Degradation of Polycyclic Aromatic Hydrocarbons by a Newly Discovered Enteric Bacterium, Leclercia adecarboxylata

A bacterial strain, PS4040, capable of degrading polycyclicaromatic hydrocarbons for use as the sole carbon source wasisolated from oily-sludge-contaminated soil. The 16S rRNA geneshowed 98.8% homology to that of Leclercia adecarboxylata. Comparativemolecular typing with the clinical strain of L. adecarboxylatarevealed that there were few comigrating and few distinct amplimersamong them.

INTRODUCTION

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Introduction

Metabolic diversity of bacterial flora is a well-establishedphenomenon, and a consequence of this diversity is the degradationof various biohazardous or persistent anthropogenic compoundsby microbial activities. Among these recalcitrant compounds,polycyclic aromatic hydrocarbons (PAHs) represent a unique classof petroleum hydrocarbons due to their pyrogenic nature andthe complexity of the assemblages in which they occur (9). Althoughmany genera of microorganisms have the ability to degrade theserecalcitrant compounds and use them as a source of carbon orenergy, such phenomena are not commonly encountered in entericbacteria (3).

The enteric bacteria in the family Enterobacteriaceae are mainlyregarded as inhabitants of animal guts (3). The ability of thisgroup to degrade high-molecular-weight PAH compounds appearsto be an unusual feature, as this phenomenon has been associatedwith typical soil bacteria. However, very few reports have indicatedutilization of aromatic compounds by enterobacteria, particularlythose of the genera Klebsiella, Enterobacter, Escherichia, andHafnia (3, 5, 7). Although there are several reports of bioremediationof high-molecular-weight PAHs, research pertaining to biodegradationof these substances by enteric bacteria has been relativelyrare (3, 9).

In this article we report the isolation of an enteric bacterialstrain, PS4040, which can degrade the high-molecular-weight,four-benzene-ring PAH pyrene for use as a sole source of carbon.Phenotypic profiling and sequence analysis identified the strainas Leclercia adecarboxylata. We report for the first time thedegradation of PAHs by L. adecarboxylata.

Source, enrichment, and isolation of bacteria.

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Source, enrichment, and...

PAH-degrading bacterial strains were isolated from subsurfacesoil collected from an oily-sludge storage pit at the Digboioil refinery, situated in the northeastern region of India (27°15'N,98°15'E). The site has a history of petroleum hydrocarboncontamination of over 100 years. A defined mineral salt medium(MSM) (11) was used for isolation and enrichment of PAH-degradingbacteria. An oily-sludge-contaminated soil sample (10 g) wasused to inoculate 200 ml of MSM containing pyrene (200 mg/liter),and the culture was incubated at 30°C on a rotary shaker(200 rpm) for 7 days. After 10 such cycles of enrichment, 1ml of the culture was diluted 10⁸-fold and the diluted culturewas plated on MSM agar containing pyrene (200 mg/liter). Thenine bacterial colonies that gave a zone of clearance on pyrene-coatedMSM agar plates were further purified on MSM agar with pyreneas the carbon and energy source.

Sequencing of 16S rRNA gene and phylogenetic analysis.

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Sequencing of 16s rrna...

Sequencing of the full-length 16S rRNA genes of the nine PAH-degradingbacterial isolates was done as described previously (2). Alignmentof the 16S rRNA gene sequences of these isolates with sequencesobtained by doing a BLAST search of the National Center forBiotechnology Information (NCBI), Ribosomal Database Project(RDP), and Microseq (Microseq Analysis software version 1.40,Microseq 16S rRNA Sequence Database version 1.01; PE AppliedBiosystems, Inc.) databases revealed up to 98 or 99% similarityto different bacterial species. The highest degree of identitywas shown by strain PS4040; it was revealed to be 98.8% identicalto L. adecarboxylata in a BLAST search. The 16S rRNA gene sequenceof isolate PS4040 differs by only 1.2% from the most closelyrelated sequence, that of L. adecarboxylata, when examined withthe RDP SIMILARITY_RANK program (12). A phylogenetic tree, constructedby using the TREEVIEW program (15), illustrates the phylogeneticrelatedness of strain PS4040 to the selected Enterobacteriaceaestrains obtained from GenBank (NCBI) and RDP (Fig. 1). For furthervalidation of the tree, the sequences were aligned using CLUSTALW(19)and the alignments were analyzed using PAUP, version 3.0 (16),and neighbor-joining (SEQBOOT) programs obtained from the PHYLIPpackage (4). The results supported the positioning of strainPS4040 in the phylogenetic tree over the alternative positionsin a majority of the bootstrap resamplings, indicating a distinctlineage within the Enterobacteriaceae.

