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Burkholderia fungorum DBT1: a promising bacterial strain for bioremediation of PAHs-contaminated soils

Marco Andreolli, Silvia Lampis, Elena Zenaro, Mirja Salkinoja-Salonen, Giovanni Vallini
DOI: http://dx.doi.org/10.1111/j.1574-6968.2011.02259.x 11-18 First published online: 1 June 2011


An extensive taxonomic analysis of the bacterial strain Burkholderia sp. DBT1, previously isolated from an oil refinery wastewater drainage, is discussed here. This strain is capable of transforming dibenzothiophene through the ‘destructive’ oxidative pathway referred to as the Kodama pathway. Burkholderia DBT1 has also been proved to use fluorene, naphthalene and phenanthrene as carbon and energy sources, although growth on the first two compounds requires a preinduction step. This evidence suggests that the strain DBT1 exerts a versatile metabolism towards polycyclic aromatic hydrocarbons other than condensed thiophenes. Phylogenetic characterization using a polyphasic approach was carried out to clarify the actual taxonomic position of this strain, potentially exploitable in bioremediation. In particular, investigations were focused on the possible exclusion of Burkholderia sp. DBT1 from the Burkholderia cepacia complex. Analysis of the sequences of 16S, recA and gyrB genes along with the DNA–DNA hybridization procedure indicated that the strain DBT1 belongs to the species Burkholderia fungorum, suggesting the proposal of the taxonomic denomination B. fungorum DBT1.

  • Burkholderia fungorum DBT1
  • Burkholderia cepacia complex
  • bioremediation
  • degradation


Polycyclic aromatic hydrocarbons (PAHs) represent an extended class of organic compounds containing two or more condensed aromatic rings. Their molecular stability and hydrophobicity are among the prominent factors that contribute to the persistence of these pollutants in the environment. Moreover, their low aqueous solubility and, consequently, their low bioavailability are the main obstacles to microbial degradation (Cerniglia, 1992). The presence of PAHs in environmental contexts depends on both natural processes (either biogenic or geochemical) and anthropogenic activities (Mueller et al., 1996). Of the PAHs occurring in soils and groundwaters, about 0.04–5%w/w are sulphur heterocycles (Thompson, 1981), among which dibenzothiophene (DBT) represents the prevailing species.

Burkholderia sp. DBT1, which was first isolated from an oil refinery sewage drainage, has been proved to lead, within 3 days, to the nearly complete decay of DBT added to the growth substrate, through the so-called Kodama oxidative pathway (Di Gregorio et al., 2004). A preliminary genomic study carried out on this strain revealed that the genes responsible for DBT transformation are actually harboured in two operons (p51 and pH1A) and show low similarity to both nah-like and phn-like genes (Di Gregorio et al., 2004). However, recent in situ molecular investigations on soils contaminated by different PAHs have ascertained the presence of a sequence corresponding to a dioxygenase closely related to that found in Burkholderia DBT1 (Chadhain et al., 2006; Sipilä, 2006; Brennerova et al., 2009). Thus, Burkholderia sp. DBT1 can be claimed to be a degrader of PAHs, often occurring along with condensed thiophenes in oil-contaminated sites; however, its taxonomic identity remains largely unknown.

The existence of Burkholderia cepacia strains causing life-threatening infections in humans with cystic fibrosis (Govan et al., 1996) has led to the rejection of bacteria belonging to this genus as possible biological agents by the US Environmental Protection Agency (Davison, 2005). Furthermore, as Burkholderia sp. can be involved in food poisoning (Jiao et al., 2003) or act as pathogens for plants and domesticated animals (Graves et al., 1997; Brett et al., 1998; Srinivasan et al., 2001; Lee et al., 2010), some concerns exist about the intentional release of potentially hazardous strains into the environment for biotechnological applications (Vandamme et al., 1997; Parke & Gurian-Sherman, 2001). The present study aims to provide new insights into the phenotypic traits and the phylogenetic relationships of strain DBT1 for a proper taxonomic positioning within the genus Burkholderia.

