5-Chloropicolinic acid is produced by specific degradation of 4-chlorobenzoic acid by Sphingomonas paucimobilis BPSI-3

We have previously shown that the bacterium Sphingomonas paucimobilis BPSI-3, isolated from PCB-contaminated soil, can degrade halogenated biphenyls, naphthalenes, catechols and benzoic acids. However, before such an organism can be used in bioremediation, it is important to characterise the degradation products and determine the degradation pathways to ensure that compounds more toxic or mobile than the original contaminants are not produced. In the degradation of 4-chlorobiphenyl, S. paucimobilis BPSI-3 produces a novel chlorinated picolinic acid. In this paper, we show that 4-chlorobenzoate is an intermediate in this degradation and, through ¹⁵N-labelling, that 5-chloropicolinate is the only nitrogenous metabolite isolated under the extraction conditions used. The position of the chlorine indicates that degradation of 4-chlorocatechol occurs exclusively via a 2,3-extradiol cleavage. These data allow us to postulate a more definitive catabolic pathway for the biodegradation of 4-chlorobiphenyl to 5-chloro-2-hydroxymuconic acid semialdehyde via 4-chlorobenzoate in S. paucimobilis BPSI-3. Received 19 April 1999/ Accepted in revised form 23 July 1999     

Hydrolytic dehalogenation of 4-chlorobenzoic acid by an Acinetobacter sp

Acinetobacter sp. strain ST-1, isolated from garden soil, can mineralize 4-chlorobenzoic acid (4-CBA). The bacterium degrades 4-CBA, starting with dehalogenation to yield 4-hydroxybenzoic acid (4-HBA) under both aerobic and anaerobic conditions, suggesting that the dehalogenating enzyme in the strain is not an oxygenase; the enzyme may catalyze halide hydrolysis. To identify the oxygen source of the C(4)-hydroxy groups in the dehalogenation step, we used H(2)(18)O as the solvent under anaerobic conditions. When resting cells were incubated in the presence of 4-CBA and H(2)(18)O under a nitrogen gas stream, the hydroxy group on the aromatic nucleus of the 4-HBA produced was derived from water, not from molecular oxygen. This dehalogenation was hydrolytic, because analysis of the mass spectrum of the trimethylsilyl derivative of one of the metabolites, (18)O-labeled 4-HBA, showed that 80% of the C4-hydroxy groups were labeled with (18)O. Hydrolytic dehalogenation of 4-CBA in intact cells has not been reported earlier. To identify substrate specificity, we next examined the ability of the strain to dehalogenate 4-CBA analogues and dichlorobenzoic acids. The results of metabolite analysis by high-pressure liquid chromatography showed that the strain dehalogenated 4-bromobenzoic acid and 4-iodobenzoic acid, yielding 4-HBA, suggesting that these compounds could be further degraded and mineralized by the strain via the beta-ketoadipate pathway, as occurs with 4-CBA. This strain, however, did not dehalogenate 4-fluorobenzoic acid, 2- and 3-chlorobenzoic acids, or 2,4-, 3,4-, and 3,5-dichlorobenzoic acids during 4 days of incubation, implying that the dehalogenating enzyme of the strain has high substrate specificity.     

Cloning of the Arthrobacter globiformis fcbA gene for dehalogenase and construction of a hybrid pathway of 4-chlorobenzoic acid degradation in Pseudomonas putida

The Artrobacter globiformis KZT1 fcbA gene responsible for dehalogenase (4-chlorobenzoate-4-hydroxylase) activity was cloned in Escherichia coli and Pseudomonas putida cells. The character of the fcbA gene expression was studied. Notwithstanding amplification of the gene dose and control of the inducible Plac promoter, the level of substrate dehalogenation by recombinant E. coli strains was lower, as compared with that in the original KZT1 strain. Cloning of the fcbA gene in P. putida KZ6R cells utilizing 4HBA resulted in a recombinant pathway of 4CBA degradation, which proved more effective for substrate consumption, in comparison with the original KZT1 strain.     

