Introduction

Anthracene is a low molecular weight polycyclic aromatic hydrocarbon. It is a regulated contaminant at sites polluted with petroleum. It also results from anthropogenic pyrolysis of petroleum products and incomplete combustion of organic matter (Moody et al. 2001). High concentrations of anthracene (up to 13,000 mg/kg) have been detected in agricultural soils in the oil-rich Niger Delta region of Nigeria (Oviasogie et al. 2006). Anthracene is highly recalcitrant, in part because of its low solubility in water (0.015 mg/l) accounted for by its high aromaticity compared with phenanthrene or pyrene (Cerniglia 1992; Garcia-Cruz and Martinez-Magadan 2007).

Although not known to exhibit any genotoxic or carcinogenic effects in humans, anthracene shares structure with known carcinogens such as benzo(a)anthracene and is useful in modeling the metabolism of such counter-part PAH (Cerniglia and Shuttleworth 2002). Anthracene is degraded in the environment mainly by bacteria and fungi. It has been demonstrated long ago that bacteria capable of degradation of anthracene can easily be isolated from soil (Evans et al. 1965 Cerniglia 1992). Degraders of anthracene reported in the literature span wide range of genera of both Gram-positive and Gram-negative bacteria, including Alcaligenes, Arthrobacter, Beijerinckia, Micrococcus, Mycobacterium, Nocardia, Pseudomonas, Rhodococcus and Sphingomonas (Kastner et al. 1994; Bastiaens et al. 2000; Ilori and Amund 2000; Dean-Ross et al. 2001; Mrozik et al. 2003). It is worthy of note that many of the early reports detailed degradation by Pseudomonas species such as P. putida, P. cepacia, P. paucimobilis and P. fluorescence (Foght and Westlake 1988; Mueller et al. 1990; Cerniglia 1992; Dagher et al. 1997).

However, in recent years, there has been increase in the number of reports of novel anthracene degraders, including actinomycetes, and even rhizosphere bacteria such as Bacillus circulans and Kurthia sp. SBA4 (Bisht et al. 2010). Others include E. coli and Thiobacter subterraneus (Abd-Elsalam et al. 2009).

Actinomycetes are a group of diverse bacteria that are Gram positive with high guanine plus cytosine in the DNA (>55 mol %). They are good candidates for bioremediation of polluted soils due to their production of extracellular enzymes that degrade a wide range of complex organic compounds, production of spores that are impervious to dessication and filamentous growth that favors colonization of soil particles (Ensign 1992). Particularly, actinomycetes are good candidates for degradation of hydrophobic compounds such as PAHs due to their surfactant-producing activity. The biosurfactant may be extracellular such as glycolipids (trehalose lipid and lipopeptide produced by Rhodococcus and Arthrobacter species) or cellular biosurfactants such as mycolic acid, which allow adherence of the microbial cells to hydrophobic phases in two-phase systems (Singer and Finnerty 1990; Morikawa et al. 1993; Neu 1996).

The initial reactions in the aerobic degradation of anthracene are catalysed by a multi-component enzyme system and involve the incorporation of other atoms of oxygen molecules into the PAH nucleus to produce a dihydrodiol. The dihydrodiol is re-aromatised and the dihydroxylated product is cleaved to products, which are further processed through the pathways of naphthalene degradation (Moody et al. 2001; Mrozik et al. 2003; Van Herwijnen et al. 2003). In some of the anthracene degraders so far reported, the genes for anthracene metabolism have been found to be localized on plasmids (Sanseverino et al. 1993; Ilori and Amund 2000; Kumar et al. 2010).

In recent years, there has been a remarkable shift in focus from isolation to demonstration of organisms capable of degrading PAHs in soil and other environmental compartments using molecular methods and other non-culture dependent techniques (Stach and Burns 2002). However, this has not obfuscated the need for continuous search for culturable organisms. As noted by Hilyard et al. (2008), on the one hand, such isolates offer opportunity for examination in order to elucidate links between phylogeny and specific catabolic capability and could as well be sources of novel or highly divergent PAH degrading enzymes. Secondly, such new strains may be useful in bioaugmentation of PAH-contaminated sites for bioremediation. In this paper, we report for the first time, to the best of our knowledge, the isolation and characterization of a Microbacterium species strain able to use anthracene as sole source of carbon and energy.

