Draft Genome Sequence of Bacillus thuringiensis SS2, from Dibenzofuran-Contaminated Soil, Reveals Putative Aromatic Compound Degradation Genes.

The draft genome sequence of strain Bacillus thuringiensis SS2 consists of 426 contigs assembled at the scaffold level, totaling 5,030,306 bp, and contains 5,288 putative PATRIC protein-coding genes, including genes responsible for total benzoate consumption, degradation of halogenated compounds, heavy metal tolerance/resistance, biosynthesis of secondary metabolites, and microcin C7 self-immunity protein.

Soil microbiome response to 2-chlorodibenzo-p-dioxin during bioremediation of contaminated tropical soil in a microcosm-based study.

A pristine soil was artificially contaminated with 2-chlorodibenzo-p-dioxin (2-CDD) and separated into three portions. Microcosms SSOC and SSCC were seeded with Bacillus sp. SS2 and a three-member bacterial consortium respectively; SSC was untreated, while heat-sterilized contaminated soil served as overall control. Significant degradation of 2-CDD occurred in all microcosms except for the control where the concentration remained unchanged. Degradation of 2-CDD was highest in SSCC (94.9%) compared to SSOC (91.66%) and SCC (85.9%). There was also a notable reduction in the microbial composition complexity both in species richness and evenness following dioxin contamination, a trend that nearly lasted the study period; particularly in setups SSC and SSOC. Irrespective of the bioremediation strategies, the soil microflora was practically dominated by the Firmicutes and at the genus level, the phylotype Bacillus was the most dominant. Other dominant taxa though negatively impacted were Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteria. Overall, this study demonstrated the feasibility of microbial seeding as an effective strategy to cleanup tropical soil contaminated with dioxins and the importance of metagenomics in elucidating the microbial diversities of contaminated soils. Meanwhile, the seeded organisms, owed their success not only to metabolic competence, but survivability, adaptability and ability to compete favourably with autochthonous microflora.

Heavy metals assessment of ecosystem polluted with wastewaters and taxonomic profiling of multi-resistant bacteria with potential for petroleum hydrocarbon catabolism in nitrogen-limiting medium.

The coexistence of heavy metals (HMs) and petroleum hydrocarbons (PHs) exacerbates ecotoxicity and impair the drivers of eco-functionalities that stimulate essential nutrients for the productivity of the impacted environment. Profiling the bacteria that stem the ecological impact via HMs sequestration and PHs catabolism with nitrogen fixation is imperative to bioremediation of the polluted sites. The sediment of site that was consistently contaminated with industrial wastewaters was analysed for ecological toxicants and the bacterial strains that combined HMs resistance with PHs catabolism in a nitrogen-limiting system were isolated from the sediment and characterized. The geochemistry of the samples revealed the co-occurrence of the above-benchmark concentrations of HMs with the derivatives of hydrocarbons. Notwithstanding, nickel and mercury (with 5% each of the total metal concentrations in the polluted site) exhibited probable effect concentrations on the biota and thus hazardous to the ecosystem. Approx. 31% of the bacterial community, comprising unclassified Planococcaceae, unclassified Bradyrhizobiaceae, Rhodococcus, and Bacillus species, resisted 160 µmol Hg2+ in the nitrogen-limiting system within 24 h post-inoculation. The bacterial strains adopt volatilization, and sometimes in combination with adsorption/bioaccumulation strategies to sequester Hg2+ toxicity while utilizing PHs as sources of carbon and energy. Efficient metabolism of petroleum biomarkers (> 87%) and Hg2+ sequestration (≥ 75% of 40 µmol Hg2+) displayed by the selected bacterial strains portend the potential applicability of the bacilli for biotechnological restoration of the polluted site.

Draft Genome Sequence of Serratia marcescens SSA1, Isolated from Dioxin-Polluted Soil.

Serratia marcescens SSA1 was isolated from a dump site with a history of incineration. Its DNA of 5.05 Mbp has a GC content of 59.65%, with 77 tRNA genes and 3 rRNA genes. Its 4,909 putative PATRIC protein-coding genes include genes responsible for the degradation of dioxins and other xenobiotics and total consumption of benzoate.

