Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Interactions between benzene, toluene, and p-xylene (BTX) during their biodegradation

A microbial consortium and Pseudomonas strain (PPO1) were used in studying biodegradation of benzene, toluene, and p-xylene under aeorbic conditions. Studies involved removal of each compound individually as well as in mixture with the others. Both cultures exhibited a qualitatively similar behavior toward each compound. Both the pure culture and the consortium grew on benzene following Monod kinetics, on toluene following inhibitory (Andrews) kinetics, whereas neither could grow on P-xylene. Benzene and toluene mixtures were removed under cross-inhibitory (competitive inhibition) kinetics. In the presence of benzene and/or toluene, p-xylene was cometabolically utilized by both cultures, but was not completely mineralized. Metabolic intermediates of p-xylene accumulated in the medium and were identified. Benzene and toluene were completely mineralized. Cometabolic removal of p-xylene reduced the yields on both benzene and toluene. Except for cometabolism, kinetic constants were determined from data analysis and are compared with values published recently by other researchers. (c) 1994 John Wiley & Sons, Inc.     

Isolation and Metabolic Characterization of a Pseudomonas stutzeri Mutant Able To Grow on the Three Isomers of Xylene

From an o-xylene-degrading Pseudomonas stutzeri strain (OX1), we previously isolated mutant M1, which had acquired the ability to grow on m-xylene and p-xylene but lost the ability to utilize the ortho isomer. From M1 cultures we have now isolated a revertant strain (R1) which grows on o-xylene and retains the ability to grow with the meta and para isomers regardless of the selective pressure applied. In P. stutzeri R1, o-xylene is degraded through two successive monooxygenations of the aromatic ring, while m-xylene and p-xylene catabolism proceeds through the progressive oxidation of a methyl substituent, although unquantifiable amounts of these two substrates are transformed into the corresponding dimethylphenols, which are not utilized for further growth. The two catabolic pathways are inducible by all three xylene isomers.     

The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate

Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological conversion into valuable biodegradable plastic polymers-polyhydroxyalkanoates (PHAs). We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX) compounds as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% cell dry weight for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA was accumulated to 24% and to 36% of the cell dry weight of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350 degrees C and a glass transition temperature of -48.5 degrees C.     

Utilization of Alkylbenzenes during Anaerobic Growth of Pure Cultures of Denitrifying Bacteria on Crude Oil

Four pure cultures of denitrifying bacteria, which had previously been isolated on defined alkylbenzenes, were capable of anaerobic growth with crude oil as the only source of organic substrates. Chemical analyses after growth revealed that the known growth substrates toluene, ethylbenzene, and m-xylene were selectively consumed from the oil. o-Xylene and p-xylene, which as pure compounds did not support growth, were consumed to a lesser extent.     

Regulation of the enzyme activity of para-xylene catabolism in a spontaneous para-xylene-negative variant of Pseudomonas aeruginosa 2x79

A spontaneous variant of Pseudomonas aeruginosa 2x unable to grow on p-xylene as the sole source of carbon and energy has been isolated. p-Xylenenegative variant of P. aeruginosa 2x79 differs from the wild type strain by the character of growth on the p-xylene oxidation intermediates p-toluate and protocatochuate. The cell of 2x79 variant inability to grow on p-xylene has been shown to be accompanied by the elimination of the activities of three enzymes - p-xylene methylhydroxylase, p-cresol methylhydroxylase and metapyrocatechase and by the considerable alteration in the regulation of orto-cleavage aromatic ring enzymes activity pyrocatechase and protocatechuate-3,4-dioxygenase. Possible reasons for appearing spontaneous variants 2x79 in the population of P. aeruginosa 2x growing on the p-xylene are being discussed.     

Isolation of a Pseudomonas stutzeri strain that degrades o-xylene.

A Pseudomonas stutzeri strain capable of growing on o-xylene was isolated from enrichment cultures. The organism grew on 2,3- and 3,4-dimethylphenol but not on 2-methylbenzyl alcohol, o-tolualdehyde, or o-toluate. P. stutzeri was not able to utilize m-xylene, p-xylene, or 1,2,4-trimethylbenzene, but growth was observed in the presence of the corresponding alcohols and acids. From the Pseudomonas cultures supplied with o-xylene, 2,3-dimethylphenol was isolated and identified. When resting P. stutzeri cells were incubated with 2,3-dimethylphenol, the reaction mixture turned greenish yellow and showed spectral properties identical to those of the 3,4-dimethylcatechol meta ring fission product. Catechol 2,3-oxygenase was induced by growth on o-xylene or on 2,3- or 3,4-dimethylphenol. The suggested hypothesis is that the first metabolic steps of growth on o-xylene involve the direct oxygenation of the aromatic nucleus, followed by meta pathway reactions.     