Materials and methods

Sampling

Soil samples for this study were collected from a Mechanical engineering workshop at Okokomaiko, Lagos. The coordinates of the sampling site was latitude 6° 79′N, longitude 3° 31′E, respectively. The site has a long history of contamination with indiscriminately disposed spent oils spanning a period of more than 10 years. Soil samples were collected at a depth of 10–12 cm with sterile trowel after clearing debris from the soil surface. Samples for physico-chemical analyses were collected in polyethylene bags, while those for microbiological analyses were collected in sterile screw-capped bottles. Samples were analyzed immediately upon arrival in the laboratory. Leftover samples were refrigerated at 4 °C.

Physico–chemical analysis of soil samples

The pH of the soil samples was determined with a pH meter (Jenway 3051) in 1:1 soil solution in distilled water. The moisture content, organic content, total nitrogen content, potassium content and available phosphorous were determined as described previously (Bray and Kurtz 1945; Black 1965; Chopra and Kanwar 1998). Conductivity was determined using a conductivity cell (PW 9504 Philips) with a cell constant of 1.2 while the heavy metal content of the soils was determined using atomic absorption spectrophotometer (α 4, AAS) following mixed acid digestion and extraction of the soil samples. The total hydrocarbon content of the soil was extracted using n-hexane:dichloromethane solvent systems (1:1).

Enrichment and isolation of anthracene-degrading bacteria

Bacteria able to degrade anthracene were isolated on anthracene mineral salts medium (MSM) by continual enrichment method. The mineral salts medium described by Mills et al. (1978) and modified by Okpokwasili and Amanchukwu (1988) was used. The medium contained the following in g/l of distilled water: NaCl, 10.0; KCl, 0.29; MgSO4∙7H2O, 0.42; KH2PO4, 0.83; K2HPO4, 1.25; NaNO3, 0.42; and Na2HPO4, 1.25. The medium was supplemented with yeast extract (0.005 g/l) as source of growth factors. After adjusting the pH to 7.0, the medium was fortified with 50 μg/ml and 20 μg/ml of nystatin and nalidixic acid for selective isolation of actinomycetes.

Air-dried contaminated soil (5 g) was added to 45 ml of MSM containing 50 ppm of anthracene. Enrichment was carried out by incubation with shaking (180 rpm) at room temperature (29 ± 2 °C) in the dark for 4–5 weeks until there was turbidity. After five consecutive transfers, anthracene degraders were isolated by plating out dilutions from the final flasks on Luria–Bertani (LB) agar. The colonies that appeared were further purified by subculturing once onto LB agar. Ability to degrade anthracene was confirmed by inoculating washed LB broth grown culture in fresh MSM flask supplemented with 50 ppm anthracene as sole carbon source. One isolate out of the four screened was singled out for further study based on its extensive degradative ability. The isolate was maintained at −20 °C in glycerol: LB broth medium (50:50).

Identification and characterization of isolate

Pure culture of bacterial isolate was identified based on their colonial morphology, cellular morphology and biochemical characteristics according to the identification scheme of Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994).

Antibiotic sensitivity of the isolate was carried out using multidiscs. Salt tolerance was tested in LB broth amended with NaCl ranging from 1 to 10 % (w/v). Incubation was carried out at room temperature (27 ± 2 °C) for one week with shaking and daily observation for growth.

Genomic DNA was isolated from the isolate using standard protocols (Ausubel et al. 1990). Using the purified genomic DNA as template, the gene coding for 16S rRNA was amplified using the universal primers 27f (5′-AGAGTTTGATC{A/C}TGGCTCAG-3′) and 1378r (5′-CGGTGTGTACAAGGCCCGGGAACG- 3′) (Heuer et al. 1997). The amplification reaction mixture contained 20 pmol each of universal primers, 10 μl of Ex Taq buffer (Mg2+ plus), 2.5 mm of each dNTPs, 2.5 U (0.5 μl) of Ex Taq polymerase (Takara) and 1.0 μl of purified genomic DNA in a total volume of 100 μl. Thermocycling conditions consisted of an initial denaturation step at 95 °C for 3 min, followed by 30 amplification cycles of 95 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min. The final elongation temperature was maintained at 72 °C for 7 min before cooling to 4 °C.