Aerobic degradation of 2,4,6-trinitrophenol by Proteus sp. strain OSES2 obtained from an explosive contaminated tropical soil.

A 2,4,6-trinitrophenol (TNP) degrading bacterial strain isolated from a site polluted with explosives was identified as Proteus sp. strain OSES2 via 16S rRNA gene sequencing. Metabolic investigation showed that the organism grew exponentially on 100 mg l-1 of TNP as a source of carbon, nitrogen, and energy. In addition, the growth of the organism was sustainable on 3-nitrotoluene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, 4-nitrophenol, methyl-3-nitrobenzoate, 4-nitroaniline, aniline and nitrobenzene. Strain OSES2 was able to utilize TNP within a concentration range of 100 mg l-1 to 500 mg l-1. The specific growth rate and degradation rates on TNP were 0.01043 h-1 and 0.01766 mg l-1 h-1 respectively. Effective degradation of TNP in a chemically defined medium was evident with a gradual reduction in the concentration of TNP concomitant with an increase in cell density as well as the substantial release of ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-) as metabolites in 96 h. Degradation competence of the organism was enhanced in the presence of starch and acetate. On starch-supplemented TNP, the highest specific growth rate and degradation rates were 0.02634 h-1 and 0.04458 mg l-1 h-1, respectively, while the corresponding values on acetate were 0.02341 h-1 and 0.02811 mg l-1 h-1. However, amendment with nitrogen sources yielded no substantial improvement in degradation. TNP was utilized optimally at pH 7 to 9 and within the temperature range of 30 °C to 37 °C. The enzyme hydride transferase II [HTII], encoded by the npdI gene which is the first step involved in the TNP degradation pathway, was readily expressed by the isolate thus suggesting that substrate was utilized through the classical metabolic pathway.

Aerobic degradation of 2,4-dinitrotoluene: Effect of raw organic wastes and nitrogen fortification.

2,4-Dinitrotoluene (2,4-DNT), a principal derivative generated in the synthesis of 2,4,6-trinitrotoluene, is widely used as a waterproofer, plasticizer, and gelatinizer in propellants and explosives. This compound has been documented as a priority pollutant because of its toxicity. Therefore, its removal from contaminated systems is a major focus of research and environmental attention. The presence of 2,4-DNT bacterial-degrading strains that could utilize 2,4-DNT as growth substrate in polluted sites in Ibadan, Nigeria, was determined using continual enrichment techniques on nitroaromatic mixtures. Proteus sp. strain OSES2 isolated in this study was characterized by phenotypic typing and 16S ribosomal RNA gene sequencing. Growth of the strain on 2,4-DNT resulted in an exponential increase in biomass and complete substrate utilization within 72 h, accompanied by NO3 - elimination. Degradation competence was enhanced in the presence of corn steep liquor, molasses, and Tween 80 compared with incubation without amendment. Conversely, amendment with nitrogen sources yielded no significant improvement in degradation. Use of these organic wastes as candidates in a bioremediation strategy should be exploited. This would provide a less-expensive organic source supplement for cleanup purposes, with the ultimate aim of reducing the cost of bioremediation while reducing wastes intended for landfill.

Aerobic degradation of dichlorinated dibenzo-p-dioxin and dichlorinated dibenzofuran by bacteria strains obtained from tropical contaminated soil.

