Browsing by Author "Picardal, Flynn W."

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    Aerobic Degradation of Di- and Trichlorobenzenes by Two Bacteria isolated from Polluted Tropical Soils.
    (Pergamon, 2007-01-01) Adebusoye, Sunday A.; Picardal, Flynn W.; Ilori, Matthew O.; Amund, Olukayode O.; Fuqua, Clay; Grindle, Nathan
    Two polychlorinated biphenyl (PCBs)-degrading bacteria were isolated by traditional enrichment technique from electrical transformer fluid (Askarel)-contaminated soils in Lagos, Nigeria. They were classified and identified as Enterobacter sp. SA-2 and Pseudomonas sp. SA-6 on the basis of 16S rRNA gene analysis, in addition to standard cultural and biochemical techniques. The strains were able to grow extensively on dichloro- and trichlorobenzenes. Although they failed to grow on tetrachlorobenzenes, monochloro- and dichlorobenzoic acids, they were able to utilize all monochlorobiphenyls, and some dichlorobiphenyls as sole sources of carbon and energy. The effect of incubation with axenic cultures on the degradation of 0.9 mM 1,4-dichlorobenzene, 0.44 mM 1,2,3- and 0.43 mM 1,3,5-trichlorobenzene in mineral salts medium was studied. Approximately, 80–90% of these xenobiotics were degraded in 200 h, concomitant with cell increase of up to three orders of magnitude, while generation times ranged significantly (P < 0.05) from 17–32 h. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were detected in crude cell-free extracts of cultures pre-grown with benzoate, with the latter enzyme exhibiting a slightly higher activity (0.15–0.17 lmol min1 mg of protein1) with catechol, suggesting that the meta-cleavage pathway is the most readily available catabolic route in the SA strains. The wider substrate specificity of these tropical isolates may help in assessing natural detoxification processes and in designing bioremediation and bioaugmentation methods.
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    Characterization of multiple novel aerobic polychlorinated biphenyl (PCB)-utilizing bacterial strains indigenous to contaminated tropical African soils
    (Springer Netherlands, 2008-02) Adebusoye, Sunday A.; Picardal, Flynn W.; Ilori, Matthew O.; Amund, Olukayode O.; Fuqua, Clay
    Contaminated sites in Lagos, Nigeria were screened for the presence of chlorobiphenyl-degrading bacteria. The technique of continual enrichment on Askarel fluid yielded bacterial isolates able to utilize dichlorobiphenyls (diCBs) as growth substrates and six were selected for further studies. Phenotypic typing and 16S rDNA analysis classified these organisms as species of Enterobacter, Ralstonia and Pseudomonas. All the strains readily utilized a broad spectrum of xenobiotics as sole sources of carbon and energy. Growth was observed on all monochlorobiphenyls (CBs), 2,2'-, 2,3-, 2,4'-, 3,3'- and 3,5-diCB as well as di- and trichlorobenzenes Growth was also sustainable on Askarel electrical transformer fluid and Aroclor 1221. Time-course studies using 100 ppm of 2-, 3- or 4-CB resulted in rapid exponential increases in cell numbers and CB transformation to respective chlorobenzoates (CBAs) within 70 h. Significant amounts of chloride were recovered in culture media of cells incubated with 2-CB and 3-CB, suggesting susceptibilities of both 2- and 3-chlorophenyl rings to attack, while the 4-CB was stoichiometrically transformed to 4-CBA. Extensive degradation of most of the congeners in Aroclor 1221 was observed when isolates were cultivated with the mixture as a sole carbon source. Aroclor 1221 was depleted by a minimum of 51% and maximum of 71%. Substantial amounts of chloride eliminated from the mixture ranged between 15 and 43%. These results suggest that some contaminated soils in the tropics may contain exotic micro-organisms whose abilities and potentials are previously unknown. An understanding of these novel strains therefore, may help answer questions about the microbial degradation of polychlorinated biphenyls (PCBs) in natural systems and enhance the potential use of bioremediation as an effective tool for cleanup of PCB-contaminated soils.
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    Co-metabolic Degradation of Polychlorinated Biphenyls (PCBs) by Axenic Cultures of Ralstonia sp. Strain SA-5 and Pseudomonas sp. Strain SA-6 obtained from Nigerian Contaminated Soils
    (Springer Netherlands, 2008-01-01) Adebusoye, Sunday A.; Ilori, Matthew O.; Picardal, Flynn W.; Amund, Olukayode O.