Biological degradation of 4-chlorobenzoic acid by a PCB-metabolizing bacterium through a pathway not involving (chloro)catechol

Cupriavidus sp. strain SK-3, previously isolated on polychlorinated biphenyl mixtures, was found to aerobically utilize a wide spectrum of substituted aromatic compounds including 4-fluoro-, 4-chloro- and 4-bromobenzoic acids as a sole carbon and energy source. Other chlorobenzoic acid (CBA) congeners such as 2-, 3-, 2,3-, 2,5-, 3,4- and 3,5-CBA were all rapidly transformed to respective chlorocatechols (CCs). Under aerobic conditions, strain SK-3 grew readily on 4-CBA to a maximum concentration of 5 mM above which growth became impaired and yielded no biomass. Growth lagged significantly at concentrations above 3 mM, however chloride elimination was stoichiometric and generally mirrored growth and substrate consumption in all incubations. Experiments with resting cells, cell-free extracts and analysis of metabolite pools suggest that 4-CBA was metabolized in a reaction exclusively involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoic acid, which was then hydroxylated to protocatechuic acid (PCA) and subsequently metabolized via the β-ketoadipate pathway. When strain SK-3 was grown on 4-CBA, there was gratuitous induction of the catechol-1,2-dioxygenase and gentisate-1,2-dioxygenase pathways, even if both were not involved in the metabolism of the acid. While activities of the modified ortho- and meta-cleavage pathways were not detectable in all extracts, activity of PCA-3,4-dioxygenase was over ten-times higher than those of catechol-1,2- and gentisate-1,2-dioxygenases. Therefore, the only reason other congeners were not utilized for growth was the accumulation of CCs, suggesting a narrow spectrum of the activity of enzymes downstream of benzoate-1,2-dioxygenase, which exhibited affinity for a number of substituted analogs, and that the metabolic bottlenecks are either CCs or catabolites of the modified ortho-cleavage metabolic route.     

4株苯系物降解菌菌株的筛选鉴定、降解特性及其降解基因研究

分别以苯、甲苯为碳源,从厦门污水处理厂活性污泥中富集筛选获得了2株苯降解菌B1、B2和2株甲苯降解菌J2、J6。16S rRNA基因鉴定结果表明B1、J2属于假单胞菌属(Pseudomonassp.),B2、J6属于不动杆菌属(Acinetobactersp.)。研究表明,这些菌在pH7~10的碱性范围内能很好生长。在以0.1%(V/V)苯或甲苯为唯一碳源的无机盐培养基中,B1、B2菌在72小时内对苯的降解率分别为67.7%、94.2%,J2、J6菌对甲苯的降解率分别为92.4%、84.8%。简并PCR扩增、序列分析表明,这些菌含有相同的苯双加氧酶基因,表明苯降解基因在这些降解菌中可能存在水平转移。此外,J2,J6两株菌还含有甲苯双加氧酶基因,而且J2能在甲苯浓度为70%(V/V)的LB培养基中生长。这些降解菌在苯、甲苯污染的生物治理中有应用前景。     

Optimized cultivation of highly-efficient degradation bacterial strains and their degradation ability towards pyrene

Two bacterial strains,Pyl and Py4,have been tamed and isolated through long cultivation with polycyclic aromatic hydrocarbon-pyrene as the single carbon source.It has been proven that they are both highly-efficient pyrene degrading bacteria and both Bacillus sp..The pyrene degradation ability of separated Pyl,Py4 and the consortium of equal Pyl and Py4 was studied in this project.It is shown that pyrene degradation rates were 88% in 10hr by Py1,84% in 14hr by Py4,and 88% in 8hr by the consortium.It was also determined that the best degradation temperatures were 37℃ and pH 7.0 respectively.The influence of different nutrient substrates added in the degradation experiments was also studied.It was shown that sodium salicylate,sodium acetate and yeast exuact had obvious simulative effect,but glucose had no obvious effect.     

