Isolation and Genetic Characterization of Bacteria That Degrade Chloroaromatic Compounds

Bacteria were isolated from a landfill site previously used for disposal of chlorinated organic wastes. These soil isolates were capable of utilizing various chloroaromatic compounds. One such bacterial strain, designated Pseudomonas cepacia HCV (2,6-DCT) and growing on 2,6-dichlorotoluene, transferred this trait to a catechol-1,2-oxygenase mutant of Pseudomonas aeruginosa.     

Isolation of a Bacterial Strain Able To Degrade Branched Nonylphenol

Conventional enrichment of microorganisms on branched nonylphenol (NP) as only carbon and energy source yielded mixed cultures able to grow on the organic compound. However, plating yielded no single colonies capable, alone or in combination with other isolates, of degrading the NP in liquid culture. Therefore, a special approach was used, referred to as “serial dilution-plate resuspension,” to reduce culture complexity. In this way, one isolate, TTNP3, tentatively identified as a Sphingomonas sp., was found to be able to grow on NP in liquid culture. Remarkably, this isolate was able to be filtered through a 0.45-μm-pore-diameter filter. Moreover, isolate TTNP3 did not form visible colonies on mineral medium with NP, and it formed visible colonies on R₂A agar only after a prolonged incubation of 1 week. High-performance liquid chromatography and gas chromatography-mass spectroscopy analysis of the culture media indicated that the strain starts the degradation of NP with a fission of the phenol ring and preferably uses the para isomer of NP and not the ortho isomer. No distinct accumulation of an intermediary product could be observed.     

Isolation of Lightning-Competent Soil Bacteria

Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl₂) or an electrical (electroporation) method. However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil. In this paper, we report on the isolation of two “lightning-competent” soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tc^r, Sp^r, Sm^r). The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E. coli DH10B and Pseudomonas fluorescens C7R12. The electrotransformation frequencies measured reached 10⁻³ to 10⁻⁴ by electroporation and 10⁻⁴ to 10⁻⁵ by simulated lightning, while no transformation was observed in the absence of electrical current. Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp. strains.     

Psychrotolerant Bacteria Isolated from Arctic Soil That Degrade Polychlorinated Biphenyls at Low Temperatures

Psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria were isolated at 7°C from PCB-contaminated Arctic soil by using biphenyl as the sole organic carbon source. These isolates were distinguished from each other by differences in substrates that supported growth and substrates that were oxidized. 16S ribosomal DNA sequences suggest that these isolates are most closely related to the genus Pseudomonas. Total removal of Aroclor 1242, and rates of removal of selected PCB congeners, by cell suspensions of Arctic soil isolates and the mesophile Burkholderia cepacia LB400 were determined at 7, 37, and 50°C. Total removal values of Aroclor 1242 at 7°C by LB400 and most Arctic soil isolates were similar (between 2 and 3.5 μg of PCBs per mg of cell protein). However the rates of removal of some individual PCB congeners by Arctic isolates were up to 10 times higher than corresponding rates of removal by LB400. Total removal of Aroclor 1242 and the rates of removal of individual congeners by the Arctic soil bacteria were higher at 37°C than at 7°C but as much as 90% lower at 50°C than at 37°C. In contrast, rates of PCB removal by LB400 were higher at 50°C than at 37°C. In all cases, temperature did not affect the congener specificity of the bacteria. These observations suggest that the PCB-degrading enzyme systems of the bacteria isolated from Arctic soil are cold adapted.     

Effect of Humic Fractions and Clay on Biodegradation of Phenanthrene by a Pseudomonas fluorescens Strain Isolated from Soil

The mineralization of phenanthrene in pure cultures of a Pseudomonas fluorescens strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite. Humic acid and clay, either separately or in combination, shortened the acclimation phase. A higher mineralization rate was measured in treatments with humic acid at 100 μg/ml. Humic acid at 10 μg/ml stimulated the transformation only in the presence of 10 g of clay per liter. We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.     