Bacterial diversity and aerobic catabolic competence of dioxin-degrading bacterial strains isolated from a polluted soil in the tropics were explored. Isolation of bacteria occurred after 12 months of consecutive enrichment, with dioxin congeners serving as the only sources of carbon and energy. Seventeen strains that were isolated were subsequently screened for dioxin metabolic competence. Among these isolates, five had unique amplified ribosomal DNA restriction analysis (ARDRA) patterns out of which two exhibiting good metabolic competence were selected for further investigation. The two strains were identified as Bacillus sp. SS2 and Serratia sp. SSA1, based on their 16S rRNA gene sequences. Bacterial growth co-occurred with dioxin disappearance and near stoichiometric release of chloride for one ring of the chlorinated congeners. The overall percentage removal of dibenzofuran (DF) by strain SS2 was 93.87%; while corresponding values for 2,8-dichlorodibenzofuran (2,8-diCDF) and 2,7-dichlorodibenzo-p-dioxin (2,7-diCDD) were 86.22% and 82.30% respectively. In the case of strain SSA1, percentage removal for DF, 2,8-diCDF and 2,7-diCDD were respectively 98.9%, 80.97% and 70.80%. The presence of two dioxin dioxygenase catabolic genes (dxnA1 and dbfA1) was investigated. Only the dbfA1 gene could be amplified in SS2 strain. Results further revealed that strain SS2 presented higher expression levels for the alpha-subunit of DF dioxygenase (dbfA1) gene during growth with dioxins. The expression level for dbfA1 gene was higher when growing on DF than on the other chlorinated analogs. This study gives an insight into dioxin degradation, with the catabolic potential of strains SS2 and SSA1 (an enteric bacterium) within the sub-Sahara Africa. It further shows that dioxin catabolic potential might be more prevalent in different groups of microorganisms than previously believed. Few reports have demonstrated the degradation of chlorinated congeners of dioxins, particularly from sub-Saharan African contaminated systems.

Structural dynamics of microbial communities in polycyclic aromatic hydrocarbon-contaminated tropical estuarine sediments undergoing simulated aerobic biotreatment.

Coastal sediments contaminated by polycyclic aromatic hydrocarbons (PAHs) can be candidates for remediation via an approach like land farming. Land farming converts naturally anaerobic sediments to aerobic environments, and the response of microbial communities, in terms of community structure alterations and corresponding effects on biodegradative activities, is unknown. A key goal of this study was to determine if different sediments exhibited common patterns in microbial community responses that might serve as indicators of PAH biodegradation. Sediments from three stations in the Lagos Lagoon (Nigeria) were used in microcosms, which were spiked with a mixture of four PAH, then examined for PAH biodegradation and for shifts in microbial community structure by analysis of diversity in PAH degradation genes and Illumina sequencing of 16S rRNA genes. PAH biodegradation was similar in all sediments, yet each exhibited unique microbiological responses and there were no microbial indicators of PAH bioremediation common to all sediments.

Biological degradation of 4-chlorobenzoic acid by a PCB-metabolizing bacterium through a pathway not involving (chloro)catechol.

Cupriavidus sp. strain SK-3, previously isolated on polychlorinated biphenyl mixtures, was found to aerobically utilize a wide spectrum of substituted aromatic compounds including 4-fluoro-, 4-chloro- and 4-bromobenzoic acids as a sole carbon and energy source. Other chlorobenzoic acid (CBA) congeners such as 2-, 3-, 2,3-, 2,5-, 3,4- and 3,5-CBA were all rapidly transformed to respective chlorocatechols (CCs). Under aerobic conditions, strain SK-3 grew readily on 4-CBA to a maximum concentration of 5 mM above which growth became impaired and yielded no biomass. Growth lagged significantly at concentrations above 3 mM, however chloride elimination was stoichiometric and generally mirrored growth and substrate consumption in all incubations. Experiments with resting cells, cell-free extracts and analysis of metabolite pools suggest that 4-CBA was metabolized in a reaction exclusively involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoic acid, which was then hydroxylated to protocatechuic acid (PCA) and subsequently metabolized via the ß-ketoadipate pathway. When strain SK-3 was grown on 4-CBA, there was gratuitous induction of the catechol-1,2-dioxygenase and gentisate-1,2-dioxygenase pathways, even if both were not involved in the metabolism of the acid. While activities of the modified ortho- and meta-cleavage pathways were not detectable in all extracts, activity of PCA-3,4-dioxygenase was over ten-times higher than those of catechol-1,2- and gentisate-1,2-dioxygenases. Therefore, the only reason other congeners were not utilized for growth was the accumulation of CCs, suggesting a narrow spectrum of the activity of enzymes downstream of benzoate-1,2-dioxygenase, which exhibited affinity for a number of substituted analogs, and that the metabolic bottlenecks are either CCs or catabolites of the modified ortho-cleavage metabolic route.

Microbial Communities in Sediments of Lagos Lagoon, Nigeria: Elucidation of Community Structure and Potential Impacts of Contamination by Municipal and Industrial Wastes.

