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Applied and Environmental Microbiology, January 2003, p. 275-284, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.275-284.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils

Mikael Eriksson,1,2 Erik Sodersten,1,2 Zhongtang Yu,1 Gunnel Dalhammar,2 and William W. Mohn1*

Departmentof Microbiology and Immunology, University of British Columbia,Vancouver, British Columbia V6T 1Z3,Canada,1 Department of Biotechnology,Royal Institute of Technology, KTH, SE-100 44 Stockholm,Sweden2

Received 1 July 2002/ Accepted 8 October 2002


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ABSTRACT
 
Thepotential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not wellunderstood, but such biodegradation would be very useful forremediation of polluted sites. Biodegradation of a mixture of 11different PAHs with two to five aromatic rings, each at a concentrationof 10 µg/ml, was studied in enrichment cultures inoculated withsamples of four northern soils. Under aerobic conditions, lowtemperature severely limited PAH biodegradation. After 90 days, aerobiccultures at 20°C removed 52 to 88% of the PAHs. The mostextensive PAH degradation under aerobic conditions at 7°C,53% removal, occurred in a culture from creosote-contaminatedsoil. Low temperature did not substantially limit PAH biodegradationunder nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene weredegraded. The most extensive PAH degradation under nitrate-reducingconditions at 7°C, 39% removal, occurred in a culturefrom fuel-contaminated Arctic soil. In separate transfer cultures fromthe above Arctic soil, incubated anaerobically at 7°C, removalof 2-methylnaphthalene and fluorene was stoichiometrically coupled tonitrate removal. Ribosomal intergenic spacer analysis suggested thatenrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, andVariovorax. Predominant populations from different soils oftenincluded phylotypes with nearly identical partial 16S rRNA genesequences (i.e., same genus) but never included phylotypes withidentical ribosomal intergenic spacers (i.e., different species orsubspecies). The composition of the enriched communities appeared to bemore affected by presence of oxygen, than by temperature or source oftheinoculum.


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INTRODUCTION
 
Biodegradation of polycyclic aromatic hydrocarbons (PAHs) is a possibleway to clean up polluted soils and water systems(4,5). Biological treatmentsare cheaper than alternatives such as incineration, storage, or soilwashing (12). PAHs areoften found in oil spills and in soil at old gasworks sites and sitesformerly used for wood preservation (creosote spills). Some PAHs arepotential carcinogenic and mutagenic substances and are therefore onthe pollutant priority lists of most countries’ environmentalprotection agencies. Degradation of PAHs in situ is often slow, andresearch over the last decades has shown that these compounds veryoften are persistent (4,9,25). This persistence maybe due to several factors such as nutrients, bioavailability of PAHs(sorption to particles), temperature, oxygen, and presence ofPAH-degrading microorganisms. The water solubilities of most PAHs arein the lower parts-per-million range, and this is a major problem whenstudying and implementing aerobic degradation of PAHs. The use ofsurfactants may increase PAH solubility but may also be toxic tomicroorganisms (13,43). In some Arctic andtemperate regions, soil temperature remains below 10°Cyear-round, and wet conditions limit oxygen availability. The cost ofincreasing the temperature may be prohibitive, so it is desirable tooptimize a treatment system for low temperature. The cost of aerationmay also be prohibitive, and it may be more practical and economical toadd nitrate, which is very water-soluble, as an electron acceptor.Bioagumentation with PAH-degrading bacteria and fungi has been triedwith both successes and failures(4,23), and it is still notclear why inoculation sometimes fails.

Despite the potentialapplications, very little is known about low-temperature degradation ofPAHs (26), and even lessis known about anaerobic degradation at low temperatures. There arereports of low-temperature degradation of jet fuel hydrocarbons andstraight-chain aliphatic compounds by psychrotolerant organisms(47) and by polar soilcommunities (2,6,31,32). There are only a fewreports of growth on PAHs or PAH biodegradation at low temperature(1,27,41,45,46). Reports concerninganaerobic degradation of PAHs under sulfate-reducing(11,29,37,40) and nitrate-reducingconditions (3,20,28,30,36,37,40) exist, but theseprocesses were studied at temperatures between 20 and30°C.