The amplicon was analyzed on 1 % agarose gel in 1xTAE, ran at 100 V for 30–35 min. The PCR product (~1,500 bp DNA fragment) was cloned into the plasmid vector pT7Blue® (Novagen, USA) after purification by centrifugation using Wizard® SV Gel and PCR Clean-up System (Promega, Madison, Wis.). The purified PCR product was transformed into E coli strain DH5α (Toyobo). The cloned fragment was partially sequenced using T7 (5′-TAATACGACTCACTATAGGG- 3′) (Sigma Genosys, Japan) and U-19mer (5′-GTTTTCCCAGTCACGACGT- 3′) (Invitrogen) sequencing primers along with f2L’ and R2L’ primers (Sigma Genosys, Japan) that anneals with the inside of the insert.

Nucleotide sequencing was carried out by the chain termination method using the ABI Prism 3730xl DNA Sequencer (Applied Biosystems, UK) according to the manufacturer’s instructions. The 16S rRNA nucleotide sequence obtained from both strands was aligned (CLUSTAL W) and the homology search for 16S rRNA were performed in the DDBJ/EMBL/GenBank database using the Basic Local Alignment Search Tool (BLAST) program (Altschul et al. 1990). Phylogenetic tree with retrieved sequences from NCBI database was constructed using neighbor joining algorithm within the program MEGA 5.1 (The Biodesign Institute) and bootstrapped with 100 repetitions.

Evaluation of anthracene biodegradation

Anthracene degradation potential of the pure isolate was assayed by inoculating 250-ml replicate flasks containing 50 ml of MSM supplemented with 50 ppm of anthracene as sole source of carbon. Flasks were inoculated with 0.5 ml of MSM-washed 18–24 h LB agar-grown cells and subsequently incubated at 180 rpm in the dark for 21 days at room temperature (29 ± 2 °C).

Controls consisted of flasks supplied with anthracene as carbon and energy source as described above but inoculated with heat-killed cells. Samples were withdrawn from each flask at 3 days interval and aliquots of appropriate dilutions were plated (in triplicates) onto nutrient agar for total viable counts

Extraction of residual anthracene

Residual anthracene was extracted by liquid–liquid extraction. Briefly, broth culture (20 ml) was extracted twice with an equal volume of hexane. After removing the aqueous phase with separating funnel, the organic fraction was concentrated to 1 ml and the residual anthracene concentration was determined by gas chromatography. Control flasks were also extracted similarly.

Analytical method

Hexane extracts (1.0 μl) of residual anthracene were analyzed with Hewlett Packard 5890 Series II gas chromatograph equipped with flame ionization detector (FID) and 30 m long HP-5 column (internal diameter, 0.25 mm; film thickness, 0.25 μm). The carrier gas was nitrogen. The injector and detector temperatures were maintained at 300 and 320 °C respectively. The column temperature was programmed from 60 to 500 °C for 27 min. The gas chromatograph column was programmed at an initial temperature of 60 °C; this was held for 2 min, and then ramped at 12 °C/min to 205 °C and held for 16 min. Nitrogen column pressure was 37 psi, the hydrogen pressure was 9 psi and compressed air pressure was 13 psi. The software was Chem Station. Rev. A. 05. 01.

Statistical analysis

Mean generation times (Td) and growth rate (K) of the isolate on anthracene was calculated using non-linear regression of growth curves for the period when growth rates were maximal using Prism version 5.0 (Graphpad software, San Diego, CA).

Results

Physico-chemical properties of the soil

Physico-chemical analysis of the soil sample used for enrichment revealed a weakly acidic pH (6.10) with a moisture content of 6.85 %. It also showed an unusually high total hydrocarbon content (157 g/kg). The detection of various heavy metals such as lead, zinc, iron, manganese and nickel in significant concentration further suggest heavy contamination of the sampling site (Table 1).