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FIG. 1. 16S rRNA gene phylogenetic analysis of PS4040 and other members of the family Enterobacteriaceae. The tree was constructed by the TREEVIEW program after an initial analysis of the sequences by using the PAUP program and SEQBOOT software.

Degradation of PAH compounds by strain PS4040 under aerobic conditions.

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Degradation of pah compounds...

Growth of the selected bacterial strain PS4040 was verifiedby demonstrating an increase in bacterial cell protein concentrationconcomitant with a decrease in the PAH concentration. StrainPS4040 was grown in batch cultures in 500-ml flasks containing200 ml of MSM supplemented with pyrene (200 mg/liter) as thesole carbon source. The flasks were inoculated with 5% (vol/vol)inoculum (10⁸ CFU/ml) and were incubated at 30°C in thedark on a rotary shaker (200 rpm) for 20 days. At specific intervals,one flask was taken out and 1 ml of culture was withdrawn forprotein estimation by the protocol described by Itzaaki andGill (8). The residual pyrene from the same culture flask wasextracted first with an equal volume of toluene and then twicewith an equal volume of chloroform. Evaporation of solventswas done under a gentle nitrogen stream in a fume hood. Theresidual pyrene was dissolved in 5 ml of acetone, and 1 µlof the resultant solution was analyzed by gas chromatography(GC; Hewlett-Packard 5890 series II gas chromatograph fittedwith flame ionization detectors and a 30-m-long DB 5.625 column[internal diameter, 0.25 µm; film thickness, 0.25 µm]).

The concentration of pyrene in the culture decreased from 200to 77 mg/liter, indicating 61.5% degradation of pyrene by strainPS4040, in 20 days (Fig. 2). The bacterial cell protein concentrationincreased from 0.4 µg/ml at the time of inoculation toa maximum of 10.3 µg/ml at day 15. There was no disappearanceof pyrene in the uninoculated control flasks (Fig. 2). StrainPS4040 was also examined for its ability to degrade naphthalene,catechol, anthracene, fluorene, and fluoranthene when used separatelyas a sole source of carbon under conditions similar to thosedescribed above. GC analysis revealed 73.2, 53.1, 40.6, and47.6% degradation of catechol, naphthalene, fluorene, and fluoranthene,respectively, by strain PS4040 in 20 days, while anthracenedid not support growth (data not shown).

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FIG. 2. Degradation of pyrene by L. adecarboxylata PS4040 grown in MSM with pyrene as the sole carbon and energy source. Growth was monitored by estimation of bacterial cell protein (•), while pyrene concentration ( ${diamondsuit}$ ) was determined by GC analysis of the residual pyrene in culture broth. The pyrene concentration in uninoculated flasks ( ${blacktriangleup}$ ) is also shown.

Extensive research pertaining to the degradation of PAHs hasresulted in documentation of a diverse microflora that can eithermineralize PAHs or use them as a sole source of carbon and energy(6, 9, 22). The ability of an enteric bacterium to degrade high-molecular-weightPAH compounds appears to be an uncommon finding, as there havebeen no reports indicating the ability of any enteric bacterialstrain to degrade high-molecular-weight PAHs such as pyrene.

L. adecarboxylata strains have been reported to cause a fewpathogenic manifestations in humans (1, 17, 18, 24) and to havemercury resistance genes (13), but there have been no reportsindicating that this bacterium is capable of degrading PAHs.As discussed by Diaz et al. (3), upon analyzing the ecologyof enteric bacteria it can be seen that an enteric bacteriummay easily encounter aromatic compounds. Although PAH compoundsmay not be expected to be normal substrates for enteric bacteria,there is evidence that competition for nutrients results inselection of substrates and shifts in nutrient preference (20).Manonmani et al. (14) also reported a role for substrate concentrationin adaptation of, and acquisition of degradative ability by,bacterial flora. Since strain PS4040 was isolated from a sitewith a contamination history of over 100 years, organic pollutantscan be postulated to have exerted effects leading to adaptationof this strain to utilize specific recalcitrant compounds assources of carbon or energy.