The purpose of this study was to evaluate thepossibility of obtaining enrichment cultures capable of efficient PAHdegradation at low temperature under aerobic or anaerobic conditions.Four different northern soils were used to enrich for mixed communitiesof PAH degraders at 7 and 20°C under both aerobic and anaerobic(nitrate-reducing) conditions. A mixture of 11 PAHs was used. Complexmixtures of PAHs typically occur at polluted sites and may permitcometabolic PAH degradation. The populations enriched in the cultureswere characterized and compared by analysis of ribosomal intergenicspacers and 16S rRNA genesequences.


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MATERIALS ANDMETHODS
 
Soils.
Four different soils were collectedfrom two Arctic sites and two other northern sites to inoculateenrichment cultures. Alert soil was from the Canadian Forces StationAlert, on Ellesmere Island, Nunavut, Canada (82°30'N,62°19'W). Alert soil was contaminated with Arcticdiesel fuel at a concentration of ~2,000 mg/kg of soil(42). Saglek soil wasfrom a radar installation at Saglek, Labrador, Canada(58°30'N, 63°0'W). Saglek soil wascontaminated with PCBs (>50 mg/kg) and associated oil(32). Värta soilwas from the former gasworks site, Värtagasverket, Husarviken,Stockholm, Sweden (59°20'N, 18°3'E).Värta soil was contaminated with creosote-PAH (~300mg/kg) (15). Wesbrooksoil was from near the Wesbrook Building, University of BritishColumbia, Vancouver, Canada (49°16'N,123°7'W). Wesbrook soil was not contaminated with PAHsand is not known to be contaminated by other pollutants. All soils weresandy with low organic content and had a similar texture and particlesize (<4 mm), a water content of approximately 10 to15%, and a pH between 7 and 8. The pollutants in thecontaminated soils were present in the soils for periods of years, butthe exact histories of the soils areunknown.

Chemicals.
The following chemicals were used(purities indicated in parentheses): naphthalene (99%),2-methylnapthtalene (97%), 1,4-dimethylnaphthalene (95%),fluorene (99%), phenanthrene (99%),9,10-dimethylanthracene (99%), fluoranthene (98%), pyrene(99%), 1,2-benzanthracene (99%), chrysene (98%),benzo[a]pyrene (98%), anthraquinone(97%), phenanthrenequinone (99%),phenanthrene-9-carboxaldehyde (97%), 9-anthracenecarboxylic acid(99%), 2-naphthyl acetic acid (99%), 2-methylanthracene(98%), 2-methylphenanthrene (95%), sulfanilamide(99%), zinc dust (<10 µm; 98%), andN-(1-naphthyl)ethylenediamine dihydrochloride (98%)(all from Aldrich Chemical Co.). Methylene chloride and ethyl acetate(both high-performance liquid chromatography grade) were from FisherScientific.

Enrichmentcultures.
Aerobic cultureswere grown in Bushnell-Haas mineral medium (Difco).Anaerobic cultures were grown in Bushnell-Haas medium plus nitrate asan electron acceptor (0.75 g/liter of KNO3).Primary enrichment cultures were prepared in 240-ml dark bottles withTeflon-septum-sealed screw caps (Supelco) by adding 2.0 g ofsoil to 20 ml of mineral medium containing a mixture of 11 differentPAHs as the only organic substrates. The 11 PAHs were naphthalene,2-methylnapthtalene, 1,4-dimethylnaphthalene, fluorene, phenanthrene,9,10-dimethylanthracene, fluoranthene, pyrene, 1,2-benzanthracene,chrysene, and benzo[a]pyrene. The PAHs were added atfinal concentrations of 100 to 500 mg/liter in pure crystal or liquidform to avoid solvents as potential substrates. Anaerobic cultures werepurged with sterile filtered (pore size, 0.4 µm) nitrogen gasfor 5 min. The enrichment cultures were incubated at 7 or 20°Con shakers at 150 rpm in darkness. Thus, there were four incubationconditions, aerobic and anaerobic cultures, each at 7 and 20°C.After 45 days, secondary cultures were established by transferring 200µl from each primary culture to fresh, homologous medium. Afteran additional 45 days, tertiary cultures were established and monitoredfor PAH degradation as described below.

