Dennis D. Focht

Terpene-utilizing isolates and their relevance to enhanced biotransformation of polychlorinated biphenyls in soil

Orange peels, eucalyptus leaves, pine needles and ivy leaves were addedseparately to soil spiked with Aroclor 1242 (100 mgkg-1.Polychorinated biphenyls (PCBs) disappeared after six months in all theamended soils, but not in unamended soils. Although biphenyl was not addedto any of the soils, all four amended soils had much higher levels(108/g) of biphenyl-utilizing bacteria than the unamendedcontrol (103/g). Ten random isolates obtained from these soilswere identified as coryneform bacteria. Five isolates, that were distinctlydifferent, were studied further with respect to growth on pure terpenes andmetabolism of PCBs. The most effective strains were Cellulomonas sp. T109and R. rhodochrous T100, which metabolized 83% and 80% ofAroclor 1242, respectively, during a six day period of growth on cymene andlimonene, respectively. The bphA gene, cloned as a 2.8 Kb Sa/I fragment ofpAW6194 from cbpA (Walia et al. 1990) hybridized to total DNA of allcoryneform isolates, and to the well-established PCB degrader Rhodococcusgloberulus. In contrast, a 5 Kb XhoI-SmaI fragment of the bphA gene(Furukawa & Miyazaki 1986) did not show any homology to the genomic DNAof any of the isolates or to R. globerulus, but did hybridize to two otherwell-known PCB degraders Pseudomonas sp. LB400, and Alcaligenes eutrophusH850. The data presented herein indicate that terpenes may be naturalsubstrates for biphenyl-degrading bacteria and may enhance substantialtransformation of Aroclor 1242.

Strategies for the improvement of aerobic metabolism of polychlorinated biphenyls

Abstract Recent research on the aerobic transformation of polychlorinated biphenyls and other chlorinated aromatic hydrocarbons has provided some encouraging results. The current paradigm is that highly chlorinated polychlorinated biphenyls are recalcitrant to bacterial dioxygenation and subjected only to reductive dehalogenation by anaerobic bacteria. The recent discovery of spontaneous dehalogenation by bacterial dioxygenases on aryl carbons bearing a chlorine substituent necessitates that this paradigm be reconsidered.

Catabolic characteristics of biphenyl-utilizing isolates which cometabolize PCBs

As there are at least three types of bacteria involved in the aerobic mineralization of polychlorinated biphenyls (PCBs), this study was undertaken to determine what catabolic features are lacking in biphenyl-degraders and to determine if chlorobenzoate- and chloroacetate-utilizing bacteria are as indigenous to soil as biphenyl-degraders. Bacteria were tested for their ability to utilize chlorinated acids and to cometabolize Aroclor 1254 and dibenzo-p-dioxane (dioxin). The broad and variable substrate specificity of the biphenyl dioxygenase among strains was noted by the range of <1 to 53% cometabolism of total PCB congeners and by the oxidation of dioxin, which was not a growth substrate. Growth on chloroalkanoic acids was more frequent with 2-chloropropionate (87% of all strains), 3-chloropropionate (72%), 4-chlorobutyrate (66%), and less frequent (28%) withtrans-3-chlorocrotonate. However, only one strain,Pseudomonas fluorescens K3, could utilize chloroacetate. No biphenyl-utilizers grew on 2- or 4-chlorobenzoate, and only five strains grew on 3-chlorobenzoate. Acetate and benzoate-utilizers were found in all three soils tested at levels near 106/g, whereas chloroacetate- or chlorobenzoate-utilizers were not detected. The inability of biphenyl-degraders to dehalogenate the products of PCB cometabolism is clearly unrelated to metabolism of saturated chloroaliphatic acids, with the notable exception of chloroacetate, since most strains grew on them. Thus, the inability to utilize chloroacetate, a central intermediate in the meta fission pathway, may be relevant to the incomplete catabolism of PCBs by biphenyl-utilizers.

Genetic construction of PCB degraders

Genetic construction of recombinant strains with expanded degradative abilities may be useful for bioremedation of recalcitrant compounds, such as polychlorinated biphenyls (PCBs). Some degradative genes have been found either on conjugative plasmids or on transposons, which would facilitate their genetic transfer. The catabolic pathway for the total degradation of PCBs is encoded by two different sets of genes that are not normally found in the same organism. ThebphABCD genes normally reside on the chromosome and encode for the four enzymes involved in the production of benzoate and chlorobenzoates from the respective catabolism of biphenyl and chlorobiphenyls. The genes encoding for chlorobenzoate catabolism have been found on both plasmids and the chromosome, often in association with transposable elements. Ring fission of chlorobiphenyls and chlorobenzoates involves themeta-fission pathway (3-phenylcatechol 2,3-dioxygenase) and theortho-fission pathway (chlorocatechol 1,2-dioxygenase), respectively. As the catecholic intermediates of both pathways are frequently inhibitory to each other, incompatibilities result. Presently, all hybrid strains constructed by in vivo matings metabolize simple chlorobiphenyls through complementary pathways by comprising thebph, benzoate, and chlorocatechol genes of parental strains. No strains have yet been verified which are able to utilize PCBs having at least one chlorine on each ring as growth substrates. The possible incompatibilities of hybrid pathways are evaluated with respect to product toxicity, and the efficiency of both in vivo and in vitro genetic methods for the construction of recombinant strains able to degrade PCBs is discussed.

METHODS FOR ANALYSIS OF DENITRIFICATION IN SOILS

Many different methods have been devised and are currently being used to evaluate either qualitatively or quantitatively the process of denitrification. A descriptive and critical discussion of these methods is presented in the text. Basically the following five approaches are considered: 1) indirect analyses (available carbon, Eh, bacterial numbers), 2) disappearance of nitrate, 3) 15N tracers, 4) 13N tracers, 5) evolution of N2O and N2. Considerable attention is given to the importance of N2O, the direct precursor to N2, in soil atmospheres. The most important kinetic factors that effect the formation and destruction of N2O are nitrate concentration and aeration. Kinetic analyses of these factors coupled with the use of inhibitors which block the reduction of N2O to N2 in situ offer the best hope for direct measurement of denitrification in soil.