Comparison of strain PS4040 with a clinical strain of L. adecarboxylata by phenotypic tests and molecular typing.

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Comparison of strain ps4040...

Isolate PS4040 is a short, rod-shaped, motile, gram-negativebacterium. It produces yellowish, circular (2-mm-diameter),convex colonies that have an odor similar to that of Escherichiacoli when grown on Luria-Bertani (LB) agar plates for 18 h.The substrate utilization pattern generated on Biolog GN2 plates(10) (Biolog Inc., Hayward, Calif.) identified strain PS4040as L. adecarboxylata. Biolog tests were done for a clinicalstrain of L. adecarboxylata (a kind gift from Mario Vaneechoutte,Ghent University Hospital, Ghent, Belgium). Resistance to antibioticswas determined by growing the isolate in LB broth containingdifferent concentration of antibiotics (5 to 40 µg/ml).A comparative representation of the substrate utilization patternand antibiotic profile is shown in Table 1.

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TABLE 1. Phenotypic comparison and antibiotic profiles of strain PS4040 and the clinical strain of L. adecarboxylata

Molecular typing of strain PS4040 was done so that a whole-genomecomparison with the clinical strain of L. adecarboxylata couldbe performed using tRNA intergenic spacer length polymorphism(ILP) analysis, as described by Welsh and McClelland (23), andrepetitive-element PCR (Rep-PCR)-based DNA fingerprinting withprimer sets for repetitive-element (enterobacterial repetitiveintergenic consensus, repetitive extragenic palindromic, andBOXAIR) sequences as described elsewhere (2, 23). Rep-PCR andtRNA ILP have been extensively used for strain-level differentiation(1, 2, 21). PCR-based DNA fingerprinting by tRNA ILP and Rep-PCRyielded unique fingerprint patterns for strain PS4040 and theclinical strain of L. adecarboxylata. tRNA ILP resulted in ninedistinct bands for strain PS4040, whereas there were six distinctamplimers for the clinical strain. When comparative tRNA ILPanalysis of strain PS4040 and the clinical strain of L. adecarboxylatawas performed, four of the amplimers comigrated while therewas nine distinct amplimers in both strains. ERIC-PCR showedfour amplimers for strain PS4040 and seven amplimers for theclinical strain of L. adecarboxylata. Comparison of the bandingpatterns revealed that two amplimers comigrated while eightunique amplimers resulted for both strains. Similarly, REP-PCRand BOXAIR-PCR respectively generated eight and six amplimersfor strain PS4040 but nine and three amplimers for the clinicalstrain. Comparative analysis of REP-PCR and BOXAIR-PCR bandingpatterns showed four and two common amplimers for the clinicalstrain and three and six distinct amplimers for PS4040, respectively.Thus, banding patterns with few comigrating amplimers and fewdistinct amplimers were observed for both strains. This indicateda specific strain-level genomic difference between these twostrains, as discussed previously in several reports (2, 21).

In summary, an enteric bacterium, L. adecarboxylata, with therare ability to degrade PAHs as a sole source of carbon andenergy was isolated, identified, and characterized. It is genotypicallydifferent from a clinical strain of L. adecarboxylata. Althoughisolated from a pyrene-enriched culture, it can degrade othertwo- and three-benzene-ring PAH compounds. This study providesnew insights into the degradation of PAH compounds by entericbacteria and demonstrates a need for further biochemical andgenetic studies of PAH metabolism and transformation

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The 16S rRNA gene sequence of strain PS4040 has been depositedin the GenBank database under accession number AY451327.

ACKNOWLEDGMENTS

We are thankful to R. K. Pachauri, Director General, TERI, andto T. P. Singh, Director, TERI School of Advanced Studies, forproviding the infrastructure for carrying out the present study.We express our gratitude to Mario Vaneechoette, Ghent UniversityHospital, Ghent, Belgium, for providing clinical isolates ofL. adecarboxylata. Thanks are due to Neeti Chauhan and TheoSmiths (EPFL, Lausanne, Switzerland) for their contributionsto the phylogenetic analysis.

We also thank the Department of Biotechnology, Government ofIndia, for funding the research.

FOOTNOTES

* Corresponding author. Mailing address: The Energy and Resources Institute, Habitat Place, Lodhi Rd., New Delhi, India 110003. Phone: 91-011-24682100. Fax: 91-011-24682144. E-mail: banwaril{at}teri.res.in.

REFERENCES

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