Characterization of the temperature dependent catabolism of 2,4'-dichlorobiphenyl by a polychlorinated biphenyl degrading psychrotolerant Hydrogenophaga sp. IA3-A

Lambo, Adewale Jibowu (2006) Characterization of the temperature dependent catabolism of 2,4'-dichlorobiphenyl by a polychlorinated biphenyl degrading psychrotolerant Hydrogenophaga sp. IA3-A. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

A biphenyl-utilizing bacterium isolated from polychlorinated biphenyls (PCBs)-contaminated soil grew on tryptic soy or biphenyl at 4 to 40°C and at 5 to 35°C, respectively. Though the bacterium utilizes selected aromatic compounds as sole carbon and energy source, it only cometabolized chlorinated biphenyls. Analysis of the profile of cellular fatty acids showed that the bacterium is most closely related to Hydrogenophaga taeniospiralis (Willems et al., 1989). The gram-negative rod, tentatively identified as Hydrogenophaga sp. IA3-A, formed yellow colonies on nutrient agar and denitrified nitrate to nitrogen. The optimal temperature, pH, and substrate concentration for oxidation of 2,4'-dichlorobiphenyl (2,4'-diCB) in 0.05M sodium phosphate buffer was 30°C, 7.5, and 0.25 mM, respectively. The extracts of cells grown on biphenyl or in the presence of 2,4' -diCB contained the activity of biphenyl-2,3-dioxygenase (BPDO). -- The bacterium degraded mostly mono- to trichlorinated biphenyls (i.e. monoCBs, diCBs, and triCBs) and few tetrachlorinated biphenyls in commercial mixtures. The extents of removal of many of the PCBs were similar at 5° and 30°C. The PCBs that were removed in Aroclor 1221, Aroclor 1232, and Aroclor 1248 was dependent on the composition of the PCB mixtures as well as the number and pattern of chlorination of the congeners. -- Chlorinated benzoates (CBAs) were end-products of transformation of 2,3-, 2,4'- diCBs, and 2,4,4'-triCB but cells accumulated significant levels of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids (HOPDAs) from 2,4'-diCB and 2,4,4'-triCB. The formation of HOPDAs from PCB mixtures or 2,4-diCB and 2,4,4'-triCB indicates that the bacterium preferentially attacked PCBs at the rings bearing para-chlorine. This could account for the accumulation of HOPDA from 2,4'-diCB or 2,4,4'-triCB. Further transformation studies at selected temperatures using 2,4'-diCB indicated that although the parent compound was significantly degraded at low or moderate temperatures (10°,25°, 37 °C), excess level of HOPDA(s) accumulated from 2,4'-diCB leading to poor production of 2- and 4-CBA. The degradation of 2,4' -diCB was inhibited at a higher temperature (45°C), but most of the degraded compound was recovered as CBAs without excess production of HOPDA. Trimethylsilyl-derivatized culture extracts contained multiple isomeric intermediates with mass spectra that are similar to those of monochlorohydroxybiphenyls, monochlorodihydroxybiphenyls, dichlorodihydrodiols, dichlorodihydroxybiphenyls, and dichlorinated HOPDAs. The detection of some of these products in culture extracts could not be accounted for on the basis of the previously proposed routes of transformation 2,4' -diCBs. Nonetheless, it is suggested that multiple pathways are used for the transformation of 2,4'-diCB and that the pathways are regulated differently at different temperature. This novel findings is the first reported case in which production of excess level of potentially toxic HOPDA from 2,4' -diCB could be avoided under specific conditions in a bacterium that preferentially attacked parasubstituted rings of PCBs. -- Three chromatographic fractions from crude cell extracts separated using a Fast-Flow DEAE Sepharose column were required together to restore maximum activity of partially purified BPDO. The enzyme was specific for (chloro)biphenyls but it shows less activity against 2,4,4'- and 2,2' ,4-triCBs. BPDO was active against benzene, toluene, and xylenes, and it retained activity against (chloro )biphenyls at 7 °C, but not against 4-CBA or benzene. It was stable at 0° to 63 °C and its activity is enhanced in the presence of 1 ,μM FAD. Unlike other metal ions, mercury (II) and copper (II) severely inhibited the activity of BPDO. The optimal pH, temperature, and substrate concentration for BPDO activity in air-saturated 50 mM MES buffer was 6.0, 25°C, and 150 μM, respectively. The apparent Km of partially purified BPDO for biphenyl was 77.5 μmol and the V max value for NADH was 0.04 μmol per min⁻¹ mg⁻¹ protein. The activity of BPDO was inhibited in the presence of excess biphenyl.

Item Type: Thesis (Doctoral (PhD))
URI: http://research.library.mun.ca/id/eprint/10844
Item ID: 10844
Additional Information: Includes bibliographical references (leaves 209-239).
Department(s): Science, Faculty of > Biology
Date: 2006
Date Type: Submission
Library of Congress Subject Heading: Aromatic compounds--Biodegradation; Polychlorinated biphenyls--Biodegradation.

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