Identification of naphthalene carboxylase as a prototype for the anaerobic activation of non-substituted aromatic hydrocarbons

Polycyclic aromatic hydrocarbons such as naphthalene are recalcitrant environmental pollutants that are only slowly metabolized by bacteria under anoxic conditions. Based on metabolite analyses of culture supernatants, carboxylation or methylation of naphthalene have been proposed as initial enzymatic activation reactions in the pathway. However, the extremely slow growth of anaerobic naphthalene degraders with doubling times of weeks and the little biomass obtained from such cultures hindered the biochemical elucidation of the initial activation reaction, so far. Here, we provide biochemical evidence that anaerobic naphthalene degradation is initiated via carboxylation. Crude cell extracts of the sulfate-reducing enrichment culture N47 converted naphthalene and (13) C-labelled bicarbonate to 2-[carboxyl-(13) C]naphthoic acid at a rate of 0.12?nmol min(-1) mg protein(-1) . The enzyme, namely naphthalene carboxylase, catalysed a much faster exchange of (13) C-labelled bicarbonate with the carboxyl group of 2-[carboxyl-(12) C]naphthoic acid at a rate of 3.2?nmol min(-1) mg protein(-1) , indicating that the rate limiting step of the carboxylation reaction is the activation of the naphthalene molecule rather than the carboxylation itself. Neither the carboxylation nor the exchange reaction activities necessitate the presence of ATP or divalent metal ions and they were not inhibited by avidin or EDTA. The new carboxylation reaction is unprecedented in biochemistry and opens the door to understand the anaerobic degradation of polycyclic aromatic hydrocarbons which are among the most hazardous environmental contaminants.     

Feasibility of using prokaryote biosensors to assess acute toxicity of polycyclic aromatic hydrocarbons

The aim of this study was to assess the acute toxicity of polycyclic aromatic hydrocarbons using lux-marked bacterial biosensors. Standard solutions of phenanthrene, pyrene and benzo[a]pyrene were produced using 50 mM hydroxpropyl-β-cyclodextrin solution which contained each respective polycyclic aromatic hydrocarbon at 6.25 times the aqueous solubility limit of the compound. The polycyclic aromatic hydrocarbon solutions were incubated with each of the biosensors for 280 min and the bioluminescence monitored every 20 min. Over the incubation time period, there was no significant decrease in bioluminescence in any of the biosensors tested with the exception of Rhizobium leguminosarum biovar trifolii TA1 luxAB. In this series of incubations, there was a dramatic increase in bioluminescence in the presence of phenanthrene (2.5 times) and benzo[a]pyrene (3 times) above that of the background control (biosensor without polycyclic aromatic hydrocarbon) after 20 min. Over the next 3 h, bioluminescence decreased to that of the control. An ATP assay was carried out on the biosensors to assess if uncoupling of the oxidative phosphorylation mechanisms in the respiratory chain of the cells had occurred. However, it was found that the polycyclic aromatic hydrocarbons had no effect on the organisms indicating that there was no uncoupling. Additionally, mineralisation studies using ¹⁴C-labelled polycyclic aromatic hydrocarbons showed that the biosensors could not mineralise the compounds. This study has shown that the three polycyclic aromatic hydrocarbons tested are not acutely toxic to the prokaryotic biosensors tested, although acute toxicity has been shown in other bioassays. These results question the rationale for using prokaryote biosensors to assess the toxicity of hydrophobic chemicals, such as polycyclic aromatic hydrocarbons.     

Differential exposure biomarker levels among cigarette smokers and smokeless tobacco consumers in the National Health and Nutrition Examination Survey 1999–2008

Assessment of biomarkers is an appropriate way to estimate exposure to cigarette mainstream smoke and smokeless tobacco (SLT) constituents in tobacco consumers. Using the US National Health and Nutrition Examination Survey (NHANES, 1999–2008), biomarkers of volatile organic compounds, halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), acrylamide, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and metals were evaluated. In general, biomarker levels in SLT consumers were significantly lower than in smokers (excluding NNK and some HAHs) and were not significantly different compared with nonconsumers (excluding NNK and some PAHs). These results provide useful information for science-based risk assessment and regulation of tobacco products.     