Materials and methods

Bacterial strains

Burkholderia fungorum LMG 16225T, Burkholderia caledonica LMG 19076T, Burkholderia graminis LMG 18924T and B. cepacia LMG 1222T were purchased from the German Collection of Microorganisms and Cell Cultures [Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)]. Burkholderia sp. DBT1 was isolated from a drain collecting oil refinery discharges near Leghorn, Tuscany, Italy (Di Gregorio et al., 2004).


DBT, naphthalene, fluorene and phenanthrene were purchased from Sigma-Aldrich (Milan, Italy). All the compounds were analytical grade. They were dissolved in N-N-dimethylformamide (Sigma-Aldrich) before addition to the bacterial cultures.

Microbiological techniques

Growth tests with PAHs

All the growth tests were carried out in 100-mL Erlenmeyer flasks containing 50mL of minimal defined medium (DM; Frassinetti et al., 1998), supplemented with different organic compounds (naphthalene, phenanthrene, fluorene and DBT, at a final concentration of 100mgL−1) as the sole carbon source, and finally incubated at 27°C on an orbital shaker (200r.p.m.). Each flask was inoculated with aliquots from stationary-phase cultures of the Burkholderia sp. DBT1 strain until a final OD of 0.01 was reached. Culture samples collected at different times during the experiment were monitored for microbial growth by measuring the OD600nm. Afterwards, cells were pelleted by centrifugation (10000g for 10min) and absorbance of the resulting solutions was checked at 600nm to evaluate the possible contribution of coloured metabolites to the final OD.

Growth tests starting from both nonpretreated and pretreated cells were arranged. In tests with nonpretreated cells, a preinoculum of the DBT1 was obtained in YMB medium (0.5gL−1 K2HPO4; 0.1gL−1 MgSO4·7H2O; 0.1gL−1 NaCl; 0.4gL−1 yeast extract; 10gL−1 mannitol) after 48h of incubation. Conversely, in tests with pretreated cells, a preinoculum of DBT1 was grown in DM supplied with DBT or phenanthrene (500mgL−1) for 72h to induce the PAH-degrading genes. The cells were then collected by centrifugation (5000g for 5min at 4°C) and washed twice with physiological solution (NaCl 0.9%).

DBT1 reactivity to PAHs

Tests were performed on YMA media (YMB added to 1.5% bacteriological agar). Naphthalene, phenanthrene, fluorene and DBT were supplied as a vapour by incubating Petri dishes containing PAH crystals placed in their base. Plates were then incubated at 27°C and colonies were picked and restreaked on fresh media every week for a month.

DNA extraction and PCR amplification of 16S rRNA, recA and gyrB gene sequences

Total DNA for PCR amplification was prepared as follows: overnight bacterial cultures were pelleted and resuspended in 567μL TE buffer, 3μL of 10% sodium dodecyl sulphate and 3μL of 20mgmL−1 proteinase K and incubated for 1h at 37°C. A 100-μL aliquot of 5M NaCl and 80μL CTAB/NaCl solution were then added and incubated again for 10min at 65°C. Samples were extracted with an equal volume of phenol/chloroform/isoamyl alcohol mixture. DNA was obtained after precipitation with 0.6 volumes of isopropanol and finally resuspended in 50μL TE buffer.

All PCR reactions were carried out in 25μL of total volume containing 0.8μM of each primer, 0.4mM of dNTPs, 1U of GoTaq DNA polymerase (Promega, Milan, Italy) and 5μL of 5 × PCR buffer. The gene encoding for 16S rRNA gene (1500bp) was amplified using FD1 and rp2 primers (Weisburg et al., 1991). PCR conditions were as follows: 95°C for 5min, then 30 cycles of 95°C for 1min, 50°C for 1min and 72°C for 2min, with a final extension step at 72°C for 5min. A specific B. fungorum recA PCR-amplification assay was performed using the primers FunF and FunR as described by Chan et al. (2003). PCR amplification for an 869-bp ORF recA was carried out according to Payne et al. (2005), while gyrB amplification was performed as described by Ait Tayeb et al. (2008).

Cloning, sequencing and phylogenetic analysis

PCR products were transformed in Escherichia coli Xl1-blue using the Promega pGEM-T vector system according to the manufacturer's instructions, sequenced on both strands and finally searched for homology using the blastn database (Altschul et al., 1997).