Estuarine sediments are significant repositories of anthropogenic contaminants, and thus knowledge of the impacts of pollution upon microbial communities in these environments is important to understand potential effects on estuaries as a whole. The Lagos lagoon (Nigeria) is one of Africa's largest estuarine ecosystems, and is impacted by hydrocarbon pollutants and other industrial and municipal wastes. The goal of this study was to elucidate microbial community structure in Lagos lagoon sediments to identify groups that may be adversely affected by pollution, and those that may serve as degraders of environmental contaminants, especially polycyclic aromatic hydrocarbons (PAHs). Sediment samples were collected from sites that ranged in types and levels of anthropogenic impacts. The sediments were characterized for a range of physicochemical properties, and microbial community structure was determined by Illumina sequencing of the 16S rRNA genes. Microbial diversity (species richness and evenness) in the Apapa and Eledu sediments was reduced compared to that of the Ofin site, and communities of both of the former two were dominated by a single operational taxonomic unit (OTU) assigned to the family Helicobacteraceae (Epsilonproteobacteria). In the Ofin community, Epsilonproteobacteria were minor constituents, while the major groups were Cyanobacteria, Bacteroidetes, and Firmicutes, which were all minor in the Apapa and Eledu sediments. Sediment oxygen demand (SOD), a broad indicator of contamination, was identified by multivariate analyses as strongly correlated with variation in alpha diversity. Environmental variables that explained beta diversity patterns included SOD, as well as levels of naphthalene, acenaphthylene, cobalt, cadmium, total organic matter, or nitrate. Of 582 OTU identified, abundance of 167 was significantly correlated (false discovery rate q≤ 0.05) to environmental variables. The largest group of OTU correlated with PAH levels were PAH/hydrocarbon-degrading genera of the Oceanospirillales order (Gammaproteobacteria), which were most abundant in the hydrocarbon-contaminated Apapa sediment. Similar Oceanospirillales taxa are responsive to marine oil spills and thus may present a unifying theme in marine microbiology as bacteria adapted for degradation of high hydrocarbon loads, and may represent a potential means for intrinsic remediation in the case of the Lagos lagoon sediments.

Catabolic plasmid specifying polychlorinated biphenyl degradation in Cupriavidus sp. strain SK-4: mobilization and expression in a pseudomonad.

Strain SK-4, a polychlorinated biphenyl (PCB) degrader previously reported to utilize di-ortho-substituted biphenyl, was genotypically re-characterized as a species of Cupriavidus. The bacterium harbored a single plasmid (pSK4), which resisted curing and which, after genetic marking by a transposon (SK4Tn5), could be mobilized into a pseudomonad. Analysis of pSK4 in both the transconjugant and the wild type revealed that it specifies the genes coding for 2-hydroxy-2,4-pentadienoate degradation in addition to those of the upper biphenyl pathway. Expression of the benzoate metabolic pathway in the transconjugant is evidence suggesting that the benzoate catabolic genes are also localized on the plasmid. This implies that pSK4 codes for all the genes involved in biphenyl mineralization. It is therefore reasonable to propose that the plasmid is the determinant for the unique metabolic capabilities known to exist in Cupriavidus sp. strain SK-4.

Characterization of multiple chlorobenzoic acid-degrading organisms from pristine and contaminated systems: mineralization of 2,4-dichlorobenzoic acid.

Multiple bacterial strains with CBA metabolic properties were isolated using a simple selective strategy. Phylogenetic analysis of the 16S rRNA gene sequences grouped them into two main clusters consisting of four bacterial phyla and belonging to 17 genera. Whereas growth was more frequent with 2-CBA (∼68%), 50% grew on 4-CBA and ∼7% utilized 3-CBA. One third of the strains exhibited 2,4-dichlorobenzoic acid (2,4-diCBA) catabolic function and were mainly representatives of α-, ß- and γ-Proteobacteria. In batch experiments, growth was concomitant with substrate disappearance and near-stoichiometric release of chloride. Doubling times for 2,4-diCBA degradation doubled those determined for mono-substituted CBAs. Out of the six 2,4-diCBA degraders submitted for enzyme assays, significant induction of catechol 1,2-dioxygenase types I and II activities in cell-free extracts were found in four while protocatechuate 3,4-dioxygenase activity was detected in the remaining two. Activities in CBA-grown cells were 20 orders-of-magnitude higher than those grown on benzoic acid.