Lignin Peroxidase Oxidation of Aromatic Compounds in Systems Containing Organic Solvents

Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations.     

Comparative analysis of the volatile fraction of fruit juice from different Citrus species

The volatile composition of fruit from four Citrus varieties (Powell Navel orange, Clemenules mandarine, and Fortune mandarine and Chandler pummelo) covering four different species has been studied. Over one hundred compounds were profiled after HS-SPME-GC-MS analysis, including 27 esters, 23 aldehydes, 21 alcohols, 13 monoterpene hydrocarbons, 10 ketones, 5 sesquiterpene hydrocarbons, 4 monoterpene cyclic ethers, 4 furans, and 2 aromatic hydrocarbons, which were all confirmed with standards. The differences in the volatile profile among juices of these varieties were essentially quantitative and only a few compounds were found exclusively in a single variety, mainly in Chandler. The volatile profile however was able to differentiate all four varieties and revealed complex interactions between them including the participation in the same biosynthetic pathway. Some compounds (6 esters, 2 ketones, 1 furan and 2 aromatic hydrocarbons) had never been reported earlier in Citrus juices. This volatile profiling platform for Citrus juice by HS-SPME-GC-MS and the interrelationship detected among the volatiles can be used as a roadmap for future breeding or biotechnological applications.     

Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons

Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.     

Protein engineering of cytochrome p450(cam) (CYP101) for the oxidation of polycyclic aromatic hydrocarbons

Mutations of the active site residues F87 and Y96 greatly enhanced the activity of cytochrome P450(cam) (CYP101) from Pseudomonas putida for the oxidation of the polycyclic aromatic hydrocarbons phenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-type P450(cam) had low (<0.01 min(-1)) activity with these substrates. Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, while fluoranthene gave mainly 3-fluoranthol. Pyrene was oxidized to 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with small amounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrene gave 3-hydroxybenzo[a]pyrene as the major product. The NADH oxidation rate of the mutants with phenanthrene was as high as 374 min(-1), which was 31% of the camphor oxidation rate by wild-type P450(cam), and with fluoranthene the fastest rate was 144 min(-1). The oxidation of phenanthrene and fluoranthene were highly uncoupled, with highest couplings of 1.3 and 3.1%, respectively. The highest coupling efficiency for pyrene oxidation was a reasonable 23%, but the NADH turnover rate was slow. The product distributions varied significantly between mutants, suggesting that substrate binding orientations can be manipulated by protein engineering, and that genetic variants of P450(cam) may be useful for studying the oxidation of polycyclic aromatic hydrocarbons by P450 enzymes.     

Substrate interactions during aerobic biodegradation of benzene.

This study dealt with the interactions with benzene degradation of the following aromatic compounds in a mixed substrate: toluene, o-xylene, naphthalene, 1,4-dimethylnaphthalene, phenanthrene, and pyrrole. The experiment was performed as a factorial experiment with simple batch cultures. The effect of two different types of inocula was tested. One type of inoculum was grown on a mixture of aromatic hydrocarbons; the other was grown on a mixture of aromatic hydrocarbons and nitrogen-, sulfur-, and oxygen-containing aromatic compounds (NSO compounds), similar to some of the compounds identified in creosote waste. The culture grown on the aromatic hydrocarbons and NSO compounds was much less efficient in degrading benzene than the culture grown on only aromatic hydrocarbons. The experiments indicated that toluene- and o-xylene-degrading bacteria are also able to degrade benzene, whereas naphthalene-, 1,,4-dimethylnaphthalene-, and phenanthrene-degrading bacteria have no or very little benzene-degrading ability. Surprisingly, the stimulating effect of toluene and o-xylene was true only if the two compounds were present alone. In combination an antagonistic effect was observed, i.e., the combined effect was smaller than the sum from each of the compounds. The reason for this behavior has not been identified. Pyrrole strongly inhibited benzene degradation even at concentrations of about 100 to 200 micrograms/liter. Future studies will investigate the generality of these findings.     

