Transfer of antibiotic multiresistant plasmid RP4 from escherichia coli to activated sludge bacteria

In situ transfer of a self-transmissible, antibiotic-multiresistant plasmid RP4 from a laboratory Escherichia coli strain C600 to indigenous activated sludge bacteria was investigated using filter mating. The transfer frequency of RP4 from the donor E. coli to the bacteria that was sampled from two wastewater treatment plants was 5.1x10(-2) to 7.5x10(-1) and 4.6x10(-3) to 1.3x10(-2)/potential recipient. The isolated transconjugants showed resistance to Ap, Km, and Tc and the presence of a plasmid with a similar size to RP4. The traG gene on RP4 was also detected from all transconjugants. Reverse-transfer experiments from the transconjugants to E. coli HB101 indicated that RP4 maintained self-transmissibility in the transconjugants. The transconjugant strains were dominant bacteria in activated sludge including Pseudomonas fluorescens, P. putida, and Ochrobactrum anthropi and minor populations of enteric bacterial strains including Citrobacter freundii, E. coli, Enterobacter cloacae, E. asburiae, and Klebsiella pneumoniae ssp. pneumoniae. The transconjugant strains K. pneumoniae ssp. pneumonia, E. cloacae, and E. asburiae had several naturally occurring plasmids. These results suggest that in situ transfer of plasmids and the exchange of antibiotic-resistant genes can occur between released and indigenous bacteria in activated sludge.     

Decolorization of heat-treatment liquor of waste sludge by a bioreactor using polyurethane foam-immobilized white rot fungus equipped with an ultramembrane filtration unit

A bench-scale bioreactor using immobilized fungal cells equipped with an ultramembrane filtration unit was developed as a means of decolorizing brown color components (melanoidins) arising from the heat-treatment liquor (HTL) of waste sludge. Artificial HTL containing 4200 color units of synthetic melanoidin supplemented with 1000 mg/l ethanol was first subjected to decolorization by the fungus Coriolus hirsutus IFO4917 immobilized onto polyurethane foam cubes. Then, the resultant biologically treated HTL was subjected to ultrafiltration to obtain the permeate (filtrate) as the effluent. The retentate (concentrate) of the filtration unit, containing the remaining melanoidin of high molecular weight and extracellular decolorizing enzymes, was returned to the fungal bioreactor to allow further decolorization. This system was operated in a sequencing batch mode under nonsterile conditions. Contamination of the bioreactor with air/water-born microbes markedly lowered the decolorization efficiency. However, this problem was solved by heating the returned concentrate at 50 degrees C for 10 min. Under the almost stable condition of a hydraulic retention time of 2 d in a 1-d cycle sequencing batch mode, about 70% decolorization was routinely achieved using the entire system (bioreactor + ultrafiltration), while the contribution of the fungal bioreactor alone to the decolorization was about 45%.     

Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.     

Characterization of Pseudomonas stutzeri NT-I capable of removing soluble selenium from the aqueous phase under aerobic conditions

Pseudomonas stutzeri strain NT-I was isolated from the drainage wastewater of a selenium refinery plant. This bacterium efficiently reduced selenate to elemental selenium without prolonged accumulation of selenite under aerobic conditions. Strain NT-I was able to reduce selenate completely at high concentrations (up to 10 mM) and selenite almost completely (up to 9 mM). In addition, higher concentrations of selenate and selenite were substantially reduced. Activity was observed under the following experimental conditions: 20–50°C, pH 7–9, and 0.05–20 g L^–1 NaCl for selenate reduction, and 20–50°C, pH 6–9, and 0.05–50 g L^–1 NaCl for selenite reduction. Under anaerobic conditions, selenate was reduced more rapidly, whereas selenite was not reduced at all. The high selenate- and selenite-reducing capability at high concentrations suggested that strain NT-I is suitable for the removal of selenium from high-strength industrial wastewater.     

Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols

The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (S)-1-phenyl-1-keto-2-methylaminopropane to d-pseudoephedrine (dPE), is inhibited by the accumulation of dPE in the reaction mixture, limiting the yield of dPE. To improve this weak point of the enzyme, random mutations were introduced into aadh, and a mutant enzyme library was constructed. The mutant library was screened with a color detectable high-throughput screening method to obtain the evolved enzymes showing the activity in the presence of a high concentration of dPE. Two mutant enzymes showed higher tolerability to dPE than the wild type enzyme. Each of these enzymes had a single amino acid substitution in a different position (G73S and S214R), and a third mutant enzyme carrying both of these amino acid substitutions was constructed. Escherichia coli transformant cells, which express mutant AADHs, showed activity in the presence of 100mg/ml dPE. A kinetic parameter analysis of the wild type and mutant enzymes was carried out. As compared with the wild type enzyme, the mutant enzymes carrying the S214R amino acid substitution or both the S214R and G73S substitutions showed higher k(cat) values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K(m) values. These results suggest that the Ser214 residue plays an important role in enzyme activity, and that the Gly73 residue participates in enzyme-substrate binding.     

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming beta-cyano-L-alanine

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable beta-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of beta-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical subunits. It was stable in the pH range of 6.0 to 10.0 and up to 70 degrees C. The enzyme also catalyzes the synthesis of various beta-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the beta-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the beta-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed beta-cyano-L-alanine synthase. Heat stable beta-cyano-L-alanine synthase can be applied to the synthesis of [4-11C]l-2,4-diaminobutyric acid as a tracer for positron emission tomography.     

Degradation of tensile strength of Ni–Ti superelastic alloy due to hydrogen absorption in methanol solution containing hydrochloric acid

Degradation of tensile strength of a Ni–Ti superelastic alloy due to hydrogen absorption has been studied in methanol solution containing 0.1 mass % hydrochloric acid (HCl). The amount of absorbed hydrogen from the solution was measured by thermal desorption analysis (TDA). Hydrogen desorption of immersed specimens appeared in the temperature range from 100 to 600 °C. The amounts of absorbed hydrogen of immersed specimens were in the range of 50–500 mass ppm when immersed in the solution for 8–120 h. Tensile strength of immersed specimens decreased abruptly up to immersion time of 24 h. In the immersion time range from 24 to 120 h, the tensile strength was coincident with the critical stress for martensite transformation. It was concluded that the degradation of tensile strength of a Ni–Ti superelastic alloy is caused by hydrogen absorption in methanol solution containing HCl.