The tertiary enrichmentcultures were established in the same way as the secondary cultures,except 10.0 ml of medium was used. These cultures had initial celldensities of 5 x 106 to 2 x 107cells/ml, estimated by total count under a light microscope. Sterilecontrols were prepared as described above but without inoculum and with3.0 mg/ml of NaN3 added. Sufficient replicate cultures wereestablished to permit whole cultures to be extracted for PAH analysisto avoid potential loss of PAHs adsorbed to the bottles. Singlecultures were analyzed on days 0, 15, and 60; duplicate cultures, onday 30; and triplicate cultures, on day 90. Additional replicatecultures of both 7°C treatments were established to analyzemetabolites by solid-phase microextraction (SPME) (see below). For eachtime point, there were two additional replicate cultures of thesetreatments.

Anaerobic nitrateconsumption at 7°C.
PAH degradation undernitrate-reducing conditions was verified in the anaerobic, 7°C,Alert soil enrichment culture. These quaternary cultures wereestablished in the same way as the tertiary cultures, except theindividual PAHs, 2-methylnaphthalene or fluorene (in methylenechloride), were each added to duplicate cultures to a finalconcentration of 40 µg/ml. Bottles were incubated as describedabove at 7°C for 40 days. Then, nitrate, nitrite, and remainingPAHs were analyzed. Sterile controls were medium withoutinoculum.

Ethyl acetateextraction.
Whole cultures(10 ml) were acidified with 1.0 ml of 3 M H2SO4and extracted with 4.0 ml of ethyl acetate in the culture bottles byshaking for 24 h at 22°C. Extracts were dried overanhydrous sodium sulfate before analysis by gas chromatography-flameionization detection (GC-FID) and GC-mass spectrometry (GC-MS). Aninternal standard of 2-methylanthracene in methylene chloride was addedto all samples before analysis, to a final concentration of 5.0µg/ml.

Analysis of metabolitesby SPME.
Samples of 2.0 mlwere removed from cultures with a sterile syringe (flushed withnitrogen gas) and placed in 5-ml vials with 0.20 ml of 3 MH2SO4. These samples were frozen at-20°C until analysis was done. A manual SPME85-µm polyacrylate fiber (Supelco) was immersed in each vialfor 10 min with stirring at room temperature. Before injection, thefibers were held in deionized water for 10 s to remove saltfrom the medium and blotted on a tissue paper to remove the waterdroplet remaining from washing. The fiber was then injected immediatelyin the GC-MS for desorption and analysis. The fibers were routinelymonitored for degradation and possible carryover of analytes to othersamples by injecting blank runs between the samples.2-methylphenanthrene in methylene chloride was added to the 2.0-mlsamples as an internal standard before analysis, to a finalconcentration of 0.50µg/ml.

GC-FID.
A Hewlett-Packard GC 5890 series IIwas used with an FID and a Hewlett-Packard HP-5 column (length,25 m; inner diameter [i.d.], 0.32 mm; filmthickness, 0.17 µm). The carrier gas was H2 at apressure of 7.5 lb/in2 and a flow rate of 1.8 ml/min. Thetemperature program was as follows: 40°C for 3 min,30°C/min to 300°C, hold for 10 min. The injector was290°C, and the detector was 300°C. Samples of 2.0µl were injected in splitless mode for 1 min. Analyticalstandards of PAHs and their metabolites were prepared in methylenechloride, at a concentration of 5.0 mg/ml for each compound. Standarddeviations for replicate samples (including variability in extractionand analysis) were from 2.4 to 7.6%, with the exceptions ofdibenzanthracene (9.3%), benzo[a]pyrene(12.5%), and 9,10-dimethylanthracene (20.7%). Differencesof less than 20% were not consideredsubstantial.

GC-MS.
A Varian 3400Cx gas chromatograph wasused with a Saturn 4D ion trap MS detector and a J&W ScientificDB5-MS column (length, 30 m; i.d., 0.25 mm; film thickness,0.25 µm). The carrier gas was helium at 10 lb/in2.The temperature program was as follows: 40°C for 5 min,10°C/min to 245°C, hold for 30 min. The injector, witha 0.8-mm-i.d. liner, was 240°C, and the transfer line was250°C. The ion trap was operated at 70 eV with a scan range ofm/z 90 to 400. Samples of 1.0 µl were injected insplitless mode for 30s.

Nitrate and nitriteanalysis.
Nitrate andnitrate were analyzed using the methods described in Methods forGeneral and Molecular Bacteriology(19). Nitrate is reducedto nitrite by zinc and the nitrite then reacts withN-(1-naphthyl)ethylenediamine to form a colored complex. Theamount of nitrite is analyzed in a spectrophotometer at 543 nm.Calibration was done by analyzing known amounts of sodium nitrite insterile water. Samples of 50 µl were withdrawn from the culturebottles for analysis.