Table 1 Physicochemical properties of hydrocarbon-contaminated sampling site
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Characterization and identification of anthracene-degrading isolate

Anthracene-degrading strain SL10 is a Gram-positive bacterium with circular, smooth, translucent, bright yellow-pigmented colonies. The organism was catalase positive but negative for oxidase, methyl red, Voges-Proskauer, indole and H2S production. It showed positive reaction to starch hydrolysis and utilized sugars such as arabinose, cellobiose, fructose, galactose, glucose, mannose, maltose, sucrose and trehalose. It also produced acids from the fermentation of xylose, rhamnose and sucrose. The isolate tolerated salt concentration of more than 4 %. It resisted cefotaxime, ciprofloxacin and streptomycin, but was susceptible to meropenem, linezolid and vancomycin (Table 2).

Table 2 Antibiotic sensitivity pattern of anthracene-degrading strain SL10
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Comparison of the 16S rDNA partial fragments of strain SL10 (1376 bp) with the nucleotide sequences in the DDBJ/EMBL/GenBank database indicates significant alignments of the strain with Microbacterium species. Strain SL10 exhibited 99 % homology with the nucleotide sequences of almost all Microbacterium spp. in the database. Worthy of note is the 99 % clustering of the nucleotide sequence of strain SL10 with the sequences of hydrocarbon-degrading Microbacterium spp. such as Microbacterium sp. PHD-5 (DQ227343.1), Microbacterium sp. CQ0110Y (DQ852355.1) and Microbacterium sp. ADAT-G (JF834172), respectively (Fig. 1). The nucleotide sequence of Microbacterium sp. strain SL10 was deposited at the DDBJ/EMBL/GenBank database and assigned the accession number AB646581.2.

Fig. 1
figure 1

Phylogenetic tree resulting from neighbour-joining (NJ) analysis of 16S rRNA showing the phylogenetic positions of anthracene-degrading Microbacterium sp. strain SL10 (AB646581.2) and related species of Microbacterium retrieved from NCBI GenBank. Accession number of each microorganism used in the analysis is shown before the species name. The scale bar indicates the numbers of nucleotide substitutions per position. Bootstrap values (expressed as percentage of 100 replicons) are shown at the branch

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Kinetics of anthracene biodegradation

The growth kinetics of strain SL10 on anthracene is depicted in Fig. 2 and Table 3. The strain exhibited a slight lag phase followed by consistent increase in cell density with concomitant decrease in anthracene concentration. Strain SL10 grew exponentially from an initial cell density of 4.2 × 105 cfu/ml to 8.2 × 109 cfu/ml in 12 days. It thereafter maintained a decreasing trend. During the exponential growth of the isolate on anthracene, it exhibited a growth rate and doubling time of 0.82/day and 0.84 days respectively.

Fig. 2
figure 2

Growth dynamic of Microbacterium sp. strain SL10 in minimal medium supplemented with 50 mg l−1 anthracene showing total viable count, TVC (diamond) and residual anthracene (square). Data points represent the mean of three replicate flasks. In the case of population counts, error bars that represent standard deviation were removed for clarity. Residual anthracene were determined with reference to anthracene recovered from heat-killed controls

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Table 3 Growth kinetics of strain SL10 on anthracene
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Gas chromatographic analysis of residual anthracene showed that strain SL10 degraded 57.5 % of the anthracene in 12 days. At the end of the 21-day cultivation period, 90.12 % of the anthracene was consumed by strain SL10. In the heat-killed control flasks, no apparent decrease of the substrate was observed, thus affirming that anthracene depletion from the MSM was due to biodegradation by strain SL10 rather than to non-specific abiotic losses. It is however noteworthy that in spite of decline in growth observed from day 15; anthracene biodegradation persists throughout the experimental period.

Discussion

PAHs are ubiquitous environmental contaminants with deleterious biological effects including acute toxicity, mutagenicity and carcinogenicity (Cerniglia 1992). Their low bioavailability, hydrophobicity, and bioaccumulation potential are premised on their high degree of conjugation and aromaticity (Nkansah et al. 2011).

In this study, anthracene, a tricyclic PAH was degraded by a Gram-positive, high G + C actinomycete, Microbacterium sp. strain SL10, isolated from a heavily polluted hydrocarbon contaminated site. Previously, Ilori and Amund (2000) isolated four anthracene-degrading bacteria namely P. aeruginosa, Alcaligenes eutrophus, Bacillus subtilis and Micrococcus luteus from crude oil polluted soil in Lagos, Nigeria.

