Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Bacterial growth and substrate degradation by BTX-oxidizing culture in response to salt stress

Interactions between microbial growth and substrate degradation are important in determining the performance of trickle-bed bioreactors (TBB), especially when salt is added to reduce biomass formation in order to alleviate media clogging. This study was aimed at quantifying salinity effects on bacterial growth and substrate degradation, and at acquiring kinetic information in order to improve the design and operation of TBB. Experiment works began by cultivating a mixed culture in a chemostat reactor receiving artificial influent containing a mixture of benzene, toluene, and xylene (BTX), followed by using the enrichment culture to degrade the individual BTX substrates under a particular salinity, which ranged 0–50 g l⁻¹ in batch mode. Then, the measured concentrations of biomass and residual substrate versus time were analyzed with the microbial kinetics; moreover, the obtained microbial kinetic constants under various salinities were modeled using noncompetitive inhibition kinetics. For the three substrates the observed bacterial yields appeared to be decreased from 0.51–0.74 to 0.20–0.22 mg mg⁻¹ and the maximum specific rate of substrate utilization, declined from 0.25–0.42 to 0.07–0.11 h⁻¹, as the salinity increased from 0 to 50 NaCl g l⁻¹. The NaCl acted as noncompetitive inhibitor, where the modeling inhibitions of the coefficients, K _T(S), were 22.7–29.7 g l⁻¹ for substrate degradation and K _T(μ), 13.0–19.0 g l⁻¹, for biomass formation. The calculated ratios for the bacterial maintenance rate, m _S, to further indicated that the percentage energy spent on maintenance increased from 19–24 to 86–91% as salinity level increased from 0 to 50 g l⁻¹. These results revealed that the bacterial growth was more inhibited than substrate degradation by the BTX oxidizers under the tested salinity levels. The findings from this study demonstrate the potential of applying NaCl salt to control excessive biomass formation in biotrickling filters.     

Simultaneous determination of benzene, toluene, ethylbenzene, and xylenes in urine by thermal desorption–gas chromatography

The determination of metabolites of benzene, toluene, ethylbenzene, and xylenes in urine has been used to assess human exposure to these compounds. The analyses of urine samples for these metabolites are tedious and time consuming. The determination of unmetabolized individual compounds in urine has been studied previously with some success. A simultaneous determination of several unmetabolized VOC compounds in urine by thermal desorption–gas chromatography was conducted to assess the exposure of smokers and nonsmokers to these compounds. The method of thermal desorption–GC was sensitive enough to detect a significant difference in exposure levels due to the contribution of light smoking in the environmentally-exposed group.     

Continuous removal of aromatic hydrocarbons by an AF reactor under denitrifying conditions

Removal of three typical aromatic hydrocarbons, benzene, biphenyl and naphthalene by an anaerobic filter (AF) reactor under continuous mode and denitrifying conditions was studied. Results showed that the AF reactor could degrade these aromatic hydrocarbons effectively under above-mentioned conditions. When influent wastewater contained 900 mg COD/l and about 60 mg (total aromatic hydrocarbons)/l, 90% and 84% removal efficiency could be achieved for them respectively. When COD/NO₃ ⁻-N ratio (C/N) was in the range 5–30, the removal of benzene was slightly influenced by C/N and it remained stable at about 90%. However, degradation of naphthalene, biphenyl and total COD was greatly influenced by C/N, and highest removal was achieved at C/N = 15, it was 90%, 85% and 82% for COD, naphthalene and biphenyl, respectively. Degradation of these three aromatic hydrocarbons followed the order: benzene > naphthalene > biphenyl.     

Utilization of toluene and xylenes by a nitrate-reducing strain of Pseudomonas maltophilia under low oxygen and anoxic conditions

Abstract A nitrate-reducing strain of Pseudomonas maltophilia isolated from sewage sludge degrades toluene, and at least two isomers of mixed xylenes, either in the presence of 2% oxygen or under anoxic conditions when nitrate is present. When individual isomers of xylene are provided only meta and para -xylene are utilized. When mixed xylenes are provided all three isomers may be utilized. In cultures limited by electron acceptor availability, succinate, when present as the major carbon source, does not prevent hydrocarbon utilization. Toluene and xylenes continued to be utilized either with limiting nitrate alone, or with limiting nitrate and oxygen present simultaneously when a hundred-fold excess of succinate is present in the medium. The data suggest that in groundwater containing low levels of oxygen and nitrate, or nitrate only as the electron acceptor, aromatic hydrocarbons may continue to be utilized even in the presence of an excess of readily-degradable non-hydrocarbon organic substrates. These data have implications for bioremediation studies. The strain of Pseudomonas maltophilia used in this study does not degrade benzene, and the presence of benzene does not affect toluene utilization.     