Ribosomalintergenic spacer analysis.
DNA was extracted and purified aspreviously described(16). A previouslydescribed (48) compositemethod was used for ribosomal intergenic spacer analysis. Universalbacterial PCR primers were used to amplify ribosomal intergenic spacersplus approximately 500 bp of the 16S rRNA gene (RIS-rDNA). Ribosomalintergenic spacer length polymorphism (RIS-LP) was analyzed byelectrophoresis of the RIS-rDNA amplicons. The samples were analyzedtwice, and the replicates yielded nearly identical fingerprints (notshown). Gelcompar II (version 2.5; Applied Maths) was used to analyzethe RIS-LP fingerprints. The similarity of entire fingerprints wasdetermined by the Pearson correlation method (2.00%). Similaritydendrograms were constructed by the unweighted-pair group method usingarithmetic averages. For selected samples, clone libraries of theRIS-rDNA amplicons were prepared. From each library, 20 clones wereanalyzed for restriction fragment length polymorphism (RIS-RFLP). Forclones representing selected RIS-RFLP phylotypes, the rDNA fragment wassequenced.

Nucleotide sequence accessionnumbers.
The partial rDNAsequences determined were deposited in the GenBank under the accessionnumbersAF532132toAF532137andAY136514toAY136545.


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RESULTS
 
PAHremoval.
Biologicalremoval of PAHs occurred under all experimental conditions in tertiaryenrichment cultures (Fig.1; Table 1). Predictably, thegreatest PAH removal consistently occurred in aerobic, 20°Ctreatments, with the highest total PAH removal being 88%. Naphthalene and2-methylnaphthalene were completely removed in all cultures, andtemperature had little effect on their removal rates (not shown). Withnotable exceptions, reducing the temperature to 7°C reducedrates and extents of removal of the other PAHs. Aerobic degradation of1,4-dimethylnaphthalene was particularly affected by the lowertemperature, being eliminated in cultures of three soils. In theaerobic cultures inoculated with Värta soil, the removal rate for1,4-dimethylnaphthalene was reduced at the lower temperature much moredramatically than were the rates for other PAHs (notshown).



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  		FIG. 1. TotalPAH degradation in cultures inoculated with soils from Alert (A),Saglek (B), Värta (C), and Wesbrook (D). Symbols: {triangleup},aerobic conditions, 7°C; {blacktriangleup}, nitrate-reducingconditions, 7°C; {square}, aerobic conditions, 20°C;{blacksquare}, nitrate-reducing conditions, 20°C; x, killedcontrol.


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  		TABLE 1. Percentremoval of PAHs from enrichment cultures during 90-dayincubations

Anaerobic conditions limited PAH removal even moredrastically than lowering temperature, particularly for PAHs with threeor more aromatic rings (Fig.1; Table1). One exception was the7°C cultures inoculated with Alert soil, in which fluorene andphenanthrene were degraded under anaerobic, but not under aerobic,conditions. In anaerobic cultures inoculated with all soils, theextents of removal of all PAHs were very similar at the low and hightemperatures.

The different soil inocula substantially affectedremoval of total PAHs in the enrichment cultures (Fig.1; Table1). Soils from Värtaand Wesbrook yielded cultures that were relatively active in aerobic,7°C treatments, with final removals of 53 and 45%,respectively. The soil from Alert yielded a culture that was relativelyactive under anaerobic conditions and actually removed more PAHs at7°C than at 20°C (39 and 31% removal,respectively).

PAH metabolites at7°C.
The metabolitesdetected during PAH degradation at 7°C, under both aerobic andanaerobic conditions, were similar in the various enrichment cultures,despite the differences in PAH removal kinetics (not shown). Majormetabolites detected in most or all cultures included1,4-dimethylnaphthol, 9-fluorenone, fluorenol,naphthalenemethanol, phenanthrenecarboxaldehyde,methoxyphenanthrene, and anthraquinone (Fig.2). The last three of these were most abundant in the anaerobic cultures.In both aerobic and anaerobic cultures, fluorenone was detected incultures that degraded fluorene efficiently, whereas fluorenol wasdetected in cultures with little fluorene removal. Concentrations ofthe metabolites ranged from a few parts per billion (trace levels) toup to 2 ppm. Low concentrations of 4-hydroxy-9-fluorenone weredetected, mainly in the anaerobic cultures. Low concentrations ofphenanthrenol were detected under anaerobic conditions, but as shown byHo et al. (22),phenanthrenol could be a GC artifact (thermal decomposition) during theGC analysis of 10-hydroxy-1-phenanthroic acid obtained from pyrenedegradation. Most of the detected compounds had a maximum concentrationafter 15 days and then declined slowly over the remaining 75days.



