Metabolism of volatile chlorinated aliphatic hydrocarbons by Pseudomonas fluorescens.

A Pseudomonas fluorescens strain designated PFL12 was isolated from soil and water that were contaminated with various chloroaliphatic hydrocarbons. The isolate was able to metabolize 1,2-dichloroethane, 1,1,2-trichloroethane, 1,2-dichloropropane, 2,2-dichloropropane, and trichloroethylene.     

Plasmid Involvement in Linalool Metabolism by Pseudomonas fluorescens

A bacterial strain was isolated from a wastewater lagoon and identified as Pseudomonas fluorescens. This isolate was able to utilize linalool as a sole carbon and energy source. The ability was found to be encoded on a 60-megadalton transmissible plasmid, pSRQ60. The plasmid was also mated into a commercial waste treatment strain, which expanded its ability to utilize other isoprenoid compounds.     

Biodegradation of individual and multiple chlorinated aliphatic hydrocarbons by methane-oxidizing cultures.

The microbial degradation of chlorinated and nonchlorinated methanes, ethanes, and ethanes by a mixed methane-oxidizing culture grown under chemostat and batch conditions is evaluated and compared with that by two pure methanotrophic strains: CAC1 (isolated from the mixed culture) and Methylosinus trichosporium OB3b. With the exception of 1,1-dichloroethylene, the transformation capacity (Tc) for each chlorinated aliphatic hydrocarbon was generally found to be in inverse proportion to its chlorine content within each aliphatic group (i.e., methanes, ethanes, and ethenes), whereas similar trends were not observed for degradation rate constants. Tc trends were similar for all methane-oxidizing cultures tested. None of the cultures were able to degrade the fully chlorinated aliphatics such as perchloroethylene and carbon tetrachloride. Of the four cultures tested, the chemostat-grown mixed culture exhibited the highest Tc for trichloroethylene, cis-1,2-dichloroethylene, tetrachloroethane, 1,1,1-trichloroethane, and 1,2-dichloroethane, whereas the pure batch-grown OB3b culture exhibited the highest Tc for all other compounds tested. The product toxicity of chlorinated aliphatic hydrocarbons in a mixture containing multiple compounds was cumulative and predictable when using parameters measured from the degradation of individual compounds. The Tc for each chlorinated aliphatic hydrocarbon in a mixture (Tcmix) and the total Tc for the mixture (sigma Tcmix) are functions of the individual Tc, the initial substrate concentration (S0), and the first-order rate constant (k/Ks) of each compound in the mixture, indicating the importance of identifying the properties and compositions of all potentially degradable compounds in a contaminant mixture.     

Metabolism of 5-hydroxylysine in Pseudomonas fluorescens.

Hydroxylysine is metabolized via two routes by a Pseudomonas fluorescens strain as shown by the oxidation of selected intermediates. Hydroxy-L-lysine is oxidized via a pathway analogous to the monooxygenase pathway for L-lysine, and data suggest that at least some of tthe enzymes are those involved in the metabolism of L-lysine. Hydroxy-L-lysine is also converted by a racemase to allohydroxy-D-lysine, which is then degraded via a pathway analogous to, but different from, that described for D-lysine, involving hydroxy-L-pipecolate, 2-amino-5-hydroxyadipate, and 2-hydroxyglutarate. Data obtained with mutants unable to oxidize L-pipecolate suggest that the enzymes for the metabolism of hydroxy-L-pipecolate are distinct from those for L-pipecolate. Studies on D- and L-lysine degradation have shown that the previously described pathways for these compounds are present in this soil pseudomonad.     

Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F

Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated. It was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine, beta-alanine or beta-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source. Only uridine, cytidine, beta-alanine, beta-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon. In solid medium, the pyrimidine analogue 5-fluorouracil or 5-fluorouridine could prevent P. fluorescens biotype F growth at a low concentration while a 20-fold higher concentration of 5-fluorocytosine, 5-fluorodeoxyuridine or 6-azauracil was necessary to block its growth. The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed. Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used. The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored. Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium. In contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol. A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NH4)2SO4 as the sole source of nitrogen.     

Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp. strain DCA1

Pseudomonas sp. strain DCA1, which is capable of utilizing 1,2-dichloroethane (DCA) as sole carbon and energy source, was used to oxidize chlorinated methanes, ethanes, propanes, and ethenes. Chloroacetic acid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCAI and did not compete for the monooxygenase. All of the tested compounds except tetrachloroethylene (PER) were oxidized by cells expressing DCA monooxygenase. Strain DCAI could not utilize any of these compounds as a growth substrate. Co-metabolic oxidation during growth on DCA was studied with 1,2-dichloropropane. Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCAI. This inhibition was not caused by competition for the monooxygenase. It was shown that the oxidation of 1,2dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol.     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.     

Regulation of the adhesion of Pseudomonas fluorescens cells to glass by culture-produced volatile compounds

The effect of the gaseous metabolites of one Pseudomonas fluorescens culture on the attachment of cells of another Pseudomonas fluorescens culture to glass was studied. Gaseous metabolites increased the number of unattached cells by 10-30% and the mean residence time of cells attached to glass by 100%. These effects were presumably due to the yet unidentified compound, which we called volatile antiadhesin. This compound could be adsorbed by activated carbon and HAYESEP-Q adsorbent.     

Degradation of polycyclic aromatic hydrocarbons by a strain of Pseudomonas fluorescens 16N2

The growth of Pseudomonas fluorescens 16N2 on naphthalene was accompanied with accumulation of salicylate in the culture medium and induction of gentisate 1,2-dioxygenase and catechol 1,2-dioxygenase. The transformation of anthracene by the cells growing on hexadecane led to the formation of 3-hydroxy-2-naphthoate and salicylate. Pathways for naphthalene and anthracene degradation are proposed.     

Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.     

Effect of chlorinated aliphatic hydrocarbons on the acetoclastic methanogenic activity of granular sludge

The toxicity of chlorinated aliphatic hydrocarbons on acetoclastic methanogens in anaerobic granular sludge was determined using a standardized anaerobic bioassay method. Most of the chloroaliphatics tested were strong inhibitors of methanogenesis. Tri- and tetrachloride derivatives of methane and ethane were the most highly toxic compounds tested, with concentrations of less than 18 mg/l resulting in 50% inhibition (IC₅₀) of the methanogenic activity. Dichlorinated compounds were less toxic, with IC₅₀ values ranging from 40 mg/l to 100 mg/l. On the other hand, perchlorinated derivatives of ethane and ethene were scarcely inhibitory at concentrations near their maximum water solubility. The toxicity caused by chlorinated aliphatic hydrocarbons was reversible. The comparison of structurally related compounds indicated that unsaturated chloroaliphatics were less toxic than their saturated counterparts. A reverse correlation between the electric dipole moment of these compounds and their methanogenic toxicity is discussed. Received: 9 July 1996 / Received revision: 11 October 1996 / Accepted: 18 October 1996     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.     

氯代脂肪烃生物与非生物共促降解机制研究进展

氯代脂肪烃(Chlorinated aliphatic hydrocarbons,CAHs)具有高毒性、高富集性、高环境残留的特点和致癌、致畸、致突变效应,对人体健康和生态环境造成了严重危害。CAHs降解是生物和非生物过程共同作用的结果,存在多种交互作用,明晰CAHs的生物与非生物共促降解机制对于强化CAHs污染场地修复具有重要意义。文中首先对CAHs降解方式进行了分类介绍,按照还原脱氯、好氧共代谢和直接氧化三种方式总结了影响CAHs降解的典型生物与非生物降解因子。从共促降解的角度出发,系统分析并提出了诱导降解机制和协同降解机制,并对基于共促机制强化CAHs降解的工程应用与存在的技术局限进行了综述和分析,最后对未来的发展方向进行了展望。     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Small RNA-dependent Expression of Secondary Metabolism Is Controlled by Krebs Cycle Function in Pseudomonas fluorescens

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.