Effects of corn steep liquor on growth rate and pyrene degradation by Pseudomonas strains.

The growth rates and pyrene degradation rates of Pseudomonas sp. LP1 and Pseudomonas aeruginosa LP5 were increased in corn steep liquor (CSL) supplemented. On pyrene alone the highest specific growth rate of LP1 was 0.018 h(-1), while on CSL-supplemented pyrene MSM, the value was 0.026 h(-)1. For LP5 the highest growth rate on CSL-supplemented pyrene-MSM was 0.034 h(-1). Conversely, on pyrene alone the highest rate was 0.024 h(-1). CSL led to marked reduction in residual pyrene. In the case of Pseudomonas sp. LP1 values of residual pyrene were 58.54 and 45.47%, respectively, for the unsupplemented and supplemented broth cultures, showing a difference of 13.09%. For LP5 the corresponding values were 64.01 and 26.96%, respectively, showing a difference of 37.05%. The rate of pyrene utilization by LP1 were 0.08 and 0.11 mg l(-1) h(-1) on unsupplemented and supplemented media, respectively. The corresponding values for LP5 were 0.07 and 0.015 mg l(-1) h(-1), respectively. These results suggest that CSL, a cheap and readily available waste product, could be very useful in the bioremediation of environments contaminated with pyrene.

Differential degradation of crude oil (Bonny Light) by four Pseudomonas strains.

Four hydrocarbon degraders isolated from enriched oil- and asphalt-contaminated soils in Lagos, Nigeria, were tested for their petroleum degradation potentials. All the isolates were identified as species of Pseudomonas. Pseudomonas putida P11 demonstrated a strong ability to degrade kerosene, gasoline, diesel, engine oil and crude oil while P. aeruginosa BB3 exhibited fair degradative ability on crude oil, gasoline, engine oil, anthracene and pyrene but weak on kerosene, diesel and dibenzothiophene. Pseudomonas putida WL2 and P. aeruginosa MVL1 grew on crude oil and all its cuts tested with the latter possessing similar polycyclic aromatic potentials as P11. All the strains grew logarithmically with 1-2 orders of magnitude and with generation time ranging significantly between 3.07 and 8.55 d at 0.05 level of confidence. Strains WL2 and MVL1 utilized the oil substrate best with more than 70% in 6 d experimental period, whereas the same feat was achieved by P11 in 12 d period. BB3 on the other hand degraded only 46% within 6 d. Interestingly, data obtained from gas chromatographic analysis of oil recovered from the culture fluids of MVL1 confirmed near-disappearance of major peaks (including aliphatics and aromatics) in the hydrocarbon mixture.

Growth and biosurfactant synthesis by Nigerian hydrocarbon-degrading estuarine bacteria

The ability of microorganisms to degrade petroleum hydrocarbons is important for finding an environmentally-friendly method to restoring contaminated environmental matrices. Screening of hydrocarbon-utilizing and biosurfactant-producing abilities of organisms from an estuarine ecosystem in Nigeria, Africa, resulted in the isolation of five microbial strains identified as Corynebacterium sp. DDv1, Flavobacterium sp. DDv2, Micrococcus roseus DDv3, Pseudomonas aeruginosa DDv4 and Saccharomyces cerevisae DDv5. These isolates grew readily on several hydrocarbons including hexadecane, dodecane, crude oil and petroleum fractions. Axenic cultures of the organisms utilized diesel oil (1.0 % v/v) with generation times that ranged significantly (t-test, P < 0.05) between 3.25 and 3.88 day, with concomitant production of biosurfactants. Kinetics of growth indicates that biosurfactant synthesis occurred predominantly during exponential growth phase, suggesting that the bioactive molecules are primary metabolites. Strains DDv1 and DDv4 were evidently the most metabolically active in terms of substrate utilization and biosurfactant synthesis compared to other strains with respective emulsification index of 63 and 78 %. Preliminary biochemical characterization indicates that the biosurfactants are heteropolymers consisting of lipid, protein and carbohydrate moieties. The hydrocarbon catabolic properties coupled with biosurfactant-producing capabilities is an asset that could be exploited for cleanup of oil-contaminated matrices and also in food and cosmetic industries. Rev. Biol. Trop. 56 (4): 16031611. Epub 2008 December 30.