    Substantial metabolism of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-tetraCB) and 2,3′,4′,5-tetraCB by axenic cultures of Ralstonia sp. SA-5 and Pseudomonas sp. SA-6 was observed in the presence of biphenyl supplementation, although, the strains were unable to utilize tetrachlorobiphenyls as growth substrate. The former was more amenable to aerobic degradation (∼70% degradation) than the latter (22–45% degradation). Recovery of 2,5-chlorobenzoic acid and chloride from 2,3′,4′,5-tetraCB assay is an indication of initial dioxygenase attack on the 3,4-dichlorophenyl ring. The PCB-degradative ability of both strains was also investigated by GC analysis of individual congeners in Aroclor 1242 (100 ppm) following 12-day incubation with washed benzoate-grown cells. Results revealed two different catabolic properties. Whereas strain SA-6 required biphenyl as inducer of the degradation activity, such induction was not required by strain SA-5. Nearly all the detectable congeners in the mixture were extensively degraded (% reduction in ECD area counts for individual congeners ranged from 50.0 to 100% and 14.2 to 100%, respectively, for SA-5 and SA-6). The two strains exhibited no noticeable specificity for congeners with varying numbers of chlorine substitution and positions. The degradative competence of these isolates most especially SA-5 makes them among the most versatile PCB-metabolizing organisms yet reported.
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    Evidence of aerobic utilization of di‐ortho‐substituted trichlorobiphenyls as growth substrates by Pseudomonas sp. SA‐6 and Ralstonia sp. SA‐4
    (Blackwell Publishing Ltd, 2008-05) Adebusoye, Sunday A.; Picardal, Flynn W.; Ilori, Matthew O.; Amund, Olukayode O.
    Robust and effective bioremediation strategies have not yet been developed for polychlorinated biphenyl (PCB)-contaminated soils. This is in part a result of the fact that otho- or ortho- and para-substituted congeners, frequent dead-end products of reductive dechlorination of PCB mixtures, have greatly reduced aerobic biodegradability. In this study, we report substantial evidence of utilization of diortho-substituted trichlorobiphenyls (triCBs) as growth substrates by Ralstonia sp. SA-4 and Pseudomonas sp. SA-6 in which ortho-substitution resulted in no obvious paterns of recalcitrance. These stains exhibited unusual preferences for growth on congeners chlorinated on both rings. Substrate uptake studies with benzoate grown cells revealed that the isolates attacked the 2-chlorophenyl rings of 2,2’,4- and 2,2’,5-triCB. Between 71% and 93% of the initial 0.23-0.34 mM dose of congeners were transformed in less than 261 h concomitant with non-stoichiometric production of respective dichlorobenzoates and chloride ion. In enzyme assays, activity of 2,3-dihydroxybiphenyl 1,2-dioxygenase was constitutive. Additionally, these strains harboured no detectable plasmids which, coupled with exponential growth on the two triCB congeners, suggested chromosomal location of PCB degradative genes. In addition to the fact that there is a paucity of information on degradation of PCBs by tropical isolates, growth on triCBs as a sole carbon and energy source has never been demonstrated for any natural or engineered microorganisms. Such isolates may help prevent accumulation of ortho-substituted congeners in natural systems and offer the hope for development of effective bioaugmentation or sequential anaerobic-aerobic bioremediation strategies.
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    Extensive Biodegradation of polychlorinated Biphenyls in Aroclor 1242 and electrical Transformer Fluid (Askarel) by natural Strains of Microorganisms indigenous to contaminated African Systems
    (Pergamon, 2008-08-01) Adebusoye, Sunday A.; Ilori, Matthew O.; Picardal, Flynn W.; Amund, Olukayode O.
    Evidence for substantial aerobic degradation of Aroclor 1242 and Askarel fluid by newly characterized bacterial strains belonging to the Enterobacter, Ralstonia and Pseudomonas genera is presented. The organisms exhibited degradative activity in terms of total PCB/Askarel degradation, degradation of individual congeners and diversity of congeners attacked. Maximal degradation by the various isolates of Askarel ranged from 69% to 86% whereas, Aroclor 1242, with the exception of Ralstonia sp. SA-4 (9.7%), was degraded by 37% to 91%. PCB analysis showed that at least 45 of the representative congeners in Aroclor 1242 were extensively transformed by benzoate-grown cells without the need for biphenyl as an inducer of the upper degradation pathway. In incubations with Aroclor 1242, no clear correlation was observed between percentage of congener transformed and the degree of chlorination, regardless of the presence or absence of biphenyl. Recovery of significant but nonstoichiometric amounts of chloride from the culture media showed partial dechlorination of congeners and suggested production of partial degradation products. Addition of biphenyl evidently enhanced dechlorination of the mixture by some isolates. With the exception of Ralstonia sp. SA-5, chloride released ranged from 24% to 60% in the presence of biphenyl versus 0.35% to 15% without biphenyl.
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    Growth on Dichlorobiphenyls with Chlorine Substitution on Each Ring by Bacteria Isolated from Contaminated African Soils.
