3-(2-hydroxyphenyl)catechol as substrate for proximal meta ring cleavage in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361.

Brevibacterium sp. strain DPO 1361 oxygenates dibenzofuran in the unusual angular position. The 3-(2-hydroxyphenyl)catechol thus generated is subject to meta ring cleavage in the proximal position, yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, which is hydrolyzed to 2-oxo-4-pentenoate and salicylate by 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase. The proximal mode of ring cleavage is definitely established by isolation and unequivocal structural characterization of a cyclization product of 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, i.e., 3-(chroman-4-on-2-yl)pyruvate.     

Dioxygenolytic cleavage of aryl ether bonds: 1, 10-dihydro-1, 10-dihydroxyfluoren-9-one, a novel arene dihydrodiol as evidence for angular dioxygenation of dibenzofuran

Two dibenzofuran degrading bacteria, Brevibacterium strain DPO 1361 and strain DPO 220, were found to utilize fluorene as sole source of carbon and energy. Cells which were grown on dibenzofuran, transformed fluorene into a number of products. For five of the seven metabolites isolated, the structure could be established unequivocally. Accumulation of one metabolite, 1,10-dihydroxy-1,10-dihydrofluoren-9-one, indicated the presence of a novel type of dioxygenase, attacking polynuclear aromatic systems in the unusual angular position. Debenzofuran degradation is proposed to likewise proceed via initial angular dioxygenation. One aryl oxygen ether bond, which normally is extremely stable, is thus transformed to a hemiacetal. After spontaneous cleavage and subsequent rearomatization by dehydration, 2,2',3-trihydroxybiphenyl [3-(2-hydroxyphenyl)-catechol] thus results as the immediate product of the first enzymatic reaction in the degradation sequence.     

Degradation of dibenzothiophene by Brevibacterium sp.DO

Dibenzothiophene, a polycyclic aromatic sulfur heterocycle, represents as a model compound the organic sulfur integrated in the macromolecular coal matrix. A pure culture of a Brevibacterium species was isolated, which is able to use dibenzothiophene as sole source of carbon, sulfur and energy for growth. During dibenzothiophene utilization sulfite was released in a stoichiometrical amount and was further oxidized to sulfate. Three metabolites of dibenzothiophene degradation were isolated and identified as dibenzothiophene-5-oxide, dibenzothiophene-5-dioxide and benzoate by cochromatography, UV spectroscopy and gas chromatographymass spectrometry analyses. Based on the identified metabolites a pathway for the degradation of dibenzothiophene by Brevibacterium sp. DO is proposed.Non-standard abbreviations DBT dibenzothiophene - PASH polycyclic aromatic sulfur heterocycle - PAH polycyclic aromatic hydrocarbons - GC-MS gas chromatography-mass spectrometry - HPLC high pressure liquid chromatography - IC ion chromatography     

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.     

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Zinc biosorption by a zinc-resistant bacterium, Brevibacterium sp. strain HZM-1

A zinc-resistant bacterium, Brevibacterium sp. strain HZM-1 which shows a high Zn²⁺-adsorbing capacity, was isolated from the soil of an abandoned zinc mine. Kinetic analyses showed that Zn²⁺ binding to HZM-1 cells follows Langmuir isotherm kinetics with a maximum metal capacity of 0.64 mmol/g dry cells and an apparent metal dissociation constant of 0.34 mM. The observed metal-binding capacity was one of the highest values among those reported for known microbial Zn²⁺ biosorbents. The cells could also adsorb heavy metal ions such as Cu²⁺. HZM-1 cells could remove relatively low levels of the Zn²⁺ ion (0.1 mM), even in the presence of large excess amounts (total concentration, 10 mM) of alkali and alkali earth metal ions. Bound Zn²⁺ ions could be efficiently desorbed by treating the cells with 10 mM HCl or 10 mM EDTA, and the Zn²⁺-adsorbing capacity of the cells was fully restored by treatment of the desorbed cells with 0.1 M NaOH. Thus, HZM-1 cells can serve as an excellent biosorbent for removal of Zn²⁺ from natural environments. The cells could grow in the presence of significant concentrations of ZnCl₂ (at least up to 15 mM) and thus is potentially applicable to in situ bioremediation of Zn²⁺-contaminated aqueous systems. Received: 1 February 2000 / Received revision: 31 March 2000 / Accepted: 1 May 2000     

Degradation of phenanthrene, fluorene, fluoranthene, and pyrene by a Mycobacterium sp.