La capacidad de los microorganismos para degradar hidrocarburos del petróleo es de gran importancia para hallar un método aceptable y ambientalmente amigable para la restauración de terrenos ambientalmente contaminados. Al investigar las capacidades de los organismos de un ecosistema de estuario que utilizan hidrocarburos y producen biosurfactantes, se produjo como resultado el aislamiento de cinco cepas microbianas identificadas como Corynebacterium sp. DDv1, Flavobacterium sp. DDv2, Micrococcus roseus DDv3, Pseudomonas aeruginosa y DDv4 Saccharomyces cerevisiae DDv5. Estas cepas crecieron fácilmente en varios hidrocarburos incluyendo hexadecanos, dodecanos, petróleo crudo y fracciones de petróleo. Los cultivos axénicos de organismos utilizaron diesel (1.0% v/v) con períodos por generación con ámbitos significativos (t-test, P <0.05) de entre 3.25 y 3.88 días, con la consiguiente producción de bio-surfactantes. La cinética del crecimiento indica que la síntesis de bio-surfactante se produjo principalmente durante la fase de crecimiento exponencial, lo que sugiere que las moléculas bioactivas son metabolitos primarios. Las cepas DDv1 y DDv4 fueron evidentemente las más metabólicamente activas en términos de utilización del sustrato y la síntesis de bio-surfactantes en comparación con otras cepas con índices respectivos de emulsificación de 63 y 78%. La caracterización bioquímica preliminar indica que los bio-surfactantes son heteropolímeros constituidos de fracciones de lípidos, proteínas y carbohidratos. Las propiedades catabólicas de los hidrocarburos, junto con las capacidades de producción de bio-surfactantes, es una ventaja que puede ser aprovechada para la limpieza de terrenos contaminados con petróleo y también en la industria alimentaria y cosmética.

Extensive biodegradation of polychlorinated biphenyls in Aroclor 1242 and electrical transformer fluid (Askarel) by natural strains of microorganisms indigenous to contaminated African systems.

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.

Evidence of aerobic utilization of di-ortho-substituted trichlorobiphenyls as growth substrates by Pseudomonas sp. SA-6 and Ralstonia sp. SA-4.

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 ortho- 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 patterns 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.

Growth and biosurfactant synthesis by Nigerian hydrocarbon-degrading estuarine bacteria.

The ability of microorganisms to degrade petroleum hydrocarbons is important for finding an environmentally-friendly method to restoring contaminated environmental matrices. Screening of hydrocarbon-utilizing and biosurfactant-producing abilities of organisms from an estuarine ecosystem in Nigeria, Africa, resulted in the isolation of five microbial strains identified as Corynebacterium sp. DDV1, Flavobacterium sp. DDV2, Micrococcus roseus DDV3, Pseudomonas aeruginosa DDV4 and Saccharomyces cerevisae DDV5. These isolates grew readily on several hydrocarbons including hexadecane, dodecane, crude oil and petroleum fractions. Axenic cultures of the organisms utilized diesel oil (1.0% v/v) with generation times that ranged significantly (t-test, P < 0.05) between 3.25 and 3.88 day, with concomitant production of biosurfactants. Kinetics of growth indicates that biosurfactant synthesis occurred predominantly during exponential growth phase, suggesting that the bioactive molecules are primary metabolites. Strains DDV1 and DDV4 were evidently the most metabolically active in terms of substrate utilization and biosurfactant synthesis compared to other strains with respective emulsification index of 63 and 78%. Preliminary biochemical characterization indicates that the biosurfactants are heteropolymers consisting of lipid, protein and carbohydrate moieties. The hydrocarbon catabolic properties coupled with biosurfactant-producing capabilities is an asset that could be exploited for cleanup of oil-contaminated matrices and also in food and cosmetic industries.

Characterization of multiple novel aerobic polychlorinated biphenyl (PCB)-utilizing bacterial strains indigenous to contaminated tropical African soils.

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.

Metabolism of chlorinated biphenyls: use of 3,3'- and 3,5-dichlorobiphenyl as sole sources of carbon by natural species of Ralstonia and Pseudomonas.

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.