BIOTECHNOLOGY
LETTERS
Volume 18 No.5 (May 1996) p.577-582 Received as revised 21st March.
DEGRADATION OF HIGH MOLECULAR WEIGHT POLYCYCLIC AROMATIC HYDROCARBONS BY PSEUDOMONAS CEPACZA. Albert L. Juhaszl, Margaret L. Britzl and Grant A. Stanley*2 lCentre for Bioprocessingand Food Technology, 2Departmentof Food Technology, Victoria University of Technology, PO Box 14428MCMC, Melbourne, Australia, 8001. fax: (+613) 9216 8284; e-mail:
[email protected] *Corresponding Author SUMMARY
When inoculated at high cell densities, three strains of Pseudomonas cepaciu degraded the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene, dibenz[u,h]anthracene and coronene as sole carbon and energy sources. After 63 days incubation, there was a 20 to 30% decreasein the concentration of benzo[u]pyrene and dibenz[u,h]anthracene and a 65 to 70% decreasein coronene concentration. The three strains were also able to degrade all the PAHs simultaneously in a PAH substrate mixture containing three-, four-, five- and sevenbenzene ring compounds. Furthermore, improved degradation of the five- and seven-ring PAHs was observedwhen low molecular weight PAHs were present. INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous environmental pollutants which have been detected in numerous aquatic and terrestrial ecosystems. There is concern about the presenceof these compoundsin the environment as they have been shown to exhibit toxic, mutagenic and carcinogenic effects (Andrews et al., 1978; Fujikawa et al., 1993; Mersch-Sundermannet al., 1992). PAHs may enter the environment through many routes. However, anthropogenic processessuch as the combustion of fossil fuels, accidental spilling of hydrocarbons and oils, coal gasification and liquifaction, wood treatment processes,open burning and incineration of wastes, are the major source of PAHs in the environment (Cerniglia and Yang, 1984; Freemanand Cattell; 1990, Guerin and Jones, 1988). There is considerable interest in the use of biological technologies as a means to remediate sites contaminated with PAHs (Grifoll et al., 1995). Bioremediation of PAH-contaminated soil has been demonstratedto be effective in removing some PAH compounds (Barratt and Harold, 1991; Erickson et al., 1993; Sims ef al., 1990). However, bioremediation is presently not effective in removing some of the high molecular weight PAHs such as fluoranthene, benz[a]anthracene,benzo[u]pyrene, dibenz[a,h]anthraceneand coronene. It is particularly important to isolate and characterise soil bacteria capable of mineralizing these high molecular weight PAHs, since such isolatescould be used in site remediation to improve the overall PAH degradation rate or, in particular, to degrade high molecular weight PAHs which may not be degraded by the indigenous microflora (Cerniglia, 1992; Grosser et al., 1991; Heitkamp and Cerniglia, 1989; Kastner et al., 1994). Since PAH-contaminated sites usually contain a mixture of PAH compounds. it is also important that the microbial degradation of high molecular weight PAHs is not adversely affected by the presenceof the more easily degradedlow molecular weight PAHs. Previously, we isolated three strains of Pseudomonas cepacio (designatedVUN 10.001. VUN 10,002 and VUN 10,003) from PAH-contaminatedsoil (Juhasz et al., 1995). which could grow on fluorene, phenanthreneor pyrene (100 mgil) as the sole carbon and energy source: the complete degradation of these compoundsoccurred within 7 to 10 days. Although the strains grew poorly on fluoranthene, benz[a]anthraceneand dibenz[a,h]anthracene, there was someevidence that thesecompoundswere degradedas sole carbon and energy sources. The low cell numbers in the inocula resulted in slow degradation rates and only small decreasesin
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the concentrationof these high molecular weight PAHs. As a continuation of this work, we use high cell density inocula to confirm the degradationof dibenz[u,h]anthraceneby the three strains, and to study the degradation of benzo[a]pyrene, coronene and a mixture of PAH compounds. METHODS Chemicals Phenanthrene (PHEN), pyrene (PYR), benzo[b]fluorene, benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), benzo[u]pyrene (B(a)P) and coronene (COR) were from Sigma. Fluorene (FLU), fluoranthene (FA) and all solventswere purchasedfrom Lab Chem, Ajax Chemicals, Sydney, Australia. Bacterial media and reagents were purchased from Oxoid. All of the solventsand chemicalswere high purity grade reagents. Stock Solutions, PAH-Containing Media