The sequences were initially aligned using the multiple alignment program clustal_x 1.83 (Thompson et al., 1997). A phylogenetic tree was constructed based on the neighbour-joining method using the mega version 4.0 software package (Kumar et al., 2008). Bootstrap analysis was performed on the basis of 1000 bootstrap replications.

DNA–DNA hybridization

DNA–DNA hybridization was performed by DSMZ (Braunschweig, Germany) through spectroscopic analysis in 2 × SSC+5% formamide at 70°C. DNA was isolated using a French pressure cell press (Thermo Spectronic, Rochester, NY) and purified by chromatography on hydroxyapatite (Cashion et al., 1977). The analytical protocol was according to De Le et al. (1970) as modified by Huss et al. (1983), using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6 × 6 multicell changer and a temperature controller with an in situ temperature probe (Varian, Palo Alto, CA).


Testing with the API 20NE system was performed following the manufacturer's specifications (bioMérieux Italia, Bagno a Ripoli, Italy). Substrate assimilations were checked after 24 and 48h.

Results and discussion

Phenotypic characteristics

Growth tests in the presence of different PAHs

Growth tests carried out in the presence of different PAHs demonstrated that Burkholderia sp. DBT1 is able to grow on both phenanthrene and DBT as the sole sources of carbon and energy, although the growth on this latter substrate proceeds with a lower yield (Fig. 1). Moreover, DBT1 is also capable of utilizing naphthalene and fluorene provided after a 3-day induction on phenanthrene (Fig. 1) or DBT (data not shown).

Figure 1

Time courses of microbial growth by Burkholderia sp. DBT1 in the presence of 100mgL−1 of (a) phenanthrene (phe) or DBT, (b) naphthalene (nah) and (c) fluorene (flu) with or without preinduction (p.t.) with phenanthrene. Each curve shows means based on the results of three experiments.

When strain DBT1 was grown on YMA plates added with crystals of different PAHs, a change in the colour of the colonies was detected. Briefly, DBT1 colonies became red in the presence of DBT, yellow when treated with fluorene and orange/pink and weakly yellow when phenanthrene and naphthalene were added to Petri dishes, respectively (Fig. 2). This change in colour may be attributed to PAH cleavage. In particular, DBT1 colonies became red when treated with DBT, owing to the transformation of DBT to oxidized intermediates (Kodama et al., 1970, 1973). When fluorene crystals were added to Petri dishes, DBT1 colonies acquired a yellow colour, as already observed by Casellas et al. (1997) and Seo et al. (2009). On the other hand, when grown in the presence of phenanthrene, the strain DBT1 produced an orange/pink pigment. This phenotype has also been reported in Alcaligenes faecalis AFK2, which degrades phenanthrene via o-phthalate by a protocatechuate pathway (Kiyohara et al., 1982). Finally, with the addition of naphthalene crystals, DBT1 colonies became weakly yellow, as already observed in a Pseudomonas strain (Kiyohara & Nagao, 1977).

Figure 2

Burkholderia DBT1 grown on YMA supplied with (a) DBT, (b) phenanthrene, (c) fluorene and (d) naphthalene crystals amended on the Petri dish bottom. (e) Burkholderia DBT1 grown on YMA without PAH crystals (negative control).

These results suggest that the strain DBT1 may rely on a broad substrate specificity towards different PAHs. Interestingly, enzymes for the degradation of naphthalene and fluorene can be induced by either phenanthrene or DBT. This indicates that these compounds, chiefly phenathrene, may act as major substrates for Burkholderia sp. DBT1.

Biochemical tests

API 20NE tests were carried out on the following strains: Burkholderia sp. DBT1, B. fungorum LMG 16225T and B. cepacia LMG 1222T. Burkholderia fungorum and B. cepacia were compared with DBT1 as they represent, respectively, the closest phylogenetically related species and the most representative species listed in the Burkholderia cepacia complex (Bcc), whose members are often responsible for opportunistic human infections (Govan et al., 1993). Furthermore, sometimes, B. fungorum isolates can be misidentified as Bcc organisms (Coenye et al., 2001, 2002). Strains DBT1, LMG 16225T and LMG 1222T were capable of utilizing d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetylglucosamine, gluconate, malate, citrate and phenylacetate. None of the strains considered was positive for indole production, arginine dihydrolase, glucose acidification, urease activity or maltose assimilation. In fact, strain DBT1 showed almost the same biochemical traits as both B. fungorum and B. cepacia type strains (Table 1). Nevertheless, the findings on LMG 1222T were consistent with previous studies (Fain & Haddock, 2001). On the other hand, LMG 16625T is listed as positive for the assimilation of caprate and adipate in Coenye et al. (2001).