Isolation and Characterization of Dehalogenases from 2,2-Dichloropropionate-Degrading Soil Bacteria

Five bacterial strains were isolated from polluted soils capable of degrading 2,2-dichloropropionate. In crude extracts, dehalogenase activity against haloacetates and longer-chained 2-haloalkanoic acids could be detected. Results from activity staining indicated that all bacterial strains expressed a single dehalogenase. In further biochemical characterization, two types of D,L-specific 2-haloalkanoic acid dehalogenases were described, which are different from each other not only in molecular weight and electrophoretic mobility, but also in sensitivity towards thiol reagents. Dehalogenases of these strains have been shown to be inducible and are catalyzing halide hydrolysis with inversion of product configuration. Received: 5 July 1996 / Accepted: 1 August 1996     

Inability of Microorganisms To Degrade Cellulose Acetate Reverse-Osmosis Membranes

Operational cellulose acetate reverse-osmosis membranes were examined for evidence of biological degradation. Numerous fungi and bacteria were isolated by direct and enrichment techniques. When tested, most of the fungi were active cellulose degraders, but none of the bacteria were. Neither fungi nor bacteria were able to degrade cellulose acetate membrane in vitro, although many fungi were able to degrade cellulose acetate membrane after it had been deacetylated. Organisms did not significantly degrade powdered cellulose acetate in pure or mixed cultures as measured by reduction in acetyl content or intrinsic viscosity or production of reducing sugars. Organisms did not affect the performance of cellulose triacetate fibers when incubated with them. The inability of the organisms to degrade cellulose acetate was attributed to the high degree of acetate substitution of the cellulose polymer. The rate of salt rejection decline was strongly correlated with chlorination of feed water and inversely with densities of microorganisms. These data suggest that microbial degradation of operational cellulose acetate reverse-osmosis membranes is unlikely.     

Isolation of Gemmata-Like and Isosphaera-Like Planctomycete Bacteria from Soil and Freshwater

New cultured strains of the planctomycete division (order Planctomycetales) of the domain Bacteria related to species in the genera Gemmata and Isosphaera were isolated from soil, freshwater, and a laboratory ampicillin solution. Phylogenetic analysis of the 16S rRNA gene from eight representative isolates showed that all the isolates were members of the planctomycete division. Six isolates clustered with Gemmata obscuriglobus and related strains, while two isolates clustered with Isosphaera pallida. A double-membrane-bounded nucleoid was observed in Gemmata-related isolates but not in Isosphaera-related isolates, consistent with the ultrastructures of existing species of each genus. Two isolates from this study represent the first planctomycetes successfully cultivated from soil.     

Degradation of Soil Humic Extract by Wood- and Soil-Associated Fungi, Bacteria, and Commercial Enzymes

Abstract An alkaline humic extract (HE) of a black calcareous forest mull was exposed to 36 fungal and 9 eubacterial isolates in liquid standing culture. At 21 d in fungi, and 4 d in bacteria, the groups of wood-degrading basidiomycetes, terricolous basidiomycetes, ectomycorrhizal fungi, soil-borne microfungi, and eubacteria had reduced the absorbance (A ₃₄₀) of HE media by 57, 28, 19, 26 and 5%, respectively. Gel permeation chromatography revealed that the large humic acid molecules were more readily degraded than the smaller fulvic acid molecules and served as a sole source of carbon and energy. The more active HE degraders reduced the overall molecular weight of humic and fulvic acids by 0.25 to 0.47 kDa. They also reduced the chemical reactivity of HE to tetrazotized o-dianisidine, indicating the degradation of hydroxylated aromatic molecules (which are responsible for this reaction). Decreases in absorbance, molecular weight, and reactivity were caused by fungal manganese peroxidase, horseradish peroxidase, β-glucosidase, and abiotic oxidants such as H₂O₂ and Mn(III) acetate. It is concluded that fungi, some of which are propagated in contaminated soils to control xenobiotics, metabolize HE compounds enzymatically. They use enzymes which are also involved in the degradation of soil xenobiotics. Because of reductions in the molecular weight of HE, which is a potential carrier of heavy metal ions and xenobiotics, solubility and motility of humic substances in soil and surface waters are increased. Received: 4 March 1998; Accepted: 1 June 1998     

Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil

[¹³C₆]salicylate, [U-¹³C]naphthalene, and [U-¹³C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the “heavy” and “light” DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [¹³C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the ¹³C during the incubation did not appear to be an issue in this experiment.     

两株苯酚降解菌的分离及降解特性的初步研究

从南昌钢铁公司焦化污水处理厂的活性污泥中分离出64株能降解苯酚的细菌,通过耐受性试验从中筛选出2株降解活性较高的苯酚降解菌,编号为F-38和F-64。研究表明:F-38和F-64都为G—菌,苯酚浓度越高,生长延滞期越长;两株菌降解苯酚基本发生在对数期,其对苯酚降解适宜条件为温度30℃,pH值8-9,通气有利于苯酚的降解。     

