Degradation of Anthracene by Bacteria Isolated from Oil Polluted Tropical Soils Matthew O. N. Ilori*s and Dan-Israel Amund Department of Botany and Microbiology, University of Lagos Akoka-Yaba, Lagos, Nigeria * Author for correspondence and reprint requests Z. Naturforsch. 55c, 890-897 (2000); received June 6 /July 11, 2000 Anthracene, Crude Oil, Dioxygenases Four bacteria, identified as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus subti- lis and Micrococcus'luteus were isolated from crude oil polluted soils using anthracene as the sole carbon and energy source. All the organisms utilized n-hexadecane, n-tetradecane, diesel oil, engine oil and naphthalene as sole carbon sources. None could utilize hexane, cycloheptane, xylene, benzene, toluene, phenol, fluoranthene,and kerosene as carbon sources. Highest cell density obtained with 0.1% (w/v) anthracene were 4.5 x 107 (cfu/ml), 8 . 6 x 106 (cfu/ml), 5.4 x 106 and 2.4 x 106 (cfu/ml) respectively, for P. aeruginosa, A. eutrophus, B. sub- tilis and M. luteus after 30 days incubation. Growth of the organisms on a Nigerian crude oil resulted in a residual oil concentration of 22.2%, 33.3%, 39.3% ,44% and 91.7% respectively, for P. aeruginosa, A. eutrophus, B. subtilis, M. luteus and the noninoculated control on the 14 th day. Ring fission enzymes of the meta pathway were detected in induced cells of P. aeruginosa and A. eutrophus while ortho pathway enzymes were detected in B. subtilis and M. luteus. P. aeruginosa and A. eutrophus had specific catechol-2,3-dioxygenase activities of 3.8 ± 0.183 and 0.64 ± 0.032 (.imol / min x mg protein respectively while catechol-1,2-dioxy- genase activities of 1.95 ± 0.029 and 1.89 ± 0.026 jimol / min x mg protein were detected in B. subtilis and M. luteus respectively. This work , highlights the capability of these unreported tropical strains of A. eutrophus, B. subtilis and M. luteus as anthracene degraders. Introduction in the environment as they have been shown to exhibit toxic, mutagenic and carcinogenic effects. Polycyclic aromatic hydrocarbons (PAHs) are Many PAHs are known to function as precarcino­ organic molecules that consist of two or more gens that require metabolic activation before they fused benzene rings in linear, angular or cluster are able to bind to DNA, RNA or proteins (Suth­ arrangement. They are components of coal, crude erland et al., 1995). and refined oils and are also formed during pyrro- Anthracene is one of the low molecular weight lytic processes. Consequently, they have been PAHs with unaided or natural water solubility of detected in numerous aquatic and terrestrial eco­ 0.07-0.08 mg/1 at 25-30 °C (Cerniglia, 1992; Wil- systems especially in industrial sites, at concentra­ lumsen and Karlson, 1997). There is, therefore, in­ tions high enough to warrant concern about bioac­ terest in the metabolic fate of the compound, firstly cumulation. For example, PAH ranges from a low because of its recalcitrance which is due to poor concentration of 5ng/g of soil in an undeveloped aqueous solubility and secondly the fact that the area to 1.79 x 106 ng/g at an oil refinery (Cerniglia, anthracene structure is found in carcinogenic PAHs 1992). The most frequently occuring PAHs include such as benz(a)pyrene, benz(a)anthracene and 3- naphthalene, phenanthrene, anthracene, pyrene, methyl-cholanthrene (Cerniglia, 1984). Aqueous benz(a)pyrene and benz(a)anthracene. There is solubility of anthracene is even poorer than some concern about the presence of these compounds higher molecular weight PAHs such as fluoran­ thene (0.26 mg/1) and pyrene (0.14 mg/1). This, therefore, gives anthracene a higher environmental § Present address: persistence over some of the high molecular weight Rheinische Westfalische Technische Hochschule, PAHs because of its poorer bioavailability. Lehrstuhl für Biologie 5, Worringerweg 1, 52074, Aachen. Germany. Studies have shown that microbiological degra­ E-mail: [email protected] dation of PAHs is the major process that results in 0939-5075/2000/1100-0890 $ 06.00 © 2000 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D M. O. N. Uori and D.