Isolation of a Pseudomonas stutzeri strain that degrades o-xylene

A Pseudomonas stutzeri strain capable of growing on o-xylene was isolated from enrichment cultures. The organism grew on 2,3- and 3,4-dimethylphenol but not on 2-methylbenzyl alcohol, o-tolualdehyde, or o-toluate. P. stutzeri was not able to utilize m-xylene, p-xylene, or 1,2,4-trimethylbenzene, but growth was observed in the presence of the corresponding alcohols and acids. From the Pseudomonas cultures supplied with o-xylene, 2,3-dimethylphenol was isolated and identified. When resting P. stutzeri cells were incubated with 2,3-dimethylphenol, the reaction mixture turned greenish yellow and showed spectral properties identical to those of the 3,4-dimethylcatechol meta ring fission product. Catechol 2,3-oxygenase was induced by growth on o-xylene or on 2,3- or 3,4-dimethylphenol. The suggested hypothesis is that the first metabolic steps of growth on o-xylene involve the direct oxygenation of the aromatic nucleus, followed by meta pathway reactions.     

Microbial Hydrocarbon Co-oxidation. II. Use of Ion-Exchange Resins

Anion-exchange resins, a weakly basic polystyrene-polyamine type and a macro-reticular type, IR-45 and IRA-93, respectively, were shown to significantly increase yields of acidic products in co-oxidation systems. p-Toluic, 2,3-dihydroxy-p-toluic, and alpha,alpha-cis,cis dimethylmuconic acids, resulting from the oxidation of p-xylene by three cultures of Nocardia, accumulated on the resin in shaken flasks or agar plates during the cultivation. Final product concentration increased with increasing resin concentration. Mineral balances were not affected if the resin was properly conditioned before use.     

Pure bacterial isolates that convert p-xylene to terephthalic acid

Bacteria that grow on p-xylene, p-toluic acid, and terephthalic acid (TPA) were isolated from a wastewater bioreactor that is used to treat a waste stream that contains all three of these compounds. Although previously described aerobic bacteria degrade p-xylene by initially oxidizing a single methyl group to form p-toluic acid and then cleaving the aromatic ring, some of the bacteria isolated during this study transformed p-xylene by oxidizing both methyl groups to produce TPA.     

Combination of the tod and the tol pathways in redesigning a metabolic route of Pseudomonas putida for the mineralization of a benzene, toluene, and p-xylene mixture.

Construction of a hybrid strain which is capable of mineralizing components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture. Consequently, a hybrid strain was constructed by cloning todC1C2BA genes encoding toluene dioxygenase on RSF1010 and introducing the resulting plasmid into P. putida mt-2. The hybrid strain of P. putida TB105 was found to mineralize a benzene, toluene, and p-xylene mixture without accumulation of any metabolic intermediate.     

Biotransformation of p-xylene and 2,6-dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate

Sphingomonas strain ASU1 was isolated from an industrial wastewater bioreactor and grew on 2,6-dimethylnaphthalene (2,6-DMN) as the sole carbon/energy source. The genes for a xylene monooxygenase were cloned from strain ASU1. Expression of the ASU1 xylene monooxygenase was compared to expression of the pWWO xylene monooxygenase in Escherichia coli. Both monooxygenases transformed p-xylene and 2,6-DMN by initially hydroxylating one methyl group. In addition, the ASU1 monooxygenase also hydroxylated the second methyl group on p-xylene and 2,6-DMN whereas the pWWO monooxygenase hydroxylated the second methyl group only on p-xylene. Endogenous E. coli enzymes contributed to further oxidation of the resulting aromatic alcohols to form aromatic carboxylates.     

Physiological properties of a Pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium.