Substrate interactions during aerobic biodegradation of benzene

This study dealt with the interactions with benzene degradation of the following aromatic compounds in a mixed substrate: toluene, o-xylene, naphthalene, 1,4-dimethylnaphthalene, phenanthrene, and pyrrole. The experiment was performed as a factorial experiment with simple batch cultures. The effect of two different types of inocula was tested. One type of inoculum was grown on a mixture of aromatic hydrocarbons; the other was grown on a mixture of aromatic hydrocarbons and nitrogen-, sulfur-, and oxygen-containing aromatic compounds (NSO compounds), similar to some of the compounds identified in creosote waste. The culture grown on the aromatic hydrocarbons and NSO compounds was much less efficient in degrading benzene than the culture grown on only aromatic hydrocarbons. The experiments indicated that toluene- and o-xylene-degrading bacteria are also able to degrade benzene, whereas naphthalene-, 1,,4-dimethylnaphthalene-, and phenanthrene-degrading bacteria have no or very little benzene-degrading ability. Surprisingly, the stimulating effect of toluene and o-xylene was true only if the two compounds were present alone. In combination an antagonistic effect was observed, i.e., the combined effect was smaller than the sum from each of the compounds. The reason for this behavior has not been identified. Pyrrole strongly inhibited benzene degradation even at concentrations of about 100 to 200 micrograms/liter. Future studies will investigate the generality of these findings.     

Aromatic hydrocarbon degradation by Sphingomonas yanoikuyae B1

Sphingomonas yanoikuyae B1 is able to grow on a wide variety of aromatic compounds including biphenyl, naphthalene, phenanthrene, toluene, m-, and p-xylene. In addition, the initial enzymes for degradation of biphenyl have the ability to metabolize a wide variety of different polycyclic aromatic hydrocarbons. The catabolic pathways for the degradation of both the monocyclic and polycyclic aromatic hydrocarbons are intertwined, joining together at the level of (methyl)benzoate and catechol. Both upper branches of the catabolic pathways are induced when S. yanoikuyae B1 is grown on either class of compound. An analysis of the genes involved in the degradation of these aromatic compounds reveals that at least six operons are involved. The genes are not arranged in discrete pathway units but are combined in groups with genes for the degradation of both classes of compounds in the same operon. Genes for multiple dioxygenases are present perhaps explaining the ability of S. yanoikuyae B1 to grow on a wide variety of aromatic compounds. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Enrichment and isolation of non-specific aromatic degraders from unique uncontaminated (plant and faecal material) sources and contaminated soils

Microbial analysis of contaminated soil and uncontaminated plant and faecal material resulted in the enrichment of a number of microbial communities capable of utilizing a range of environmental pollutants. Growth was observed on polycyclic aromatic hydrocarbons, polychlorinated biphenyls, heterocyclic aromatic compounds and organochlorine pesticides. However, none of the communities could grow on pentachlorophenol. Pure cultures were isolated from microbial communities using phenanthrene and pyrene as the sole carbon and energy source. Isolates were also obtained using DDT, DOH, DBH and PCPA when peptone was supplemented to the medium. Strain AJR39,504, isolated using DDT and peptone, could not be positively identified on the basis of substrate utilization tests. However, it most closely resembled Stenotrophomonas maltophilia (0.424 similarity) using the Microlog 3 database software. Isolate AJR39, 504 could also grow on polycyclic aromatic hydrocarbons, chlorinated- and nitro-aromatic compounds. In addition, the degradation of DDT (100 mg l(-1)) by isolate AJR39,504 resulted in a 35% decrease in DDT concentration after 28 days with a concomitant increase in DDD concentration.     

Assessment of occupational exposure to PAHs in an Estonian coke oven plant- correlation of total external exposure to internal dose measured as 1-hydroxypyrene concentration