A simple method to evaluate the concentration of pentachlorophenol degraders in contaminated soils

A new most probable number (MPN) method for the determination of pentachlorophenol (PCP) degraders in soil using the change in pH due to PCP degradation is compared with a well documented MPN method using radiolabeled PCP. The results of all MPN counts were similar within a 95% confidence limit. The results obtained in MPN per gram of dry soil using pH measurements were 1.8 (+3.1, -1.03) x10 (4) compared to 0.64 (+1.34, -0.42) x 10(4) when using production of [(14)C]CO(2).     

一株苯系物降解真菌筛选及降解特性

采用逐量分批驯化的方法以污水处理厂污泥作为菌源,苯、甲苯、二甲苯为唯一碳源,驯化、分离、筛选能够有效降解苯系物的真菌,命名为B1。采用单因素以及正交实验方法并对真菌降解环境影响因素及降解效率进行了测定和研究。结果表明:真菌B1对苯系物降解的最佳条件为C:N=5:1,pH5,温度30℃,菌种接种量为5.5ml(50ml培养基)。采用GC对初始液相浓度0~90mg/L范围内的苯系物降解效果进行测定,未发现苯系物对真菌降解活性产生抑制作用。真菌对苯系物的降解效率为:甲苯>苯>二甲苯,最高降解效率分别达到87.39%,85.21%,81.47%。混合物降解效果略高于单一底物的降解效果。     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Interrelationships between metabolic hormones,leptin and ghrelin,and oil-related contaminants in control of oxytocin and prostaglandin F release by feline ovaries

We examined the effects of metabolic hormones leptin and ghrelin, and the oil-related environmental contaminants toluene and xylene on the release of ovarian hormones by gravid and non-gravid cats, as well as the functional interrelationships between metabolic hormones and contaminants. Ovarian fragments of non-gravid cats were cultured with and without leptin and toluene. Next, ovarian fragments of either non-gravid or gravid animals were cultured with and without ghrelin and xylene. Oxytocin (OT) and prostaglandin F (PGF) release was measured using ELISA.We confirm ovarian OT and PGF production by feline ovary, demonstrate the involvement of leptin and ghrelin in controlling OT and PGF release, show the direct influence of toluene and xylene on feline ovarian secretory activity, indicate the ability of leptin and ghrelin to mimic and promote the main contaminant effects, demonstrate that oil-related contaminants can prevent and even invert the effects of leptin and ghrelin on the ovary, and suggest the gravidity-associated changes in ability of ghrelin to promote xylene action on PGF (but not to OT), but not in basic ovarian OT and PGF release and their response to ghrelin or xylene.     

Isolation,gene detection and solvent tolerance of benzene,toluene and xylene degrading bacteria from nearshore surface water and Pacific Ocean sediment

BTX (benzene, toluene and xylene) degrading bacteria were isolated from Pacific Ocean sediment and nearshore surface water. In the seawater near a ferry dock, degrading bacteria of a relatively wide diversity were detected, including species of Pseudomonas, Rhodococcus, Exiguobacterium and Bacillus; while species of Bacillus only have been detected from the deep-sea sediment. Most of the isolates showed degradation to more than one compound. Generally better growth was obtained with p-xylene and ethylbenzene than with the other two. All the bacteria could tolerate and grow with the compounds at 5–20% (v/v). Both benzene and toluene degradation related genes had been successfully PCR cloned from the isolates of nearshore water, the detected benzene dioxygenase gene was identical among all the species and close to its soil counterpart. However, they were not detected in all the isolates from deep sea. Results in this report suggested that BTX degrading bacteria widely spread in marine environments and they might be of potentials in biotreatment of BTEX in saline environments.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.     