-I. Amund • Biodegradation of Anthracene 891 the decontamination of sediment and surface soils Soil samples (Cerniglia, 1992). Some bacteria and fungi have Samples were taken from 2 experimental soil been reported to have the ability to grow on sites within the University of Lagos polluted for anthracene. They include Beijerinckia sp., M yco­ over 1 year with crude oil. Composite samples bacterium sp., Pseudomonas sp., Arthrobacter sp., were placed into sterile bottles and transported Cunninghamella sp. and Trametes sp. (Cerniglia, immediately to the laboratory for further work. 1992). Aerobic biodegradation mechanism re­ quires the presence of molecular oxygen to initiate enzymatic attack on the rings. The initial reaction Isolation and identification of PAH degrading in the bacterial degradation of anthracene involves bacteria oxygenation at the 1,2 positions with the forma­ tion of cis l,2-dihydroxy-l, 2-dihydroanthracene Bacteria capable of degrading anthracene as which is then oxidized to 1 ,2-dihydroxyanthracene sole carbon and energy source were isolated from prior to ring fission (Menn et al., 1993). The 1,2- the soil samples using minimal salts agar medium dihydroxyanthracene is further metabolized to 2- as described by Kästner et al. (1994). The medium hydroxy-3-naphthoic acid, 2,3 dihydroxynaphtha- contained per liter: Na 2H P 0 4, 2.13 g; KH 2P 0 4, lene, salicylic acid and then to catechol. The resul­ 1.3 g; NH 4C1, 0.5 g; MgSC>4. 7H 20 , 0.2 g. Steriliza­ tant catechol is degraded in what is referred to as tion was carried out by autoclaving at 121 °C for the lower pathway through meta or ortho cleavage 15 min. Trace elements solution (1 ml per liter) as depending on the bacterial strain and other factors described by Bauchop and Elsden (1960) was ster­ while the degradative steps up to salicylate is re­ ilized separately and then added aseptically to the ferred to as the upper pathway. Catechol metabo­ medium. The plates were poured and allowed to lism through the meta and ortho pathways are me­ dry overnight after which 0.2 ml of a filtered etha­ diated by intracellular dioxygenases which include nol solution (containing 0.5 g anthracene per catechol 2,3 dioxygenase and catechol 1,2 dioxy­ 100 ml of ethanol) was aseptically pipetted and genase respectively. Organisms utilizing the two uniformly spread on the agar surface as described pathways form products which are capable of en­ by West et al. (1984). The ethanol was allowed to tering the tricarboxylic acid cycle (Cerniglia, dry under sterile condition before inoculation. 1984). Plates prepared in this manner had an opaque film Microbial seeding of polluted soils and sediment of anthracene on the agar surface. Diluted soil is gaining worldwide acceptance as one of the en­ samples were inoculated onto the agar surface ,the vironmentally acceptable alternatives to physical plates were kept in clean polythene bags to con­ remediation such as chemical washing and inciner­ serve moisture and incubated in the dark for 12 to ation. The search for suitable PAH degrading bac­ 15 days. Inoculated anthracene free minimal salts teria with bioremediation potential is an important agar medium were included in all cases as control project in every environmental contigency plan. In for comparison with test plates. Colonies on the this paper, we report the degradation of anthra­ control plates were counted and taken as oli- cene up to the lower pathway by four genera of gothrophs able to grow on medium components tropical bacteria, three of which to our knowledge without any further carbon source. The colonies has not been reported previously. that appeared on the anthracene plates with crys­ tal cleared zones were replicated onto fresh Materials and Methods anthracene coated agar plates and incubated for another 15 days. Isolates which fail to grow were Chemicals and media excluded from further tests. The anthracene de­ All chemicals were of analytical grade. PAHs graders were identified as described by Cowan such as anthracene and naphthalene were (1974) and Holt et al. (1994). Diagnostic properties purchased from Sigma (U.K). Media such as nutri­ used include Gram reaction, motility, colonial ent agar and broth were purchased from Oxoid morphology, production of oxidase, catalase, in­ (U.K). Crude oil (Escravos light) was obtained dole, gelatin liquefaction, starch hydrolysis and from Chevron Nigeria Limited. sugar utilization. 892 M. O. N. Ilori and D.-I. Amund • Biodegradation of Anthracene Preparation o f starter cultures cene crystals (0.1%, w/v) was added. The medium Cells grown for 12 days, were harvested from was sterilized and inoculated with 10 ml of a the surface of anthracene agar and resuspended in starter culture of each organism. Incubation was carried out in the dark at 28 °C on a shaker set at 10 ml sodium phosphate buffer (50 m M , pH 7.2) in a test tube. Centrifugation was carried out at 150 rpm. Growth of the organisms were assayed 10,000 xg for 10 min at 4 °C. The supernatant was by plating in triplicate aliquots of the cultures on discarded while the pelleted cells were resus­ to sterile nutrient agar. The plates were incubated pended in the same buffer and the washing re­ at 28 °C for 48 h after which the colonies were peated for 2 more times at the same condition. counted. Assays for growth was carried out on the The pelleted cells were finally resuspended in ster­ day the medium was inoculated and subsequently ile minimal salts medium (10 ml) to a final popula­ at 3 days interval for 30 days.