The exposure of cokery workers to polynuclear aromatic hydrocarbons at an Estonian oil shale processing plant was assessed by using occupational hygiene and biomonitoring measurements which were carried out twice, in midwinter and in the autumn. To assess the external dose of polynuclear aromatic hydrocarbons, pyrene and benzo[a]pyrene concentrations were measured from the breathing zone of workers during a workshift. Skin contamination with pyrene and benzo[a]pyrene was assessed by skin wipe sampling before and after the workshift. As a biomarker of overall exposure to polynuclear aromatic hydrocarbons, and as an integral of all absorption routes of pyrene, 1-hydroxypyrene concentration was measured from post shift urine samples. Of the personal air samples, 18% exceeded the Finnish threshold limit value of benzo[a]pyrene (10 μg m^-3). Mean value (two separate measurements together) for benzo[a]pyrene was 5.7 μg m^-3 and for pyrene, 8.1 μg m^-3. Based on skin wipe sample analyses, the skin contamination was also obvious. The mean value of benzo[a]pyrene in the samples collected after the shift was 1.2 ng cm^-2. Benzo[a]pyrene was not found in control samples. The mean value of urinary 1-hydroxypyrene concentration was 6.0 μmol mol^-1 creatinine for the exposed workers and 0.5 μmol mol^-1 creatinine for the controls. This study undoubtedly shows the usefulness of 1-hydroxypyrene as an indicator of internal dose of polynuclear aromatic hydrocarbons. It can be concluded that the cokery workers at the Kohtla-Järve plant are exposed to high concentrations of polynuclear aromatic compounds, and the exposure level is considerably higher during the winter measurements.     

Preparation and chromatographic performance of a multifunctional immobilized chiral stationary phase based on dialdehyde microcrystalline cellulose derivatives

A novel high‐performance liquid chromatography (HPLC) multifunctional immobilized chiral stationary phase was prepared by bonding dialdehyde microcrystalline cellulose to aminosilica via Schiff base reaction and then derivatized with 3,5‐dimethylphenyl isocyanate. The HPLC multifunctional immobilized chiral stationary phase could not only achieve chiral separation but also achieve achiral separation. Chiral separation evaluation showed that 1‐(1‐naphthyl)ethanol and mandelonitrile got separation in normal phase (NP) mode. Ranolazine, benzoin ethyl ether, metalaxyl, and diclofop were successfully separated in reversed phase (RP) mode. Aromatic compounds such as polycyclic aromatic hydrocarbons (PAHs), anilines, and aromatic acids were selected as analytes to investigate the achiral separation performance of the multifunctional immobilized chiral stationary phase in NP and RP modes. The achiral separation evaluation showed that six PAHs could get good separation within 10 minutes in NP mode. Four aromatic acids were well separated in RP mode. The retention mechanism of aromatic compounds on the stationary phase was discussed, founding that π‐π interaction, π‐π electron‐donor‐acceptor (EDA) interaction, and hydrogen bonding interaction played important roles during the achiral separation process. This multifunctional immobilized chiral stationary phase had the advantages of simple bonding steps, short reaction time, and no need for space arm.     

Hydrocarbon-degrading filamentous fungi isolated from flare pit soils in northern and western Canada

Sixty-four species of filamentous fungi from five flare pits in northern and western Canada were tested for their ability to degrade crude oil using gas chromatographic analysis of residual hydrocarbons following incubation. Nine isolates were tested further using radiorespirometry to determine the extent of mineralization of model radiolabelled aliphatic and aromatic hydrocarbons dissolved in crude oil. Hydrocarbon biodegradation capability was observed in species representing six orders of the Ascomycota. Gas chromatography indicated that species capable of hydrocarbon degradation attacked compounds within the aliphatic fraction of crude oil, n-C12-n-C26; degradation of compounds within the aromatic fraction was not observed. Radiorespirometry, using n-[1-14C]hexadecane and [9-14C]phenanthrene, confirmed the gas chromatographic results and verified that aliphatic compounds were being mineralized, not simply transformed to intermediate metabolites. This study shows that filamentous fungi may play an integral role in the in situ biodegradation of aliphatic pollutants in flare pit soils.     

Engineering cytochrome P450 BM-3 for oxidation of polycyclic aromatic hydrocarbons.

Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.     