Specific rates of substrate oxidation and product formation in autotrophically growingChromatium vinosum cultures

Eighty-six species of fungi belonging to sixty-four genera were examined for their ability to metabolize naphthalene. Analysis by thin-layer and high pressure liquid chromatography revealed that naphthalene metabolism occurred in forty-seven species belonging to thirty-four genera from the major fungal taxa. All organisms tested from the order Mucorales oxidized naphthalene with species of Cunninghamella, Syncephalastrum and Mucor showing the greatest activity. Significant metabolism was also observed with Neurospora crassa, Claviceps paspali and four species of Psilocybe. The predominant metabolite formed by most organisms was 1-naphthol. Other products identified were, 4-hydroxy-1-tetralone, trans-1,2-dihydroxy-1,2-dihydronaphthalene, 2-naphthol, 1,2-and 1,4-naphthoquinone.     

Identification,cloning, and characterization of a multicomponent biphenyl dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.     

Detection of aromatic catabolic gene expression in heterogeneous organic matter used for reduction of volatile organic compounds (VOC) by biofiltration

A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, used as biofilter support for removing volatile organic compounds, was set up and applied to detect xylene monooxygenase gene expression by RT-PCR. A DNA/RNA extraction protocol based on a combination of sample lyophilization pre-treatment and CTAB––phenol/chloroform extraction procedure was optimized for the recovery of purified nucleic acids [100–500 ng DNA (10 kb) and 0.5–2 μg of rRNA 16S from 100 mg matrix]. PCR and RT-PCR protocols were established to detect xylene monooxygenase gene expression starting from differentially induced organic matrices obtained by biofiltration technology. This work allowed the microbial degradation activities in heterogeneous organic solid media to be studied and suggests a rapid method to follow specific biological activities during solid and/or semisolid organic substrates biotransformation.     

Synthesis of cationic (arene)IronCp complexes via arene exchange

Rates and regioselectivity of arene exchange reactions in cationic fused arene Fe(II)Cp complexes were investigated. Thermal exchange of pyrene, naphthalenes, and cyclooctatetraene occurs in the temperature range of 90-140 °C. The most labile complex in the series studied is [(η⁶-(1-4,4a,8a)-1,4-dimethoxynaphthalene)FeCp][PF₆] having the FeCp coordinated to the substituted ring. Pyrene and other naphthalene complexes come next, followed by the cyclooctatetraene complex. Phenanthrene, veratrol, and dihydronaphthalene do not undergo exchange at temperatures up to 130 °C. With Me- and OMe-substituted naphthalenes, exchange is reversible and favors the product having the metal coordinated to the non-substituted ring. The X-ray crystal structures of the two regioisomeric 1,4-dimethoxynaphthalene complexes were determined. Arene exchange in fused arene complexes is shown to be a useful synthetic method and provides new arene complexes cleanly and efficiently. The method is particularly attractive for arenes that contain functionalities that are not compatible with the Lewis acid-mediated routes. The starting materials are readily accessible via the TiCl₄-assisted Cp exchange in ferrocene.     

Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker

Benzylsuccinate synthase (Bss) is the key enzyme of anaerobic toluene degradation and has been found in all anaerobic toluene degrading bacterial isolates tested. However, only a few pure cultures capable of anaerobic toluene oxidation are available to date, and it is important to understand the relevance of these model organisms for in situ bioremediation of hydrocarbon-contaminated aquifers. Due to their phylogenetic dispersal, it is not possible to specifically target anaerobic toluene degraders using marker rRNA genes. We therefore established an assay targeting a approximately 794 bp fragment within the Bss alpha-subunit (bssA) gene, which allows for the specific detection and affiliation of both known and unknown anaerobic degraders. Three distinct tar-oil-contaminated aquifer sites were screened for intrinsic bssA gene pools in order to identify and compare the diversity of hydrocarbon degraders present at these selected sites. We were able to show that local diversity patterns of degraders were entirely distinct, apparently highly specialized and well-adapted to local biogeochemical settings. Discovered at one of the sites were bssA genes closely related to that of Geobacter spp., which provides evidence for an importance of iron reduction for toluene degradation in these sediments. Retrieved from the other two sites, dominated by sulfate reduction, were previously unidentified bssA genes and also deeply branching putative bssA homologues. We provide evidence for a previously unrecognized diversity of anaerobic toluene degraders and also of other hydrocarbon degraders using fumarate-adding key reactions in contaminated aquifers. These findings enhance our current understanding of intrinsic hydrocarbon-degrading microbial communities in perturbed aquifers and may have potential for the future assessment and prediction of natural attenuation based on degradation genes.     