Pseudomonas putida Idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50% (vol/vol). Growth also occurs on Luria-Bertani medium in the presence of a wide range of organic solvents. The ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethyl-phthalate (log P(OCT) = 2.3) being the most polar solvent tolerated. During growth with p-xylene (20% [vol/vol]), there was an initial lag period accompanied by cell death, which was followed by a period of exponential growth. The stationary phase of growth was characterized by a dramatic decrease in cell viability, although cell dry weight and turbidity measurements slowly increased. Electron micrographs revealed that during growth in the presence of p-xylene, the outer cell membrane becomes convoluted and membrane fragments are shed into the culture medium. At the same time, the cytoplasmic membrane invaginates, forming vesicles, and becomes disorganized. Electron-dense intracellular inclusions were observed in cells grown with p-xylene (20% [vol/vol]) and p-xylene vapors, which are not present in cells grown with succinate. Attempts to demonstrate the presence of plasmid DNA in P. putida Idaho were negative. However, polarographic studies indicated that the organism utilizes the same pathway for the degradation of toluene, m-xylene, and p-xylene as that used by P. putida mt-2 which contains the TOL plasmid pWWO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological properties of a Pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium.

Pseudomonas putida Idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50% (vol/vol). Growth also occurs on Luria-Bertani medium in the presence of a wide range of organic solvents. The ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethyl-phthalate (log P(OCT) = 2.3) being the most polar solvent tolerated. During growth with p-xylene (20% [vol/vol]), there was an initial lag period accompanied by cell death, which was followed by a period of exponential growth. The stationary phase of growth was characterized by a dramatic decrease in cell viability, although cell dry weight and turbidity measurements slowly increased. Electron micrographs revealed that during growth in the presence of p-xylene, the outer cell membrane becomes convoluted and membrane fragments are shed into the culture medium. At the same time, the cytoplasmic membrane invaginates, forming vesicles, and becomes disorganized. Electron-dense intracellular inclusions were observed in cells grown with p-xylene (20% [vol/vol]) and p-xylene vapors, which are not present in cells grown with succinate. Attempts to demonstrate the presence of plasmid DNA in P. putida Idaho were negative. However, polarographic studies indicated that the organism utilizes the same pathway for the degradation of toluene, m-xylene, and p-xylene as that used by P. putida mt-2 which contains the TOL plasmid pWWO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Performance and macrokinetic analysis of biofiltration of toluene and p-xylene mixtures in a conventional biofilter packed with inert material

Interactions of toluene and p-xylene in air treatment biofilters packed with an inert filter media were studied. The effect of the inlet load of toluene, p-xylene and mixtures of both compounds on the biodegradation rate was analyzed in three lab-scale biofilters. A maximum elimination capacity (EC) of 26.5 and 40.3 g C m⁻³ h⁻¹ for an inlet load (IL) of 65.6 and 57.8 g C m⁻³ h⁻¹ was obtained for p-xylene and toluene biofilters, respectively. Inhibition of p-xylene biodegradation by the presence of toluene took place when the mixture was treated, whereas the presence of p-xylene had an enhancing effect on the toluene removal efficiency. Specific growth rates (μ) from 0.019 to 0.068 h⁻¹ were calculated in the mixed biofilter, where the highest values were similar to mixtures with lower p-xylene levels (IL_p-Xyl 8.84 ± 0.29 g C m⁻³ h⁻¹). Michaelis-Menten and Haldane type models were fitted to experimental EC for p-xylene and toluene biofilters, respectively.     

Regioselective oxidation of xylene isomers by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is able to utilize a variety of monocyclic aromatic hydrocarbons, including benzene, phenol, toluene, and o-xylene, as growth substrates. Although DK17 is unable to grow on m- and p-xylene, this strain could transform these two xylene isomers to some extent after induction by o-xylene. The major accumulating compounds formed during the degradation of m- and p-xylene by DK17 were isolated by high-pressure liquid chromatography and identified by gas chromatography-mass spectrometric and (1)H nuclear magnetic resonance spectral techniques. Both xylene isomers were transformed to dihydroxylated compounds by what must be two successive hydroxylation events: m-xylene was converted to 2,4-dimethylresorcinol and p-xylene was converted to 2,5-dimethylhydroquinone. The rigorous structural identification of 2,4-dimethylresorcinol and 2,5-dimethylhydroquinone demonstrates that DK17 can perform distinct regioselective hydroxylations depending on the position of the substituent groups on the aromatic ring.     

