Biodegradation ability and catabolic genes of petroleum-degrading Sphingomonas koreensis strain ASU-06 isolated from Egyptian oily soil

Polycyclic aromatic hydrocarbons (PAHs) are serious pollutants and health hazards. In this study, 15 PAHs-degrading bacteria were isolated from Egyptian oily soil. Among them, one Gram-negative strain (ASU-06) was selected and biodegradation ability and initial catabolic genes of petroleum compounds were investigated. Comparison of 16S rRNA gene sequence of strain ASU-06 to published sequences in GenBank database as well as phylogenetic analysis identified ASU-06 as Sphingomonas koreensis. Strain ASU-06 degraded 100, 99, 98, and 92.7% of 100 mg/L naphthalene, phenanthrene, anthracene, and pyrene within 15 days, respectively. When these PAHs present in a mixed form, the enhancement phenomenon appeared, particularly in the degradation of pyrene, whereas the degradation rate was 98.6% within the period. This is the first report showing the degradation of different PAHs by this species. PCR experiments with specific primers for catabolic genes alkB, alkB1, nahAc, C12O, and C23O suggested that ASU-06 might possess genes for aliphatic and PAHs degradation, while PAH-RHDαGP gene was not detected. Production of biosurfactants and increasing cell-surface hydrophobicity were investigated. GC/MS analysis of intermediate metabolites of studied PAHs concluded that this strain utilized these compounds via two main pathways, and phthalate was the major constant product that appeared in each day of the degradation period.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a group of hydrophobic organic compounds composed of two or more fused aromatic rings in their chemical structure [1]. PAHs are released into the environment from the incomplete combustion of fossil fuels and organic matter, the accidental spilling of processed hydrocarbons and oils, run-off from asphalt pavements, coal liquefaction, and gasification, and natural geological processes [2]. Due to their toxic, carcinogenic, and mutagenic properties, PAHs are of environmental and human concern, and 16 PAHs have been listed by the US Environmental Protection Agency as priority contaminants in ecosystems [3].

Microbial degradation is the most dominant and significant process for removing PAHs from the environment. Many microorganisms capable of metabolizing PAHs were isolated including bacteria [4], yeasts [5], fungi [6], and algae [7]. Most of the bacteria isolated belong to genera Pseudomonas, Burkholderia, Mycobacteria, Rhodococcus, Alcaligenes, Ralstonia, and others [4, 8].

Usually, contaminated sites are polluted by a mixture of PAHs. Thus, for an efficient remediation process, it is important that the bacteria involved have a complete degradation pathway so that no potentially toxic degradation products accumulate [9]. Genetic analyses of PAH catabolic pathways in several PAH-degrading bacteria revealed the presence of a group of genes for complete degradation of aromatic compounds [10, 11]. PCR amplification using genes specific primers or degenerate primers has been used to detect and study the diversity of aromatic-dioxygenase genes in PAH-degrading bacterial isolates [12–14].

Although many bacteria capable of degrading PAHs have already been isolated, it is still important to screen strains that can completely and rapidly decompose PAHs under the condition of Egyptian environment. Therefore, the aim of the present study was to isolate strains capable of degrading low and high molecular weight PAHs. A strain was obtained and identified as S. koreensis according to morphological characteristics and 16S rRNA gene sequence analysis and its ability to degrade naphthalene, phenanthrene, anthracene, and pyrene were studied. The production of biosurfactants and increasing cell-surface hydrophobicity, the metabolites during the degradation process, and the genetics of catabolic genes in the isolated PAH-degrading bacterium were also investigated.

2. Materials and Methods2.1. Sample Collection and Chemicals

Oil contaminated soil was collected in sterilized polyethylene bags from Oil Refinery Company in Assiut, Egypt, and stored at 4°C in the laboratory. Naphthalene, phenanthrene, anthracene, pyrene (all ≤99% purity), and mineral basal medium with trace metals were purchased from Sigma-Aldrich.

2.2. Enrichment, Isolation, and Evaluation of PAHs-Degrading Bacteria

Soil enrichment technique was used for the isolation of PAH-degrading bacteria as described in [5]. About 10 g oil contaminated soil sample was suspended in 90 mL mineral basal salt medium (MBS) containing (g/L) 1.0(NH4)2SO4, 0.8K2HPO4, 0.2KH2PO4, 0.2MgSO4·7H2O, 0.1CaCl2·2H2O, 0.005FeSO4·7H2O, and 1 mL of trace elements, pH 7.0 ± 0.2. The medium was supplemented with one of the following PAHs: naphthalene, phenanthrene, anthracene, or pyrene at concentration of 100 mg/L as a sole source of carbon. The flasks were incubated on an orbital shaker at 150 rpm at 30°C. After 7-day incubation, an aliquot of 10% enriched cultures was transferred into another 250 mL conical flask containing 90 mL fresh autoclaved MBS medium supplemented with previously mentioned PAHs. This step was repeated five times to attain well-adapted PAH-degrading enriched bacterial consortia. Bacterial strains were isolated from MBS agar plates coated with the same PAHs as the sole carbon source. Colonies with different morphologies were individually selected. All isolates were evaluated for their PAHs-degrading abilities. One pure strain of bacteria with a high PAHs degradation rate was designated as ASU-06 and selected for further study.