View this table:
Table 1

Carbon source utilization and biochemical tests that differentiate strain DBT1 from Burkholderia cepacia LMG 1222T and Burkholderia fungorum LMG 16225T

TestsB. cepacia LMG 1222TB. fungorum LMG 16225TBurkholderia sp. DBT1
Nitrate reduction++
Aesculin hydrolysis++
Caprate assimilation+
Adipate assimilation++
  • −, Negative; +, positive.

Phylogenetic analysis

Sequence analysis of 16S rRNA, recA and gyrB genes

A 1493-bp fragment of DBT1 16S rRNA gene was sequenced and nucleotide blast (NCBI) analysis was performed. Thereafter, multiple alignment and evolutionary distances were calculated with 16S rRNA genes of related and nonrelated taxa in order to construct a phylogenetic tree based on the neighbour-joining algorithm (Fig. 3). The 16S rRNA gene sequence of strain DBT1 was closely related (99.7–100% similarity) to those of different strains of B. fungorum. Burkholderia fungorum strains LMG 16225T and LMG 16307 were isolated from the white-rot fungus Phanerochaete chrysosporium and cerebrospinal fluid, respectively (Coenye et al., 2001). Strain N2P5 was isolated from a PAH-contaminated soil (Mueller et al., 1997; Coenye et al., 2001) and might have useful degradative properties similar to DBT1. Burkholderia phytofirmans LMG 22487T was ranked as the second most closely related bacterial species to DBT1, with a 98.9% similarity. Good similarities of 16S rRNA gene sequences were also found between DBT1 and B. caledonica LMG 19076T (98.5%), Burkholderia megapolitana LMG 23650T (98.4%) and Burkholderia phenazinium LMG 2247T (98.4%). Still significant similarities to DBT1 were shown by Burkholderia phenoliruptrix LMG 21445T, Burkholderia xenovorans LMG 21463T, Burkholderia terricola LMG 20594T, B. graminis LMG 18924T and Burkholderia caryophylli LMG 2155T in the range 97.9–97.3%. Finally, the similarities between DBT1 and the other Burkholderia sp. considered in this study were <97.0%. In particular, 16S rRNA gene phylogeny shows that DBT1 and B. cepacia (94.9% similarity) are not related species.

Figure 3

Neighbour-joining phylogenetic tree based on the sequence of the 16S rRNA gene of Burkholderia sp. DBT1 and related strains. Bootstrap values are given at branch nodes and are based on 1000 replicates. The scale bar indicates 0.005 substitutions per nucleotide position.

Although the analysis of the 16S rRNA gene sequence represents a basic step in the taxonomic characterization of bacterial genera (Vandamme et al., 1996), often, it is not adequate to solve uncertainties in comparisons of closely related species (Ash et al., 1991; Fox et al., 1992). In the present study, an 869-bp portion of the recA gene sequence from Burkholderia sp. DBT1 was amplified by PCR and sequenced. Related recA sequences were aligned and a phylogenetic tree was constructed (Fig. 4). The similarity of recA gene sequences between strain DBT1 and single B. fungorum strains ranged from 99.4% to 99.1%. On the other hand, the similarity for the same sequence to B. phytofirmans LMG 22487T, B. xenovorans LMG 21463T, B. caledonica LMG 19076T and B. graminis LMG 18924T declined to 95.5%, 93.9%, 92.0% and 91.4%, respectively.

Figure 4

Neighbour-joining phylogenetic tree based on the sequence of the recA gene of Burkholderia sp. DBT1 and related strains. Bootstrap values are given at branch nodes and are based on 1000 replicates. The scale bar indicates 0.02 substitutions per nucleotide position. Pseudomonas putida F1 was used as the outgroup.