The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, O-, and m-xylene by sulfate-reducing bacteria

Anaerobic sulfate-reducing bacteria were enriched from contaminated aquifer samples with naphthalene, o-, and m-xylene as sole carbon and energy source in the presence of Amberlite-XAD7, a solid adsorber resin. XAD7 served as a substrate reservoir maintaining a constantly low substrate concentration in the culture medium. In equilibration experiments with XAD7, the aromatic hydrocarbons needed up to 5 days to achieve equilibrium between the water and the XAD7 phase. The equilibrium concentration was directly correlated with the amount of added substrate and XAD7. In the enrichments presented here, XAD7 and aromatic hydrocarbons were adjusted to maintain substrate concentrations of 100 microM m-, or o-xylene, or 50 microM naphthalene. After five subsequent transfers, the three cultures were able to grow with higher substrate concentrations in the absence of XAD7 although they grew best with lower hydrocarbon concentrations. Two new xylene-degrading cultures were obtained that could not utilise toluene as carbon source. O-xylene was degraded anaerobically by a culture, which could also oxidise m-xylene but not p-xylene. Eighty-three percent of the electrons from o-xylene oxidation were recovered in the produced sulfide, indicating a complete oxidation to CO2. Another sulfate-reducing enrichment culture oxidised m-xylene completely to CO2 but not o-, or p-xylene. A naphthalene-degrading sulfate-reducing enrichment culture oxidised naphthalene completely to CO2. Metabolites of naphthalene degradation were recovered from the XAD7 phase and subjected to GC/MS analysis. Besides the metabolites 2-naphthoic acid and decahydro-2-naphthoic acid which were identified by the mass spectrum and coelution with chemically synthesised reference compounds, the reduced 2-naphthoic acid derivatives 5,6,7,8-tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. Cultivation of bacterial cultures in the presence of XAD7 and subsequent derivatisation and extraction of metabolites directly from the solid XAD7 resin provides a new method for the isolation of sensitive bacteria and identification of metabolites.     

对二甲苯对皱纹盘鲍肝胰腺细胞DNA的损伤

为研究对二甲苯对皱纹盘鲍肝胰腺的毒性作用,设置4个浓度(0.5、1.0、1.5和2.0 mg·L^-1)和对照组,开展为期21 d的对二甲苯对皱纹盘鲍的亚慢性毒性试验,采用彗星试验技术进行皱纹盘鲍肝胰腺细胞DNA损伤分析,采用CASP分析软件对拖尾率、彗星尾长、彗尾DNA相对含量、Olive矩等损伤指标进行统计。结果表明: 与对照组相比,各染毒组皱纹盘鲍肝胰腺细胞DNA均受到损伤,且损伤程度存在显著性差异。随着染毒浓度的增加,肝胰腺细胞DNA受损程度加重,高浓度甚至可以引发细胞凋亡,呈现一定的剂量-损伤效应。中浓度对二甲苯短时间暴露即可对皱纹盘鲍肝胰腺细胞造成DNA损伤,随着暴露时间延长,细胞DNA受损程度加重,呈现一定的时间-损伤效应。但长时间暴露细胞DNA各损伤指标有所减小,这可能与细胞自身的DNA修复机制和生物体解毒系统的代谢机制有关。研究表明,对二甲苯可对皱纹盘鲍肝胰腺细胞产生氧化损伤,导致DNA断裂,高浓度的对二甲苯长时间暴露可导致其细胞凋亡。     

Changes in the rat's motor behaviour during 4-hr inhalation exposure to prenarcotic concentrations of benzene and its derivatives

Group motility was recorded continuously in male rats during the inhalation of benzene, toluene, ethylbenzene, o-, m- and p-xylene vapours. The solvents were applied in at least six concentrations, up to those inducing anaesthesia. Minimum narcotic concentrations (ppm) were: 5940 (benzene), 3590 (toluene), 2180 (ethyl-benzene), 2180 (0-xylene), 2100 (m-xylene), and 1940 (p-xylene). The results indicate that prenarcotic concentrations of these structurally related aromatic hydrocarbons and also the xylene isomers elicit qualitatively and quantitatively different acute behavioral effects. Except o-xylene which caused depression only the agents produced bell-shaped concentration-action curves characteristic of the biphasic effect, i.e., activation at lower and depression at higher concentrations. The curves differed in form and magnitude depending on the stimulatory potency and on the range of effective concentrations. Based on arbitrary assessment of central excitation, the five aromatics may be ranked as follows: benzene and toluene (striking activation), p-xylene (marked activation), ethylbenzene (moderate activation), m-xylene (slight activation). At the same time, high degree of motor incoordination, and in the case of benzene and p-xylene, also marked tremor could be seen.