2.3. Physiological and Biochemical Tests of Strain ASU-06

Conventional physiological and biochemical characteristics were determined using the procedures described by John and Krieg [15].

2.4. 16S rRNA Gene Amplification and Sequence Determination

The genomic DNA was isolated from strain ASU-06 according to the method described by Hesham [16] and the 16S rRNA gene was amplified. Amplification was carried out with universal primers: 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-CGGCTACCTTGTTACGACTT-3) in a final volume of 50 μL containing 10 mM tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, each dNTP at a concentration of 0.2 mM, 1.25 IU of Taq polymerase, each primer at a concentration of 0.2 mM, and 1 μL of the DNA template. PCR was performed with the following program: 5 min denaturation at 95°C, followed by 36 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C, 1.5 min extension at 72°C, and a final extension step of 7 min at 72°C. 5 μL of the amplified mixture was then analyzed using 1.5% 0.5 × TBE agarose gel electrophoresis. The gel was stained with ethidium bromide, visualized under UV light, and photographed. Product of the correct size was purified and sequenced in both directions using an ABI automated sequencer.

2.5. Sequence Alignment and Phylogenetic Analysis

The 16S rRNA gene sequences of the isolate obtained in this study were aligned and compared with the known 16S rRNA gene sequences in Genbank database using the BLAST search at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) to determine the closest available database sequences. To determine the taxonomic position of the isolate, a phylogenetic tree was constructed with MEGA version 4.0 using a neighbor-joining algorithm; in addition, the Jukes-Cantor distance estimation method with bootstrap analyses for 1,00 replicates was performed [17].

2.6. PAHs Degradation Analysis by HPLC

Pure strain was resuspended in 50 mL MBS supplemented with either naphthalene, phenanthrene, anthracene, or pyrene at concentration of 100 mg/L for each or mixed of them PAHs (25 mg/L) for each to a turbidity of 0.2 at OD (600 nm). Abiotic controls were prepared in the same way but without addition of bacteria. Samples and controls were prepared in triplicate. Flasks were incubated at 30°C with rotation at 150 rpm for 15 days. After every three days of incubation, 25 mL aliquot of each sample was extracted three times with equal volumes of ethyl acetate, according to the method described by Manohar et al. [18]. The residual PAHs were analyzed by HPLC as described by Bishnoi et al. [19].

2.7. Detection of PAH-Degrading Enzymes

The initial dioxygenase activity catalyzing the conversion of indole to be indigo was determined following the standard method described by a previous worker [20], while Catechol 2,3-dioxygenase activity was determined according to Ornston and Stanier [21].

2.8. Detection of Aliphatic and PAH-Degrading Genes by PCR

The presence of six genes including monooxygenase and dioxygenase genes in the isolated bacterial strain ASU-06 was detected based on PCR amplification. The primers for the detection of n-alkanes monooxygenase (alkB and alkB1), dioxygenase (nahAc), Catechol dioxygenase (C12O and C23O), and PAH-ring hydroxylating dioxygenase (PAH-RHDα) genes were listed in Table 1. PCR conditions were initial denaturation for 5 min at 95°C, 35 cycles with 40 s at 94°C, 40 s at 55°C, 60 s at 72°C, and final elongation for 7 min at 72°C for the four genes alkB, alkB1, nahAc, and PAH-RHDα [12–14, 22]. However, PCR was performed for Catechol 1,2-dioxygenase (C12O) and Catechol 2,3-dioxygenase (C23O) genes with initial denaturation for 5 min at 95°C, 35 cycles with 20 s at 94°C, 30 s at 63°C, and 45 s at 72°C, and final elongation for 5 min at 72°C [23]. All PCR products were separated in 1.5% agarose gel, stained with ethidium bromide, visualized under UV light, and photographed.

PrimerSequence (5′ to 3′)Expected size (bp)ReferencealkBF
alkBR5′-AACTACMTCGARCAYTACGG-3′ 
5′-TGAMGATGTGGTYRCTGTTCC-3′100Powell et al. [22]AlkB1F
AlkB1R5′-TACGGGCACTTCGCGATTGA-3′ 
5′-CGCCCAGTTCGAMACGATGTG-3′550Kloos et al. [13]nahAc F
nahAc R5′-TGGCGATGAAGAACTTTTCC-3′ 
5′-AACGTACGCTGAACCGAGTC-3′487 Laurie and Jones [14]PAH-RHD-GPf
PAH-RHGPr5′-CGG CGC CGA CAA YTT YGT NGG-3′ 
5′-GGG GAA CAC GGT GCC RTG DAT RAA-3′292Cébron et al. [12]C12OF
C12OR5′-GCCAACGTCGACGTCTGGCAGCA-3′ 
5′-CGCCTTCAAAGTTGATCTGCGTGGTTGGT-3′350Sei et al. [23]C23OF
C23OR5′-AAGAGGCATGGGGGCGCACCGGTTCGA-3′ 
5′-TCACCAGCAAACACCTCGTTGCGGTTGCC