Mycobacterium sp. strain BB1 was isolated from a former coal gasification site. It was able to utilize phenanthrene, pyrene, and fluoranthene as sole sources of carbon and energy and to degrade fluorene cometabolically. Exponential growth with solid phenanthrene, pyrene, and fluoranthene was obtained in fermentor cultures. The growth rates were 0.069, 0.056, and 0.040 h-1, respectively. Several metabolites of phenanthrene and fluorene metabolism were identified.     

微小杆菌(Exiguobacterium sp.)对肉桂酸降解行为

【目的】为有效缓解自毒物质肉桂酸对西瓜等作物生长的危害,从宁夏中卫硒砂瓜连作土壤中分离筛选得到一株高效降解肉桂酸的菌株,研究其基本降解特性。【方法】分离筛选得到一株能有效利用肉桂酸生长的菌株,采用16S r RNA基因序列分析进行菌株鉴定,运用高效液相色谱法和西瓜幼苗生长毒性实验检测降解特性。【结果】从多年西瓜连作土壤中筛选得到一株高效降解肉桂酸的细菌R30,鉴定为Exiguobacterium sp.,其96 h内对肉桂酸的降解率可达99%以上,最适降解温度和p H分别为30°C、p H 7.0。除肉桂酸外,该菌也能够高效降解香豆酸、阿魏酸、苯甲酸等其他酚酸类物质,表现出一定的底物广谱性;检测96 h降解液对西瓜种子萌发直至幼苗生长阶段的影响表明,该菌株可有效缓解肉桂酸对西瓜幼苗的生长抑制作用。【结论】菌株R30在肉桂酸、香豆酸、阿魏酸、苯甲酸等酚酸类物质导致的农作物连作障碍治理领域具有潜在的开发应用价值。     

Decolourisation of Remazol Brilliant Blue R via a novel Bjerkandera sp. strain

A novel strain of Bjerkandera sp. (B33/3), with particularly high decolourisation activities upon Poly R-478 and Remazol Brilliant Blue R (RBBR) dyes, was isolated. The role of the ligninolytic extracellular enzymes produced by this strain on decolourisation of RBBR was studied in some depth. The basis of decolourisation is an enzyme-mediated process, in which the main enzyme responsible is a recently described peroxidase with capacity for oxidation of manganese, as well as veratryl alcohol and 2,6-dimethoxyphenol in a manganese-independent reaction.     

New metabolites in the degradation of fluorene by Arthrobacter sp. strain F101.

Identification of new metabolites and demonstration of key enzyme activities support and extend the pathways previously reported for fluorene metabolism by Arthrobacter sp. strain F101. Washed-cell suspensions of strain F101 with fluorene accumulated 9-fluorenone, 4-hydroxy-9-fluorenone, 3-hydroxy-1-indanone, 1-indanone, 2-indanone, 3-(2-hydroxyphenyl) propionate, and a compound tentatively identified as a formyl indanone. Incubations with 2-indanone produced 3-isochromanone. The growth yield with fluorene as a sole source of carbon and energy corresponded to an assimilation of about 34% of fluorene carbon. About 7.4% was transformed into 9-fluorenol, 9-fluorenone, and 4-hydroxy-9-fluorenone. Crude extracts from fluorene-induced cells showed 3,4-dihydrocoumarin hydrolase and catechol 2,3-dioxygenase activities. These results and biodegradation experiments with the identified metabolites indicate that metabolism of fluorene by Arthrobacter sp. strain F101 proceeds through three independent pathways. Two productive routes are initiated by dioxygenation at positions 1,2 and 3,4, respectively. meta cleavage followed by an aldolase reaction and loss of C-1 yield the detected indanones. Subsequent biological Baeyer-Villiger reactions produce the aromatic lactones 3,4-dihydrocoumarin and 3-isochromanone. Enzymatic hydrolysis of the former gives 3-(2-hydroxyphenyl) propionate, which could be a substrate for a beta oxidation cycle, to give salicylate. Further oxidation of the latter via catechol and 2-hydroxymuconic semialdehyde connects with the central metabolism, allowing the utilization of all fluorene carbons. Identification of 4-hydroxy-9-fluorenone is consistent with an alternative pathway initiated by monooxygenation at C-9 to give 9-fluorenol and then 9-fluorenone. Although dioxygenation at 3,4 positions of the ketone apparently occurs, this reaction fails to furnish a subsequent productive oxidation of this compound.     

Degradation of biphenyl by Mycobacterium sp. strain PYR-1

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.     

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6.

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.     