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  		FIG. 2. Metabolitesdetected during degradation of PAHs at 7°C under aerobic andnitrate-reducingconditions.

Coupling of PAH and nitrate removalat 7°C.
Quaternaryenrichment cultures from Alert soil incubated anaerobically tested thecoupling of PAH degradation to denitrification at 7°C. Expectednitrate removal was calculated for oxidation of each PAH to carbondioxide plus water coupled to nitrate reduction to nitrogen gas.Nitrate was present in twice the amounts required for PAH oxidation.Nitrate removal was consistent with 2-methylnaphthalene and fluoreneremoval in the cultures (Table2). Trace amounts of nitrite were detected in the active samples after 40days of incubation. The presence of nitrite confirms nitrate-reducingactivity. The small amounts of nitrite accumulating suggest that thereduction process was denitrification (i.e., reduction to nitrous oxideor dinitrogen gases). These results strongly support the conclusionthat anaerobic 2-methylnaphthalene and fluorene oxidation was coupledto denitrification at7°C.


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  		TABLE 2. PAHand nitrate consumption in anaerobic transfer cultures (7°Cenrichment) inoculated with Alert soil during 40-dayincubations

RIS-LP.
The RIS-LP fingerprint of each tertiaryenrichment culture at the end of the 90-day incubation wascharacterized by a few RIS-rDNA bands (Fig.3). Frequently, fingerprints from cultures with different enrichmentconditions inoculated with the same soil had bands of common sizes. Inseveral cases, enrichment cultures inoculated with different soils alsohad bands of common sizes. Cluster analysis of fingerprint similarity(Fig.4) did not consistently indicate greatest similarity between fingerprintsfrom cultures with common inocula, nor between fingerprints fromcultures incubated at a common temperature. The most clear trend is acluster containing all of the fingerprints from anaerobic cultures,which seems to be based on the presence of two predominant bands and,in many cases, the absence of many other bands. Another clustercontains four of the aerobic cultures. The initial fingerprints fromsoils prior to their use as inocula were distinct. In two cases, theinitial fingerprints lacked predominant bands, and in two cases theinitial fingerprints had predominant bands which were not present infinal fingerprints from cultures inoculated with thosesoils.



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  		FIG. 3. RIS-LPbanding patterns from enrichment cultures after 90 days of incubation.Lane identifications: I, initial soil sample; O, aerobic; A, anaerobic;7, 7°C; 20,20°C.



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  		FIG. 4. Similarityanalysis of RIS-LP banding patterns. Sample identification: letterspreceding the hyphen indicate soil (A, Alert; S, Saglek; V,Värta; W, Wesbrook); letters following the hyphen are asexplained in the legend to Fig.3.

RIS-RFLP and rDNAsequences.
At the end of the90-day incubation, clone libraries of RIS-rDNA amplicons were preparedfrom all of the tertiary cultures inoculated with Alert soil and fromthe cultures inoculated with the other soils and incubated aerobicallyat 7°C. From each library, 20 clones were screened for RIS-RFLPpatterns. The libraries consistently had two to four predominantRIS-RFLP phylotypes, with the remainder being singletons (Table3). The sequence of the rDNA fragment in the RIS-rDNA amplicon wasdetermined for a representative of each predominant RIS-rDNA phylotypeplus some singletons. The rDNA phylotypes were affiliated with sixgenera among three subdivisions, all within the Proteobacteria(Fig.5). The predominant rDNA phylotypes were affiliated with the generaPseudomonas, Sphingomonas, Bordetella, andAcidovorax. In many cases, a library had two predominantRIS-RFLP phylotypes affiliated with the same genus. The RIS-rDNAlibraries were consistent with the RIS-LP analysis in that, for eachculture analyzed, the most abundant library clones had inserts thatcorresponded in size to major bands in the RIS-LP fingerprint (compareTable 3 to Fig.3). However, there werenot clone inserts corresponding to every visible band. Only twoRIS-RFLP phylotypes occurred in more than one library from the culturesinoculated with Alert soil. The RFLP patterns were identical forA-A20-2 and A-O7-11, as well as for A-A20-19 and A-O7-12. The rDNAsequences were identical for both phylotypes in each pair, and bothpairs were affiliated with the same genus, Pseudomonas. Therewere no RIS-RFLP phylotypes that occurred in libraries from culturesinoculated with differentsoils.