Metabolism of aromatic hydrocarbons by yeasts

Six yeasts were examined for their ability to metabolize naphthalene, biphenyl and benzo(a)pyrene. All of the organisms tested oxidized these aromatic hydrocarbons. Candida lipolytica oxidized naphthalene to 1-naphthol, 2-naphthol, 4-hydroxy-1-tetralone and trans-1,2-dihydroxy-1,2-dihydronaphthalene. The major metabolite was 1-naphthol. C. lipolytica oxidized biphenyl to produce 2-, 3-, and 4-hydroxybiphenyl, 4,4′-dihydroxybiphenyl and 3-methoxy-4-hydroxybiphenyl. 4-Hydroxybiphenyl was the predominant metabolite formed. C. lipolytica oxidized benzo(a)pyrene to 3-hydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene. Metabolites were isolated and identified by absorption spectrophotometry, mass spectrometry and thin-layer, gasliquid and high-pressure liquid chromatography. Where possible the structures of these metabolites were confirmed by comparison with authenic compounds.     

Oxidation of both termini of p- and m-xylene by Escherichia coli transformed with xylene monooxygenase gene

Xylene monooxygenase (XMO) from Pseudomonas putida mt-2 catalyzes oxidation of methyl group of toluene and xylenes. While it has been postulated that this enzyme oxidizes one methyl group of xylene, we observed that both methyl groups in p- and m-xylene were oxidized to alcohol and aldehyde when the relevant genes (xylM and xylA) were co-expressed in Escherichia coli C600 and MC4100. When p-xylene was used as a substrate, p-hydroxymethylbenzaldehyde and p-xylyleneglycol were identified, in addition to p-methylbenzylalcohol and p-tolualdehyde. When m-xylene was used as a substrate, m-hydroxymethylbenzaldehyde and m-xylyleneglycol were identified, in addition to m-methylbenzylalcohol and m-tolualdehyde. Ratio of the products varied significantly according to the reaction condition and host strain, presumably reflecting the relative activity of XMO and host-derived dehydrogenase(s). Using various oxidized compounds as substrates, it was indicated that dialcohol (p- or m-xylyleneglycol) was formed via p- or m-hydroxymethylbenzaldehyde, respectively, rather than directly from corresponding monoalcohol (p- or m-methybenzylalcohol).     

Modeling aspects of hydrodesulfurization by molybdenum hydride compounds: Desulfurization of thiophene and benzothiophene and C-S bond cleavage of dibenzothiophene

The molybdenum hydride complexes Mo(PMe₃)₅H₂ and Mo(PMe₃)₄H₄ are capable of cleaving the C-S bonds of thiophene, benzothiophene and dibenzothiophene. For example, Mo(PMe₃)₅H₂ reacts with thiophene to give the η⁵-thiophene and butadiene-thiolate complexes, (η⁵-C₄H₄S)Mo(PMe₃)₃ and (η⁵-C₄H₅S)Mo(PMe₃)₂(η²-CH₂PMe₂). These complexes are also obtained from the reaction between Mo(PMe₃)₄H₄ and thiophene under photochemical conditions, whereas at elevated temperatures thiophene is desulfurized to liberate but-1-ene. Similarly, Mo(PMe₃)₄H₄ desulfurizes benzothiophene at elevated temperatures to liberate ethylbenzene, while the arylthiolate complex Mo(PMe₃)₄(SC₆H₄Et)H₃ is obtained photochemically. Furthermore, Mo(PMe₃)₄H₄ cleaves the C-S bond of dibenzothiophene to give [η⁶,κ¹-C₆H₅C₆H₄S]Mo(PMe₃)₂H.