Optimized cultivation of highly-efficient degradation bacterial strains and their degradation ability towards pyrene

Two bacterial strains, Py1 and Py4, have been tamed and isolated through long cultivation with polycyclic aromatic hydrocarbon—pyrene as the single carbon source. It has been proven that they are both highly-efficient pyrene degrading bacteria and both Bacillus sp.. The pyrene degradation ability of separated Py1, Py4 and the consortium of equal Py1 and Py4 was studied in this project. It is shown that pyrene degradation rates were 88% in 10hr by Py1, 84% in 14hr by Py4, and 88% in 8hr by the consortium. It was also determined that the best degradation temperatures were 37°C and pH 7.0 respectively. The influence of different nutrient substrates added in the degradation experiments was also studied. It was shown that sodium salicylate, sodium acetate and yeast extract had obvious simulative effect, but glucose had no obvious effect. __________ Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis (Natural Science Edition) 2006, 39: 71–74 [译自: 南开大学学报 (自然科学版)]     

Aerobic degradation of 3-chlorobenzoic acid by an indigenous strain isolated from a polluted river

An indigenous strain of Pseudomonas putida capable of degrading 3-chlorobenzoic acid as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. Aerobic biodegradation assays were performed using a 2-l microfermentor. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Lactuca sativa as test organisms. The indigenous bacterial strain degrades 100 mg l⁻¹ 3-chlorobenzoic acid in 14 h with a removal efficiency of 92.0 and 86.1% expressed as compound and chemical oxygen demand removal, respectively. The strain was capable of degrading up to 1,000 mg of the compound l⁻¹. Toxicity was not detected at the end of the biodegradation process. Besides initial concentration, the effect of different factors, such as initial pH, initial inoculum, adaptation to the compound and presence of other substrates and toxic related compounds, was studied.     

Survival of genetically engineered Escherichia coli in natural soil and river water

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased.     

Survival of genetically engineered Escherichia coli in natural soil and river water

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased. and accepted 19 August 1989     

Improved degradation of 4-chlorobiphenyl, 2,3-dihydroxybiphenyl, and catecholic compounds by recombinant bacterial strains

ThepcbC gene encoding (4-chloro-)2,3-dihydroxybiphenyl dioxygenase was cloned from the genomic DNA ofPseudomonas sp. P20 using pKT230 to construct pKK1. A recombinant strain,E. coli KK1, was selected by transforming the pKK1 intoE. coli XL1-Blue. Another recombinant strain,Pseudomonas sp. DJP-120, was obtained by transferring the pKK1 ofE. coli KK1 intoPseudomonas sp. DJ-12 by conjugation. Both recombinant strains showed a 23.7 to 26.5 fold increase in the degradation activity to 2,3-dihydroxybiphenyl compared with that of the natural isolate,Pseudomonas sp. DJ-12. The DJP-120 strain showed 24.5, 3.5, and 4.8 fold higher degradation activities to 4-chlorobiphenyl, catechol, and 3-methylcatechol than DJ-12 strain, respectively. The pKK1 plasmid of both strains and their ability to degrade 2,3-dihydroxybiphenyl were stable even after about 1,200 generations.     

能利用五碳糖和六碳糖生产乙醇的基因工程菌菌株的构建

燃料乙醇是一种极具前景的燃油代用品,近年来发展尤为迅速,为了推广这种能源和满足日益增长的需求,我们有必要开发更为高效的生产工艺和寻找更为廉价的原料。解决此问题的关键在于获得高效的工程菌,使其能利用木质纤维素水解液中的五碳糖和六碳糖发酵生产乙醇。通过代谢工程的研究和基因重组技术,几种重组细菌显示出良好的开发前景,它们是运动发酵单胞菌、大肠杆菌、产酸克雷伯氏菌和菊欧文氏菌。本文就这四种细菌的研究进展以及基因重组过程进行了介绍和评价。     

Characterization and degradation potential of diesel-degrading bacterial strains for application in bioremediation

Bioremediation of polluted soils is a promising technique with low environmental impact, which uses soil organisms to degrade soil contaminants. In this study, 19 bacterial strains isolated from a diesel-contaminated soil were screened for their diesel-degrading potential, biosurfactant (BS) production, and biofilm formation abilities, all desirable characteristics when selecting strains for re-inoculation into hydrocarbon-contaminated soils. Diesel-degradation rates were determined in vitro in minimal medium with diesel as the sole carbon source. The capacity to degrade diesel range organics (DROs) of strains SPG23 (Arthobacter sp.) and PF1 (Acinetobacter oleivorans) reached 17–26% of total DROs after 10 days, and 90% for strain GK2 (Acinetobacter calcoaceticus). The amount and rate of alkane degradation decreased significantly with increasing carbon number for strains SPG23 and PF1. Strain GK2, which produced BSs and biofilms, exhibited a greater extent, and faster rate of alkane degradation compared to SPG23 and PF1. Based on the outcomes of degradation experiments, in addition to BS production, biofilm formation capacities, and previous genome characterizations, strain GK2 is a promising candidate for microbial-assisted phytoremediation of diesel-contaminated soils. These results are of particular interest to select suitable strains for bioremediation, not only presenting high diesel-degradation rates, but also other characteristics which could improve rhizosphere colonization.     