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  		TABLE 3. RIS-RFLPphylotypes found in libraries from selected enrichmentculturesa



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  		FIG. 5. Affiliationsof the partial 16S rDNA sequences (Escherichia coli positions910 to 1360) from cloned RIS-rDNA amplicons. Reference strains are fromthe Ribosomal Database Project. Phylotype designations correspond tothose in Table 3. Solidcircles indicate branch points with >75% bootstrapvalues, and open circles indicate branch points with>50% bootstrap values. The scale bar corresponds to 0.1mutation per nucleotideposition.


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DISCUSSION
 
AerobicPAH degradation at low temperature.
The capacity of the cultures foraerobic PAH biodegradation at 7°C, relative to biodegradationat 20°C, depended very much on the soil inoculum used (Fig.1; Table1). These results suggestthat the abundance of psychrotolerant PAH degraders in the soils was afactor in the outcome of the enrichment cultures. All soils yieldedcultures that degraded naphthalene and 2-methylnaphthalene at7°C. The severe effect of low temperature on1,4-dimethylnaphthalene degradation was unexpected, as this is notconsidered a particularly recalcitrant PAH. Only Värta andWesbrook soils yielded cultures that at 7°C degraded PAHs withthree or more rings, including fluorene, phenanthrene, fluoranthene anddibenzanthracene. RISA suggests that predominant populations enrichedfrom the two soils belong to different genera. Of the four soils, onlythe Värta soil came from a site with extensive PAH contamination.The Värta and Wesbrook soils come from less cold regions than dothe Alert and Saglek soils; however, the regions of the former twosoils are cold (<10°C) for a substantial period eachyear. Removal of benzo[a]pyrene at 7°C wasanomalous, as it occurred to a similar extent (31 to 37%removal) in treatments inoculated with all inocula. This removal mayrepresent partial degradation, possibly cometabolic, although nobenzo[a]pyrene metabolite was detected. This removaldoes not appear to be abiological, since it did not occur in theuninoculated controls, nor did it occur in all of the anaerobictreatments at 7°C. This and other studies(22,34) suggest thatmesophilic PAH degraders are relatively ubiquitous in soils. However,this study suggests that psychrotolerant PAH degraders may be lessubiquitous or may require a very long time to enrich. Thus, the sourceof organisms may be critical for PAH biodegradation applications at lowtemperature.

Temperature may also affect PAH biodegradation viaits affect on the bioavailability of PAHs. This explanation isconsistent with the fact that low temperature mainly affectedbiodegradation of less-soluble, larger PAHs, having three or morearomatic rings (Table 1).However, low temperature inhibited degradation of individual PAHs tovery different extents under aerobic and anaerobic conditions, which isnot consistent with an effect on bioavailability. The balance of theevidence indicates that low temperature primarily limits PAHbiodegradation via an effect on biologicalactivity.

Accumulation of PAH metabolitesat 7°C.
Themetabolites detected during the low-temperature PAH degradation, bothaerobic and anaerobic, confirm biological transformation of the PAHs.The metabolites provided no evidence for substantially differentdegradation processes associated with different soil inocula.Naphthalenemethanol indicates oxidation of the methyl group ofmethyl-naphthalene under both aerobic and anaerobic conditions.Fluorene was reported to be transformed by an Arthrobacter sp.to 9-fluorenone and then 4-hydroxy-9-fluorenone as a dead-endmetabolite (7).Fluoranthene was also proposed to be transformed to 9-fluorenone(39). Thus, the9-fluorenone detected in this study may have come from either or bothfluorene and fluoranthene. Since some of the metabolites remained inthe system for a long period of time (90 days) they should beconsidered as possible inhibitors of growth and PAH degradation.9-Fluorenone, for example, was shown to be inhibitory todenitrification at concentrations of 10 ppm and higher for purecultures of Pseudomonas strains(14). Very little isknown about other inhibitory or possible stimulatory effects thatmetabolites may have on PAH degradation.