The ability of Microbacterium sp. strain SL10 to grow at salt concentration higher than 4 % could favour their use for bioaugmentation purpose as salinity has been regarded as a very important factor that determine the survival of bacterial inoculum seeded to soils (Kastner et al. 1998; Obayori et al. 2008). Microorganisms produce antibiotics as a survival strategy and for competitive edge in a nutrient limiting, highly compacted and diverse contaminated soil environment. As such, knowledge of sensitivity and resistance patterns of organisms with potentials for use as seeds for bioremediation is important. Resistance of strain SL10 to streptomycin, cefotaxime and ciprofloxacin may be attributed to acquisition of resistant genes to these antibiotics through gene transfer, as soil environments are replete with these antibiotics, which could allow evolution of resistance by indigenous strains (Obayori et al. 2008).

Strain SL10 degraded 90.12 % of the initial concentration of anthracene (50 ppm) in 21 days with a rate of degradation of 4.79 mg l−1 day−1 and growth rate and doubling time of 0.82 day−1 respectively. This degradation rate is lower than 92 % reported for Mycobacterium sp strain PYR-1 after 14 days of incubation (Moody et al. 2001). However, the degradation rate and rate of degradation of this strain is higher than 56 and 71 % (3.90 mg l−1 day−1) after 48 days reported for two anthracene-degrading Pseudomonas strains isolated from a petrochemical sludge landfarming site (Jacques et al. 2005). It is equally higher than the 87.5 and 86.6 % reported for rhizosphere bacteria Bacillus circulans SBA12 and Kurthia sp. SBA4 respectively by Bisht et al. (2010).

The ability of Microbacterium sp. strain SL10 to degrade anthracene may be attributed to the fact that the strain was isolated from a site where spent oils with its attendant PAHs and heavy metals are indiscriminately dumped and disposed allowing the organism to adapt and evolve necessary gene battery to degrade pollutants and also resist the presence of heavy metals. This underscores the role of vertical (point mutation, DNA rearrangement by IS elements) and horizontal evolution (plasmid transfer, conjugative transposons, integrons and genomic islands) in the emergence of degradative pathways by microorganisms (van der Meer et al. 1992; Top and Springael 2003; Obayori and Salam 2010).

The phylum Actinobacteria encompasses bacteria genera such as Mycobacterium, Rhodococcus, and Gordonia with unrivalled capability to degrade recalcitrant pollutants due to their metabolic versatility, genetic plasticity and ability to survive in harsh environments (Larkin et al. 2005; Mutnuri et al. 2005; Kanaly and Harayama 2010). In fact, Dean-Ross et al. (2001) reported anthracene degradation by a Rhodococcus sp. that utilized 53 % of 3 μg/ml within 24 h with concomitant production of two ring fission products as metabolites.

Information on biodegradative abilities of Microbacterium spp is relatively new as the genus is a known human opportunistic pathogen. However, two crude oil degrading Microbacterium spp. identified as M. oleivorans and M. hydrocarbonoxydans were isolated by Schippers et al. (2005) from oil storage cavern 126 and oil contaminated soil in Germany. Similarly, Manickam et al. (2006) reported a hexachorocyclohexane-degrading Microbacterium sp. ITRCI capable of degrading four major isomers of the toxic compound. Furthermore, four bioemulsifier-producing Microbacterium strains were isolated from oil-contaminated mangrove with heavy metals removal abilities (Aniszewski et al. 2010). In this study, phylogenetic analysis of strain SL10 shows 99 % homology with a phenol-degrading Microbacterium PHD-5 strain (DQ227343.1), a di-2-ethylhexylphthalate-degrading Microbacterium CQ0110Y strain (DQ852355.1), and acenaphthene-degrading Microbacterium ADAT-G strain (JF834172), respectively. This shows the evolutionary relationship between strain SL10 and other hydrocarbon-degrading Microbacterium strains isolated elsewhere.

Though reports on hydrocarbon degrading abilities of Microbacterium spp. are gaining momentum, to the best of our knowledge, this is the first report highlighting anthracene degradative ability of a Microbacterium strain from tropical hydrocarbon-contaminated soil. Further research works will focus on the metabolic pathway employed by strain SL10 for anthracene degradation and the degradative genes involved in the process.