Degradation of o-toluate by Pseudomonas sp. strain WR401

Abstract A Pseudomonas sp. strain WR401 was isolated for growth on 3-, 4-, and 5-methylsalicylate. The organism was capable of growth on o -toluate. The data on enzyme activities in cell-free extracts, DHB dehydrogenase and catechol 2,3-dioxygenase, as well as the cooxidation of the substrate analog 2-chlorobenzoate yielding 3-chlorocatechol indicated a pathway for o -toluate degradation through 6-methyldihydrodihydroxybenzoate, 3-methylcatechol and further through the meta -pathway. In contrast to other toluate dioxygenating enzymes found in m - and p -toluate degrading organisms, strain WR401 was able to dioxygenate a wider range of chlorobenzoates including 2-chlorobenzoate.     

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.     

Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD

The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation.     

Degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9

Abstract The aerobic degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9 was studied. Intact cells of Pseudomonas sp. BN9 grown with 2,4-dihydroxybenzoate oxidized 2,4-dihydroxybenzoate but not salicylate. Cell-free extracts of Pseudomonas sp. BN9 converted 2,4-dihydroxybenzoate after the addition of NAD(P)H. A partially purified protein fraction converted 2,4-dihydroxybenzoate with NADH to 1,2,4-trihydroxybenzene. 1,2,4-Trihydroxybenzene was converted by a 1,2-dioxygenase to maleylpyruvate, which was reduced by a NADH-dependent enzyme to 3-oxoadipate. 2,4-Dihydroxybenzoate 1-monooxygenase, 1,2,4-trihydroxybenzene 1,2-dioxygenase and maleylpyruvate reductase were induced in Pseudomonas sp. BN9 after growth with 2,4-dihydroxybenzoate.     

Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175.

An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source. During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released. Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene. It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.     

Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GP1.

A bacterium which utilizes 2,4,6-trichlorophenol (TCP) as a sole source of carbon and energy was isolated from soil. The bacterium, designated strain GP1, was identified as an Azotobacter sp. TCP was the only chlorinated phenol which supported the growth of the bacterium. Resting cells transformed monochlorophenols, 2,6-dichlorophenol, and 2,3,6-trichlorophenol. Phenol and a number of phenolic compounds, including 4-methylphenol, all of the monohydroxybenzoates, and several dihydroxybenzoates, were very good carbon sources for Azotobacter sp. strain GP1. The organism utilized up to 800 mg of TCP per liter; the lag phase and time for degradation, however, were severely prolonged at TCP concentrations above 500 mg/liter. Repeated additions of 200 mg of TCP per liter led to accelerated degradation, with an optimum value of 100 mg of TCP per liter per h. TCP degradation was significantly faster in shaken than in nonshaken cultures. The optimum temperature for degradation was 25 to 30 degrees C. Induction studies, including treatment of the cells with chloramphenicol prior to TCP or phenol addition, revealed that TCP induced TCP degradation but not phenol degradation and that phenol induced only its own utilization. Per mol of TCP, 3 mol of Cl- was released. 2,6-Dichloro-p-benzoquinone was detected in the resting-cell medium of Azotobacter sp. strain GP1. By chemical mutagenesis, mutants blocked in either TCP degradation or phenol degradation were obtained. No mutant defective in the degradation of both phenols was found, indicating separate pathways for the dissimilation of the compounds. In some of the phenol-deficient mutants, pyrocatechol was found to accumulate, and in some of the TCP-deficient mutants, 2,6-dichlorohydroquinone was found to accumulate.     

Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175

An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source. During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released. Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene. It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.     

Degradation of alkylphenol ethoxylates by Pseudomonas sp. strain TR01.

An alkylphenol ethoxylate-degrading bacterium was isolated from activated sludge of a municipal sewage treatment plant by enrichment culture. This organism was found to belong to the genus Pseudomonas; since no corresponding species was identified, we designated it as Pseudomonas sp. strain TR01. This strain had an optimal temperature and pH of 30 degrees C and 7, respectively, for both growth and the degradation of Triton N-101 (a nonylphenol ethoxylate in which the average number of ethylene oxide [EO] units is 9.5). The strain was unable to mineralize Triton N-101 but was able to degrade its EO chain exclusively. The resulting dominant intermediate was identified by normal-phase high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry as a nonylphenol ethoxylate with 2 mol of EO units. A carboxylated metabolite, [(nonylphenoxy)ethoxy]acetic acid, was detected by gas chromatography-mass spectrometry. This bacterium also metabolized alcohol ethoxylates with various numbers of EO units but not polyethylene glycols whatever their degree of polymerization. By oxygen consumption assay, the alkyl group or arene corresponding to the hydrophobic part of alcohol ethoxylates or alkylphenol ethoxylates was shown to contribute to the induction of the metabolic system of the EO chain of Triton N-101, instead of the EO chain itself, which corresponds to its hydrophilic part. Thus, the isolated pseudomonad bacterium has unique substrate assimilability: it metabolizes the EO chain only when the chain linked to bulky hydrophobic groups.