Metabolites detected inthe anaerobic cultures indicate transformations, which were notobserved under aerobic conditions. These transformations may be part ofthe primary pathways for anaerobic degradation of the PAHs or mayaccount for transformation of only a small fraction of the substrates.Phenanthrenecarboxaldehyde has not previously been reported as ananaerobic metabolite of phenanthrene, but carboxylation has beenreported as the initial step in PAH degradation under sulfate-reducingconditions (49). Sinceanthracene was not provided to the cultures, the anthraquinone detectedprobably came from oxidation of the methyl groups of9,10-dimethylantracene. Similar reactions have been reported beforewhere anthraquinone was a metabolite from anthracene(4). Methoxylation of PAHshas been reported to be catalyzed by fungi(38) and cyanobacteria(35), which can explainthe detection of methoxyphenanthrene under anaerobicconditions.

PAH degradation coupled todenitrification at low temperature.
This study demonstrated for the firsttime anaerobic PAH degradation at a low temperature. This degradationwas limited to naphthalene, 2-methylnaphthalene, fluorene,phenanthrene, and perhaps, benzo[a]pyrene(Table 1). For2-methylnaphthalene and fluorene, degradation was shown to be coupledto denitrification on the basis of the stoichiometry of removal ofthese compounds and removal of nitrate (Table2). The Arctic soils, fromAlert and Saglek, showed the greatest potential for anaerobic PAHdegradation at low temperature, despite their relatively poor potentialfor aerobic PAH removal at low temperature (Fig.1; Table1). The capacity foranaerobic degradation may be related to anoxic conditions where thosesoils were collected. We do not know whether the particular sourceareas for the Arctic soils are frequently anoxic, but we have observedthat soils from these and other Arctic sites often are wet and drainpoorly, in part due to the effect of permafrost on water flow. TheVärta and Wesbrook soils came from well-drained areas. Ingeneral, most successful enrichment cultures and isolates that degradePAHs under anaerobic conditions were obtained from contaminatedsediments, and not from contaminated soils(11,36,40). A low abundance ofanaerobic PAH degraders in aerobic soils might be the reason for thelack of anaerobic PAH degradation in other experiments(40,44). This study and thatof Hayes et al. (21)indicate that anaerobic PAH degraders can be found in environmentswithout PAH contamination.

Organisms capable of degradinghydrocarbons at low temperature tend to be psychrotolerant, rather thanpsychrophilic (46).Consistent with this, hydrocarbons were degraded in Arctic soils atincreasing rates from 7 to 20°C(31). Therefore, it issurprising that the higher temperature did not stimulate anaerobic PAHremoval by the cultures inoculated with Alert and Saglek soils (Fig.1; Table1). This could be becauseanaerobic PAH removal at low temperature was catalyzed by psychrophilicorganisms. The RIS-LP fingerprints of the anaerobic Saglek soilcultures at low and high temperatures have different predominant bands(Fig. 3 and4), which is consistentwith enrichment of psychrophilic organisms at low temperature. On theother hand, the RIS-LP fingerprints of the anaerobic Alert soilcultures at low and high temperatures generally have the samepredominant bands, suggesting that the same psychrotolerant organismswere enriched at both temperatures. Our results indicate that increasedtemperature will not always stimulate PAH biodegradation in soils fromcold regions and that the reasons for this may bemultiple.

Populations enriched.
The RIS-LP fingerprints suggest that afew predominant populations were enriched in the cultures. We haveobtained more complex RIS-LP fingerprints from wastewater treatmentsystems (48) and muchmore complex fingerprints from soil (unpublished data). Comparison ofinitial and final RIS-LP banding patterns (Fig.3 and4), suggests that thepopulations enriched were not abundant prior to incubation. The mostintense RIS-LP bands likely represent predominant populations, but itis important to note that additional predominant populations may nothave been detected for reasons such as unequal DNA recovery fromdifferent organisms, failure of the primers to amplify certain RIS-rDNAsequences, variability in rrn copy number and PCRbias.