Use of soft hydrothermal processing to improve and recycle bedding for laboratory animals

Cage bedding for laboratory rodents can influence animal wellbeing and thus the experimental data. In addition, a large amount of used bedding containing excrement is discharged as medical waste from life science institutes and breeding companies. We developed a ground-breaking system to improve fresh bedding and recycle used bedding by applying a soft hydrothermal process with high-temperature and high-pressure dry steam. The system removes both harmful organic components and aromatic hydrocarbons that can affect animals' metabolism. The purpose of the present study was to evaluate the chemical and physical properties of the improved fresh bedding and the recycled used bedding treated by the system. The results showed that 68-99% of the predominant aromatic hydrocarbons were removed from fresh bedding treated at 0.35 MPa and 140 degrees C for 120 min ('improved bedding'). In addition, 59.4-99.0% of predominant harmful organic compounds derived from excrement were removed from used bedding treated at 0.45 MPa and 150 degrees C for 60 min ('recycled bedding'). The soft hydrothermal treatment increased the number of acidic functional groups on the bedding surface and gave it the high adsorptive efficiency of ammonia gas. Harmful substances such as microorganisms, heavy metals and pesticides decreased below the detection limit. The results clearly showed that the improved and recycled bedding is safer for laboratory rodents and has the potential to ameliorate conditions in primary and secondary enclosures (e.g. cages and animal rooms) used for maintaining laboratory animals. This process may be one of the most advanced techniques in providing an alternative to softwood and other bedding, economizing through the recycling of used bedding and reducing bedding waste from animal facilities.     

Energetics of interactions of aromatic hydrocarbons with water

The thermodynamics of transfer of aromatic (benzene, toluene) and aliphatic (ethane, propane, butane) hydrocarbons from the gas phase into water in the temperature range 5–125°C have been analyzed in order to determine the net hydration effect of these compounds. In the case of the aromatic hydrocarbons the enthalpic contribution predominates over the entropic contribution to the Gibbs energy of hydration. This results in a negative value of the hydration Gibbs energy of aromatic hydrocarbons, in contrast to the positive Gibbs energy of hydration of aliphatic hydrocarbons. The different sign of the hydration Gibbs energies indicates that the mechanism causing hydrophobicity of aromatic hydrocarbons has different nature than that causing the hydrophobicity of aliphatic hydrocarbons. The comparison of hydration of aliphatic and aromatic hydrocarbons leads to the following thermodynamic parameters for these additional interactions between the benzene ring and water at 25°C: enthalpy −5.4 kJ/mol, entropy 26.8 J/K mol and Gibbs energy −13.4 kJ/mol. The large enthalpic contribution to the Gibbs energy of hydration of aromatic hydrocarbons probably comes from the ability of the aromatic ring to accept hydrogens from water, forming hydrogen bonds.     

Dioxygen activation responsible for oxidation of aliphatic and aromatic hydrocarbon compounds: current state and variants

The most significant aspect in microbial metabolisms, especially those of bacteria and archaea, is their marvelously wide acceptability of substrate electron donors and acceptors. This feature makes them to be attractive catalysts for environmental biotechnology in terms of degradation of harmful recalcitrant compounds, including hydrocarbons. Transformation of highly reduced and inert hydrocarbon compounds is with no doubt a challenging biochemical reaction for a single enzyme. However, several multi-component enzyme systems enable microorganisms to utilize hydrocarbons as carbon and energy (electron) sources. Initial biological attack to hydrocarbons is, in most cases, the hydroxylation that requires molecular dioxygen as a co-substrate. Dioxygen also contributes to the ring cleavage reaction of homo- and hetero-cyclic aromatic hydrocarbons. Although the molecular dioxygen is omnipresent and highly soluble in water, activation and splitting this triplet ground-state molecule to wed with difficult hydrocarbons need special devices. Non-heme iron, heme iron, or flavin nucleotide was designated as a major hidden dagger for this purpose.     

Nucleolipids as potential organogelators

Four different series of nucleolipids or bola-nucleolipids were synthesized or re-synthesized. Most of the compounds were studied with respect to their gelation properties toward either water or aromatic, hetero-aromatic, and aliphatic hydrocarbons. Bola-nucleolipids 6 and 7 do not gelate any solvent tested, neither as sole additive nor by adding up to 10 wt% of a 1:1 mixture. The nucleolipid 22 carrying the antiviral acyclovir as a head group proved to be a potent organogelator for aromatic hydrocarbons such as toluene, but not for hetarenes, aliphatic hydrocarbons or water. The mono-tailed nucleolipid 24 exhibits excellent organogelator properties for both aromatic and aliphatic hydrocarbons. These were studied as a function of concentration and temperature.