Two-liquid-phase slurry bioreactors to enhance the degradation of high-molecular-weight polycyclic aromatic hydrocarbons in soil

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. The addition of a water-immiscible, nonbiodegradable, and biocompatible liquid, silicone oil, to a soil slurry was studied to promote the desorption of PAHs from soil and to increase their bioavailability. First, the transfer into silicone oil of phenanthrene, pyrene, chrysene, and benzo[a]pyrene added to a sterilized soil (sandy soil with 0.65% total volatile solids) was measured for 4 days in three two-liquid-phase (TLP) slurry systems each containing 30% (w/v) soil but different volumes of silicone oil (2.5%, 7.5%, and 15% [v/v]). Except for chrysene, a high percentage of these PAHs was transferred from soil to silicone oil in the TLP slurry system containing 15% silicone oil. Rapid PAH transfer occurred during the first 8 h, probably resulting from the extraction of nonsolubilized and of poorly sorbed PAHs. This was followed by a period in which a slower but constant transfer occurred, suggesting extraction of more tightly bound PAHs. Second, a HMW PAH-degrading consortium was enriched in a TLP slurry system with a microbial population isolated from a creosote-contaminated soil. This consortium was then added to three other TLP slurry systems each containing 30% (w/v) sterilized soil that had been artificially contaminated with pyrene, chrysene, and benzo[a]pyrene, but different volumes of silicone oil (10%, 20%, and 30% [v/v]). The resulting TLP slurry bioreactors were much more efficient than the control slurry bioreactor containing the same contaminated soil but no oil phase. In the TLP slurry bioreactor containing 30% silicone oil, the rate of pyrene degradation was 19 mg L(-)(1) day(-)(1) and no pyrene was detected after 4 days. The degradation rates of chrysene and benzo[a]pyrene in the 30% TLP slurry bioreactor were, respectively, 3.5 and 0.94 mg L(-)(1) day(-)(1). Low degradation of pyrene and no significant degradation of chrysene and benzo[a]pyrene occurred in the slurry bioreactor. This is the first report in which a TLP system was combined with a slurry system to improve the biodegradation of PAHs in soil.     

Trinitrotoluene removal in a soil slurry and soil box systems by an oil-degrading mixed bacterial culture

Bioremediation of trinitrotoluene (TNT)-contaminated soil has proven difficult due to the low bioavailability of the contaminant and its resistance to biocatalytic attack, causing slow rates of biodegradation. We have previously described a mixed bacterial culture acclimated and maintained on crude oil-containing medium that is capable of high rates of TNT biotransformation activity with low production of metabolites. We investigated the ability of this culture to bioremediate TNT-spiked soil and artificially weathered soil slurry systems, as well as a soil box system. The culture was able to remove up to 302 ppm (mg/l) of TNT within 24 h in a spiked-soil slurry system, which is among the highest rates of TNT removal reported to date. The toxicity of artificially weathered TNT-spiked soil to Vibrio fischeri decreased over a period of 39 h from a 15-min EC₅₀ of 15.7 to 32.5 ppm. Preliminary results of a soil box system, in which no agitation was used, showed similar TNT removal to the soil slurry system, with 100 ppm TNT being removed within 24 h.     

Degradation of 4-chlorobenzoic acid by an Arthrobacter species (author's transl)]

An Arthrobacter sp. growing on 4-Chlorobenzoic acid as its sole source of carbon excretes 4-hydroxygenzoic acid and protocatechuic acid into the culture medium. Protocatechuic acid is further attacked by "meta"-cleavage. During growth of the Arthrobacter sp. on benzoic acid cis-cis muconic acid can be isolated from the medium, suggesting the involvement of the "ortho"-cleavage pathway. The enzymes both for the "meta"- and the "ortho"-cleavage pathway are inducible.