Pyrene and Phenanthrene Influence on Soil Microbial Populations

Two studies were conducted to evaluate microbial populations in polycyclic aromatic hydrocarbon-contaminated soil. Captina silt loam was freshly exposed to (1) 0 or 2000 mg pyrene/kg and sampled after 10- and 61-wk incubation and (2) 0 or 505 mg pyrene + 445 mg phenanthrene/kg and sampled after a 21-wk incubation. Microbial numbers were determined by plate-count techniques. Isolated bacteria, selected degraders, and wholesoil extracts were analyzed by fatty acid methyl ester analysis (FAME). In the pyrene experiment, pyrene did not affect total bacterial or fungal numbers, but pyrene degraders increased from undetectable levels to 7.09 log₁₀ degraders/g in the contaminated soil. The FAME analysis of bacterial isolates detected no pyrene effect, but wholesoil FAME indicated an increase in the contaminated soil of a fatty acid characteristic of protozoa and a major fatty acid detected in isolated degraders. In the pyrene + phenanthrene experiment, the contaminants had no impact on bacterial, fungal, or actinomycete numbers but increased degrader numbers. No effect of pyrene + phenanthrene was detected by isolate FAME, but whole-soil FAME indicated an effect similar to that in the pyrene experiment. The results indicate that pyrene, although not impacting microbial numbers, may have altered the soil microbial composition and that Captina silt loam can develop an effective degrader population under tested conditions.     

Pyrene and Phenanthrene Influence on Soil Microbial Populations

Two studies were conducted to evaluate microbial populations in polycyclic aromatic hydrocarbon-contaminated soil. Captina silt loam was freshly exposed to (1) 0 or 2000?mg pyrene/kg and sampled after 10- and 61-wk incubation and (2) 0 or 505?mg pyrene + 445?mg phenanthrene/kg and sampled after a 21-wk incubation. Microbial numbers were determined by plate-count techniques. Isolated bacteria, selected degraders, and wholesoil extracts were analyzed by fatty acid methyl ester analysis (FAME). In the pyrene experiment, pyrene did not affect total bacterial or fungal numbers, but pyrene degraders increased from undetectable levels to 7.09 log₁₀ degraders/g in the contaminated soil. The FAME analysis of bacterial isolates detected no pyrene effect, but wholesoil FAME indicated an increase in the contaminated soil of a fatty acid characteristic of protozoa and a major fatty acid detected in isolated degraders. In the pyrene + phenanthrene experiment, the contaminants had no impact on bacterial, fungal, or actinomycete numbers but increased degrader numbers. No effect of pyrene + phenanthrene was detected by isolate FAME, but whole-soil FAME indicated an effect similar to that in the pyrene experiment. The results indicate that pyrene, although not impacting microbial numbers, may have altered the soil microbial composition and that Captina silt loam can develop an effective degrader population under tested conditions.     

Isolation and Characterization of Antagonistic Bacillus Strains Capable to Degrade Ethylenethiourea

In this study, more than 150 bacteria showing antagonistic properties against bacterial and fungal pathogens of the tomato plant were isolated and characterized. The most efficient agents against these phytopathogenic microorganisms belong to the genus Bacillus: the best biocontrol isolates were representatives of Bacillus subtilis, B. mojavensis and B. amyloliquefaciens species. They intensively produced fengycin or/and surfactin depsipeptide antibiotics and also proved to be excellent protease secretors. It was proved, that the selected strains were able to use ethylenethiourea (ETU) as sole nitrogen source. These antagonistic and ETU-degrading Bacillus strains can be applied as biocontrol and also as bioremediation agents.     

Use of Aliphatic n-Alkynes To Discriminate Soil Nitrification Activities of Ammonia-Oxidizing Thaumarchaea and Bacteria

Ammonia (NH₃)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH₃ oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤20 μM concentrations of 1-alkynes C₈ and C₉. In contrast, NH₃ oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C₈ (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH₄⁺-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH₄⁺-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH₄⁺-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.     

Linking Sequence to Function in Soil Bacteria: Sequence-Directed Isolation of Novel Bacteria Contributing to Soilborne Plant Disease Suppression

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.     

Interaction between Diclofenac and Soil Humic Acids

The interaction between the non-steroidal anti-inflammatory drug diclofenac and standard humic acids (HAs) in bulk solution was studied using two complementary analytical methods: UV-Visible spectroscopy and square wave voltammetry. The observed UV-Vis spectra and Ip/V curves suggested that, at our experimental conditions, albeit both substances being negatively charged at pH 6.5, interaction between the pharmaceutical and the soil humic acids may led to the formation of diclofenac-humic acids supramolecules.

Our results could contribute to give information on the behaviour of diclofenac into the soil environment, thus suggesting its migration as HAs-micelles through the coarse soil profile.