    (Springer-Verlag, 2007) Adebusoye, Sunday A.; Picardal, Flynn W.; Ilori, Matthew O.; Amund, Olukayode O.; Fuqua, Clay; Grindle, Nathan
    Until recently, it was generally believed that the presence of more than one chlorine substituent prevented chlorinated biphenyls from serving as a sole source of carbon and energy for aerobic bacteria. In this study, we report the isolation of three aerobic strains, identified as Enterobacter sp. SA-2, Ralstonia sp. SA-4, and Pseudomonas sp. SA-6 from Nigeria polluted soils, that were able to grow on a wide range of dichlorobiphenyls (diCBs). In addition to growing on all monochlorobiphenyls (monoCBs), the strains were all able to utilize 2,2’-, 2,4’-, and 2,3-diCB as a sole source of carbon and energy. With the exception of strain SA-2, growth was also sustainable on 3,3’-, and 3,5-diCB. Washed benzoate-grown cells were typically able to degrade 68 to 100% of the diCB (100 ppm) within 188 h, concomitant with a cell number increase of up to three orders-of-magnitude and elimination of varying amounts of chloride. In many cases, stoichiometric production of a chlorobenzoate (CBA) as a product was observed. During growth on 2,2’, and 2,4’-diCB, organisms exclusively attacked an o-chlorinated ring resulting in the production of 2-CBA and 4-CBA, respectively. A gradual decline in the concentration of the latter was observed, which suggested that the product was being degraded further. In the case of 2,3-diCB, the unsubstituted ring was preferentially metabolized. Initial diCB degradation rates were greatest for 2,4’-diCB (11.2+0.91 to 30.3+7.8 nmol/min per 109 cells) and lowest for 2,2’-diCB (0.37+0.12 to 2.7+1.2 nmol/min per 109 cells).
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    Influence of Chlorobenzoic acids on the Growth and Degradation Potentials of PCB-Degrading Microorganisms.
    (Springer Netherlands, 2008-07-01) Adebusoye, Sunday A.; Picardal, Flynn W.; Ilori, Matthew O.; Amund, Olukayode O.
    The biodegradation of polychlorinated biphenyls (PCBs) by diverse bacteria including those utilized in this study is often incomplete, a concomitant accumulation of chlorobenzoic acids (CBAs) are released as dead-end products. The build-up of these metabolites in the growth medium may result in feed-back inhibition and impede PCB biotransformation. In this investigation using GC-ECD and HPLC analyses, we confirmed that CBAs inhibit growth and PCB biodegradation potentials of five tropical bacteria namely, Pseudomonas aeruginosa SA-1, Enterobacter sp. SA-2, Ralstonia sp. SA-3, Ralstonia sp. SA-5 and Pseudomonas sp. SA-6. Among the four CBAs (2-CBA, 3-CBA, 4-CBA acids and 2,3-diCBA), 3-CBA was the strongest inhibitor followed by 4-CBA. Furthermore, we found that 3-CBA heavily inhibited growth of SA-3 and SA-6 on monochlorobiphenyls by 82-90% while elimination rate was inhibited by 71-88%. In the case of 2,3-diCBA, inhibition was generally less than 60%. However, effects of both acids were stronger in SA-3 than SA-6. We also found that 3-CBA and 2,3-diCBA completely inhibited carbon-chloride cleavage of 2-CB and 3-CB since cultivation in the absence of the acids resulted in recovery of 23-50% chloride in the culture fluids of organisms. These findings may therefore, have practical and ecological significance and are useful for improving the efficiency and the stability of some biological treatment processes.
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    Metabolism of chlorinated biphenyls: Use of 3, 3′-and 3, 5-dichlorobiphenyl as sole sources of carbon by natural species of Ralstonia and Pseudomonas
    (Pergamon, 2008-01-01) Adebusoye, Sunday A.; Ilori, Matthew O.; Picardal, Flynn W.; Amund, Olukayode O.
    Ralstonia sp. SA-3, Ralstonia sp. SA-4 and Pseudomonas sp. SA-6 are natural strains with a novel capacity to utilize meta-substituted dichlorobiphenyls (diCBs) hitherto not known to serve as a sole source of carbon and energy for polychlorobiphenyl-degraders. In growth experiments, axenic cultures of isolates grew logarithmically on 3,3’-diCB with generation times that ranged insignificantly (t-test, P>0.05) from 30.4 to 33.8 h. Both 3-chlorobenzoate (3-CBA) and chloride produced as metabolites were recovered in non-stoichiometric quantities. The release of chloride by the cultures lagged substantially, indicating that the initial dioxygenase attack preceded cleavage of carbon-chloride bonds and that chloride must have been released from the chlorinated hydroxypentadienoate. In the case of 3,5-diCB, SA-3 and SA-6 metabolised this substrate primarily to 3,5-CBA. The lack of chloride in the culture media coupled with stoichiometric recovery of 3,5-CBA suggests that growth by these strains occurred predominantly at the expense of the unsubstituted phenyl ring. The unique metabolic properties of these three aerobic isolates point to their potential usefulness as seeds for bioremediation of PCBs polluted environments without the need for repeated inoculation or supplementation by a primary growth substrate such as biphenyl.