In the last few years, species-specific primers, namely FunF and FunR, have been designed for recA-based PCR assays targeted for B. fungorum (Chan et al., 2003). These primers were used to assign Burkholderia sp. DBT1 incontrovertibly to the B. fungorum species. PCR assays carried out with genomic DNA obtained from B. cepacia LMG 1222T, B. caledonica LMG 19076T and B. graminis LMG 18924T were used as negative controls, and the test carried out with DNA from B. fungorum LMG 16225T was taken as a positive control. An amplicon of 330bp was obtained through PCR analysis of DNAs from either B. fungorum LMG 16225T or strain DBT1. Afterwards, the amplicons were purified and sequenced to confirm the identity of the fragments with the correct sequence of the recA gene. No amplification products were generated with DNA from the other Burkholderia strains tested (Fig. 5).

Figure 5

recA PCR-amplification assay using species-specific primers for Burkholderia fungorum. Lanes from left to right contain a 100bp molecular marker (BioLabs) and, respectively, PCR amplicons obtained using DNA templates from B. fungorum LMG 16225T, Burkholderia sp. DBT1, Burkholderia graminis LMG 18924T, Burkholderia caledonica LMG 19076T, Burkholderia cepacia LMG 1222T and negative control (PCR carried out without a template).

Moreover, a 432-bp portion of the gyrB gene was amplified by PCR starting from the genomic DNAs of B. cepacia LMG 1222T, B. fungorum LMG 16225T and Burkholderia DBT1. The amplicons were then cloned and sequenced. In this case, the degree of similarity of DBT1 to LMG 16225T and LMG 1222T was 98.2% and 86.5%, respectively. The gyrB sequence of DBT1 was compared through the available DNA sequence databases using the blast interface (NCBI). The following similarities were found: 94.0% to B. xenovorans LMG 21463T (GenBank accession no. CP000270), 93.7% to B. phytofirmans LMG 22487T (GenBank accession no. CP001052) and 91.1% to B. graminis LMG 18924T (GenBank accession no. EU024212).

Strain DBT1, within the phylogenetic trees based on the comparison of both 16S rRNA and recA gene sequences, forms a well-substantiated clade with B. fungorum strains. Moreover, gyrB gene sequence similarity scoring also indicates that DBT1 closely fits strains of the species B. fungorum, although databases are poor in bacterial gyrB sequence information.

DNA–DNA hybridization

Clusters of bacteria sharing almost identical 16S rRNA gene sequences have sometimes been identified. However, their DNAs hybridize at significantly lower than 70%. In these cases, the microorganisms represented distinct species (Fox et al., 1992; Tønjum et al., 1998). Therefore, to clarify conclusively the taxonomic affiliation of strain DBT1, DNA–DNA hybridization was performed against B. fungorum LMG 16225T. A complementation of 78.2±2.9% demonstrated that Burkholderia DBT1 belongs to the species B. fungorum according to the definition of bacterial species by Wayne et al. (1987).

Eventually, DNA–DNA hybridization confirmed the affiliation of strain DBT1 to the B. fungorum species. Thus, on the basis of these evidences, Burkholderia DBT1 can be ascribed to B. fungorum, excluding any relationship with Bcc representatives.


Burkholderia DBT1, a bacterial strain isolated from oil refinery drainage, has been shown to be capable of degrading DBT in liquid culture oxidatively, through the Kodama pathway, within 3 days of incubation (Di Gregorio et al., 2004). Because DBT behaves as a recalcitrant compound and tends to bioaccumulate throughout the food chains, the isolation and characterization of bacterial strains able to degrade condensed thiophenes, using them as the sole source of carbon and energy, can result in applications in bioremediation protocols. Nevertheless, for the harmless exploitation of Burkholderia DBT1 in environmental biotechnology, a probative exclusion of this strain from the B. cepacia complex is a prerequisite.

The versatile metabolism of DBT1 towards PAHs such as naphthalene, phenanthrene and fluorene shown in the present study is an encouraging trait for the possible use of this strain in the clean-up of contaminated sites. Moreover, the taxonomic details gained in this study attribute the strain DBT1 to the species fungorum, excluding any possible association of this isolate to the Bcc.


The authors thank the Academy of Finland (grant no. 118637) for support.


  • Editor: Bernard Glick


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