Minoru Shimura

Novel Approach to the Improvement of Biphenyl and Polychlorinated Biphenyl Degradation Activity: Promoter Implantation by Homologous Recombination

ABSTRACT To improve the capabilities of microorganisms relevant for biodegradation, we developed a new genetic approach and applied it to the bph operon (bphEGF[orf4]A1A2A3CD[orf1]A4R) of Pseudomonas sp. strain KKS102 to enhance its biphenyl- and polychlorinated biphenyl (PCB)-degrading activity. A native promoter of the bph operon, which was under control, was replaced through homologous recombination by a series of promoters that had constitutive activity. By testing a series of promoters with various strengths, we were able to obtain strains that have enhanced degradation activity for biphenyl and PCBs. This strategy removes the rate-limiting factor associated with transcription and has the potential to improve the degradation activity of a wide variety of microorganisms involved in biodegradation.

Degradation of polychlorinated biphenyl by cells of Rhodococcus opacus strain TSP203 immobilized in alginate and in solution

Rhodococcus opacus strain TSP203, a Gram-positive biphenyl utilizer, can degrade trichloro-, tetrachloro-, and pentachlorobiphenyl congeners in PCB concentrations of 100 μg ml−1 Kaneclor 300 (equivalent to Aroclor 1242). The effect of Ca-alginate entrapment on the PCB-degrading competence of strain TSP203 was studied. Initially, the ability of immobilized cells of strain TSP203 and cells of TSP203 in solution to degrade congeners in 20 μg ml−1 Kaneclor 300, Kaneclor 400 (equivalent to Aroclor 1248), and Kaneclor 500 (equivalent to Aroclor 1254) was compared. In Kaneclor 300 and Kaneclor 400, PCB degradation by alginate immobilized and cells in solution were almost equivalent while in Kaneclor 500, degradation by cells in solution was slightly higher. The effect of increasing concentrations of Kaneclor 300 on the PCB-degrading ability of alginate-immobilized cells was evaluated. At 50, 100, and 150 μg ml−1 Kaneclor 300, an improvement in degradation by immobilized cells compared to cells in solution was observed. For degradation of 100 μg ml−1 Kaneclor 300, the optimum pH was 8.4 and optimum temperature 30°C. The biomass concentration was found to be critical for degradation. At low biomass concentration, trichloro-, tetrachloro-, and pentachlorobiphenyls were poorly degraded. In semicontinuous experiments, the immobilized cells could be used for three successive batch cultures with decreasing degrees of efficiency while cells in solution lysed after the first use. Although Ca-alginate afforded protection against toxicity of PCBs, we were unable to obtain a stable regeneration of the biocatalyst probably due to the absence of phosphate in the regeneration medium.

Analysis of changes in congener selectivity during PCB degradation by Burkholderia sp. strain TSN101 with increasing concentrations of PCB and characterization of the bphBCD genes and gene products

Abstract We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 μg Kaneclor 300/ml, a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls in medium containing 25 μg Kaneclor 300/ml. Using PCB concentrations of 50–150 μg of Kaneclor 300/ml, the congener selectivity pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 μg Kaneclor 300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2′- and 4′-positions. A 5-kb DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated 99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2,3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the cloned bph gene in biphenyl metabolism.

Complete degradation of polychlorinated biphenyls by a combination of ultraviolet and biological treatments

A method combining ultraviolet (UV) irradiation followed by microbial treatment was successfully applied to the efficient and complete degradation of polychlorinated biphenyls (PCBs). By UV irradiation, most PCB congeners in a methanol solution were transformed into lesser chlorinated compounds containing less than three chlorines. The resultant UV-irradiated PCBs were then subjected to microbial degradation by Pseudomonas alcaligenes TK102, resulting in their complete degradation within a week.