies and values of V_oc exceeding 1.0 V.     

Tuning FRET efficiency as a novel approach for improved detection of naphthalene: application to environmental samples

Naphthalene has emission in the ultraviolet (UV) region, limiting its trace level determination in biological and environmental samples due to detrimental effect of UV light on the living cell and interference from other substances having emission in the UV region. Fluorescence resonance energy transfer strategy is adopted for determination of traces naphthalene in the visible region. Significant improvement of lowest detection limit of naphthalene has been achieved through tuning of fluorescence resonance energy transfer efficiency. Anthranilic acid pyrene (ANP) conjugate provides lowest detection limit for naphthalene among three probes studied, viz. ANB, aniline‐ pyrene conjugate (APA) and ANP. ANP efficiently measures naphthalene content in river water. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis of novel bispyrene diamines and their application as ratiometric fluorescent probes for detection of DNA

Fluorescent DNA probes with 1,6-hexanediyl as the linker between two pyrenes, phenylpyrenes or phenylethynyl pyrene fluorophores were synthesized (Py-1, Py-2 and Py-3) and their interactions with DNA were studied by UV–vis absorption spectra, fluorescence spectra and viscosity measurements. The probes show red-shifted emission compared with pyrene (up to 20 nm). We found the interaction of these probes with DNA can be either intercalation or groove binding. Ratiometric fluorometry (ratio of the monomer and excimer emission intensity versus concentration of DNA) was achieved with these probes for DNA quantification (with limit of detection, LOD, up to 0.1 μg/mL). We also found that the undesired oxygen sensitivity of the emission intensity of pyrene fluorophore can be greatly suppressed by extending the π-conjugation framework of pyrene (the I_Ar/I_air value is decreased from 8.10 for pyrene to less than 2.20 for the DNA probes described herein).     

Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons.

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6'-dihydroxy-2,2'-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.     

Conformational switching of the pyridine-pyrimidine-pyridine scaffold for ion-controlled FRET

Fluorescence resonance energy transfer (FRET) has become a major tool for the static and dynamic study of conformational changes in biological systems. We report herein the investigation of a switchable pyridine-pyrimidine-pyridine scaffold as a support to ion-controlled intramolecular FRET. A dissymmetrical switch bearing naphthalene and acridine fluorophores was synthesized and its photophysical behavior studied. In the neutral state where the molecule adopts a U-shape, the emission of the naphthalene is quenched while a strong emission from the acridine fluorophore is observed, consistent with energy transfer between the naphthalene and the acridine units. The emission of the acridine is also enhanced by the pyridine-induced sensitization (excitation at 280 nm). After introduction of a copper(I) cation which switches the conformation to a W-shape, the complex formed shows the emission of both the naphthalene and acridine units when excited at 280 nm, although coordination also leads to a strong quenching of emission.     

Larval convergence in a colonial tunicate: the organization of the sarcotubular complex in <Emphasis Type="Italic">Ecteinascidia turbinata</Emphasis> (Perophoridae,Phlebobranchiata, Tunicata,Chordata)

Ecteinascidia turbinata is a colonial ascidian that as an adult shares characters with phlebobranch ascidians, whereas the larvae are similar to aplousobranch ascidian larvae. The sarcotubular complex consists of invaginations of the sarcolemma that contact the sarcoplasmatic reticulum via dyads or triads. If present, the invaginations of the sarcolemma in tunicates have been characterized as laminar or tubular. We comparatively investigated the sarcotubular complex of E. turbinata and seven other tunicate species using 3D-reconstruction techniques based on electron micrographs of serial sections. The mononucleate muscle cells in E. turbinata possess intermediate and close junctions and contain several layers of peripheral myofibrillae. The myofibrillae are surrounded by continuous cisternae of the sarcoplasmic reticulum that forms interconnected rings around the z-bands. The invaginations of the sarcolemma are laminar, contacting the sarcoplasamatic reticulum at the height of the z-bands via dyads and triads. We present a clear definition of character states encountered in Tunicata: laminar invaginations are characterized by a width to length ratio of smaller than 1:20, tubular invagination by a width to length ratio of larger than 1:10. Laminar invaginations are found in stolidobranch ascidians and E. turbinata. Tubular invaginations are present in aplousobranch ascidians and appendicularians. This character state distribution across taxa supports the hypothesis that E. turbinata should be included in Phlebobranchiata as suggested by adult characters and that the larval similarities with Aplousobranchiata arose by convergent evolution. An erratum to this article can be found at     

Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319

A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of R. ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5′-monophosphate. The enzyme was successfully expressed in Escherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.     

Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.     

Microbial degradation of high and low molecular weight polyaromatic hydrocarbons in a two-phase partitioning bioreactor by two strains of Sphingomonas sp.

A mixture of six polyaromatic hydrocarbons (naphthalene, phenanthrene, fluoranthene, pyrene, chyrysene and benzo[a]pyrene), varying in size from 2 to 5 rings, was dissolved in dodecane, and used as the delivery phase of a partitioning bioreactor. Two species of Sphingomonas were then used individually, and as a consortium, to determine which of the PAHs were degraded. Only low molecular weight PAHs (naphthalene, phenanthrene and fluoranthene) were degraded by the individual strains, but the consortium degraded all substrates either to completion or near completion.     

Simultaneous Open‐Circuit Voltage Enhancement and Short‐Circuit Current Loss in Polymer: Fullerene Solar Cells Correlated by Reduced Quantum Efficiency for Photoinduced Electron Transfer

The limits of maximizing the open‐circuit voltage V_oc in solar cells based on poly[2,7‐(9,9‐didecylfluorene)‐alt‐5,5‐(4,7‐di‐2‐thienyl‐2,1,3‐benzothiadiazole)] (PF10TBT) as a donor using different fullerene derivatives as acceptor are investigated. Bulk heterojunction solar cells with PF10TBT and [6,6]‐phenyl‐C₆₁‐butyric acid methyl ester (PCBM) give a V_oc over 1 V and a power conversion efficiency of 4.2%. Devices in which PF10TBT is blended with fullerene bisadduct derivatives give an even higher V_oc, but also a strong decrease in short circuit current (J_sc). The higher V_oc is attributed to the higher LUMO of the acceptors in comparison to PCBM. By investigating the photophysics of PF10TBT:fullerene blends using near‐IR photo‐ and electroluminescence, time‐resolved photoluminescence, and photoinduced absorption we find that the charge transfer (CT) state is not formed efficiently when using fullerene bisadducts. Hence, engineering acceptor materials with a LUMO level that is as high as possible can increase V_oc, but will only provide a higher power conversion efficiency, when the quantum efficiency for charge transfer is preserved. To quantify this, we determine the CT energy (E_CT) and optical band gap (E_g), defined as the lowest first singlet state energy E_S1 of either the donor or acceptor, for each of the blends and find a clear correlation between the free energy for photoinduced electron transfer and J_sc. We find that E_g ? qV_oc > 0.6 eV is a simple, but general criterion for efficient charge generation in donor‐acceptor blends.     

Synthesis and photophysical studies of covalently linked porphyrin-21-thiaporphyrin dyads

The synthesis and photophysical properties of four covalently linked unsymmetrical porphyrin dyads containing two different porphyrin cores such as N₄ and N₃S are reported. The covalently linked dyads were prepared by the coupling of appropriate porphyrin having ethynylphenyl functional group at meso-position with porphyrin having iodophenyl or bromo functional group at meso-position under mild palladium coupling conditions. The photophysical study indicated an intramolecular singlet-singlet energy transfer from N₄/ZnN₄ porphyrin sub-unit to N₃S porphyrin sub-unit in all four dyads with an efficiency of energy transfer process was typically ?97%. To probe the role of linker in through bond electronic communication between the two porphyrin sub-units in dyads, the linker was varied from diphenylethyne to phenylethyne and the study revealed that the energy transfer rates and efficiencies were much higher for phenylethyne-bridged porphyrin dyads compared with diphenylethyne-bridged porphyrin dyads.