It is also important to realize that one population mayyield more than one RIS-LP band, as the multiple rrn operonsof a single organism can yield distinct RIS-rDNA amplicons of differentlength or RIS sequences. This is consistent with the fact that, in mostclone libraries, we found two predominant RIS-RFLP phylotypes withidentical or nearly identical rDNA sequences (Table3; Fig.5). Thus, for example, thetwo predominant phylotypes affiliated with the genusAcidovorax in the library from the aerobic, 7°Cculture inoculated with Wesbrook soil may represent two distinctpopulations or one population with at least two distinct RISs ofapproximately the same length but of different sequences, which yieldeddifferent RIS-RFLP patterns. We have previously obtained from one tothree RIS-rDNA amplicons of different sizes from individual purecultures (16). There islittle information available to suggest the extent to which singleorganisms have RISs of common sizes that yield distinct RFLPpatterns.

Phylotypes affiliated with only a few genera weredetected as predominant populations in the enrichment cultures (Table3). These phylotypesincluded probable members of Acidovorax, Pseudomonas,and Variovorax as well as phylotypes less closely affiliatedwith Bordetella and Sphingomonas (Fig.5). All of the phylotypesidentified by rDNA sequence analysis are members of the Proteobacteria.Previously (48), we havedetected in wastewater treatment systems members of theCytophagales, Fexistipes, and low-G+Cgram-positive bacteria, using the method and PCR primers used in thepresent study. Thus, it appears that Proteobacteria wereselectively enriched in the present study.

Our analyses suggestthat members of a few proteobacterial genera are widely distributed andshare characteristics that caused their enrichment in the aerobic,7°C cultures. Members of Acidovorax were predominantin those cultures inoculated with Alert, Saglek, and Wesbrook soils(Table 3). Members ofPseudomonas were predominant in the Alert culture and detectedin the Värta and Saglek cultures. Members of these genera fromthe different soils often yielded RIS-rDNA amplicons of identical ornearly identical sizes. For example, phylotypes affiliated withAcidovorax consistently yielded 1.5-kb amplicons, andphylotypes affiliated with Pseudomonas yielded both 1.1- and1.3-kb amplicons. Further, in several cases, the rDNA fragmentsequences (ca. 500 bp) were identical for phylotypes originating fromdifferent soils. This may indicate that such phylotypes represent acommon species. However, such a short 16S rDNA fragment cannotconclusively indicate a common species, particularly for the genusPseudomonas(33). In all cases, theRIS-RFLP phylotypes affiliated with a common genus, but derived fromdifferent soils, were distinct. The RIS size and sequence is known tovary among strains of the same species(17,24), so the RIS-RFLPanalysis probably resolves strains at the species or subspecies level.Thus, the soils from distant regions appear to harbor members of theabove genera that can occupy a common niche, but the species orsubspecies of these genera occupying this niche may be endemic. Todetermine whether this is the case, it would be necessary to assay thepresence of these phylotypes (as with a PCR assay) in environmentalsamples, rather than in enrichment cultures. The endemicity ofmicroorganisms in natural environments has not been well studied, butthere are a few reports suggesting endemicity of species or subspecies(8,10,18).

Similarly,members of the same genera were enriched from Alert soil under thedifferent incubation conditions. Members of Pseudomonas werepredominant in all cultures inoculated with Alert soil, except theaerobic, 20°C culture, in which a Pseudomonas strainwas detected, but Sphingomonas was predominant (Table3). Again, most RIS-RFLPphylotypes were found only in one enrichment culture. However, twoRIS-RFLP phylotypes affiliated with the genus Pseudomonas werefound in both the aerobic, 7°C cultures and the anaerobic,20°C cultures inoculated with Alert soil (A-O7-11 =A-A20-2 and A-O7-12 = A-A20-19). This suggests that the one ortwo species or subspecies represented by these phylotypes were welladapted to both culture conditions.


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ACKNOWLEDGMENTS
 
This work was supported bya Strategic Grant from the National Science and Engineering Council ofCanada.

We thank the Environmental Sciences Group of the RoyalMilitary College of Canada for providing Arctic soil samples. We thankSara Leckie for assistance in similarity analysis of RIS-LPfingerprints.


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FOOTNOTES
 
* Correspondingauthor. Mailing address: Department of Microbiology and Immunology,University of British Columbia, 300-6174 University Blvd., BritishColumbia V6T 1Z3, Canada. Phone: (604) 822-4285. Fax: (604) 822-6041.E-mail:wmohn{at}interchange.ubc.ca. Back


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Applied and Environmental Microbiology, January 2003, p. 275-284, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.275-284.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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