and Growth Conditions Stock solutions of each PAH were prepared in dimethylformamide (DMF) at the following concentrations:5 mg/ml, DBA and B(a)P; 1 mg/ml, COR. A stock mixture of selectedPAHs (FLU, PHEN, FA, PYR, BA, B[a]P and DBA) was also prepared at a concentration of 5 mg/ml for each PAH. A basal salts medium (BSM) (Juhaszet al., 1995) was supplemented with individual PAHs to achieve final concentrationsof 50 mg/l for the five ring compounds and 20 mg/l for the seven-ring compound(COR). BSM was also supplementedwith the stock mixture of PAHs to give a final concentration of 50 mg/l for each PAH. Coronene was separatelyaddedto BSM containing the PAH mixture to achieve a final concentration of 20 mg/l. Unlessotherwise stated,cultures were incubatedin the dark at 30°C and 175 rpm. Degradation using High Cell Density Inocula Bacterial inocula for high cell density experiments were prepared as follows: VUN 10,001, VUN 10,002and VUN 10,003 were each grown in four 1.5 litre volumes of BSM containing 250 mg/l pyrene. Cultures were incubated on a rotary shaker at 3OW175 rpm. Following the complete degradationof pyrene, cells were harvestedby centrifugation at 5,000 rpm for 10 minutes. Cell pellets were washedtwice in Ringers solution and resuspendedin BSM to achieve a ten-fold concentration in cell biomass. This cell suspension(5 ml, 0.7-l .5 mg/ml protein) was inoculated into serum bottles to evaluatedegradationof single PAHs or the PAH mixture. Incubations were performed in triplicate for each set of culture conditions and sampleswere removed for analysis at 3, 7, 10, 14, 21, 28, 35, 42, 49, 56 and 63 days. Incubationscontaining VUN 10,001, VUN 10,002 and VUN 10,003 without a carbon source and uninoculated PAH-containing media served as the controls. During the course of incubations, cell biomass was monitored by measuring protein concentrations and residual PAHs were determined by gas chromatography. Degradation of PAHs were determined in terms of ug substrateusedper mg of cellular protein. Analytical Procedures PAHs were extracted from bacterial culture fluids with dichloromethane (DCM). Gas chromatographicanalysisof DCM extracts and of PAH standardswas performed in triplicate on a Varian Star 3400 gas chromatograph equipped with a flame ionisation detector (GCFID), using a BPX-5 capillary column (25 m x 0.22 mm, SGE, Melbourne, Australia). The oven temperaturewas programed at 200°C for one minute, followed by a linear increaseof lO”C/min to 32O”C, holding at 320°C for 10 minutes. Injector and detector temperatures were maintainedat 300°C. Cells for protein assays(10 ml) were collected by centrifugation (5,000 rpm for 10 minutes) and washed twice in Ringers solution. Cell pellets were resuspendedin 1.0 ml 4.6M NaOH and boiled for 10 minutes to lyse the cells. Protein concentrationswere measuredby the method of Lowry et al. (1951). RESULTS In previous studieswhich used low cell populations of VUN 10,001, VUN 10,002 and VUN 10,003, it was difficult to determine whether significant degradation of the high molecular weight PAH compounds had occurred (Juhasz et al., 1995). To firmly establish the degradationof high molecular weight PAHs by the three isolates, experimentswere prepared using inocula containing high cell densities. Figure 1 shows that strain VUN 10,003,
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Time (days) Figure 1. Time course for benzo[ajpyrcnc (A). dibcnz[N.hlanthraccnc (B) anrl coroncnc (C) degradation. Benzo[a]pyrcnc (0). dibcnz[a.hlanlhraccnc (O)and coroncnc (A)wcrc added lo BSM inoculated with high cell nunibcrs of Pseudonroms cepock~ VUN IO.WD. Dcgradalion ram per mg of protein are shown lbr bcneo[o]pyrcnc (0). dibcnz[u,ltlanlhraccnc (m) and coronene (A). Controls (%)conlained uninoculated BSM and the respcclivc PAH.
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enriched on pyrene, was able to degrade five- and seven-ring PAHs [B(a)P, DBA and COR] as sole carbon and energy sources; similar results were obtained using strains VUN 10,001 and VUN 10,002 (data not shown). The degradation of benzo[u]pyrene (Fig. 1A) and dibenz[a,h]anthracene(Fig. 1B) resulted in significant PAH concentration decreasesof 20 to 30% (10 to 14 mg/l) after 63 days. Lag periods of up to 21 days were observed for all three strains before the degradation of benzo[u]pyrene and dibenz[a,h]anthracene commenced. Protein concentrations decreased slightly (5 %) over the incubation period. Coronene degradation (Fig. 1C) by the three pure cultures was extensive, with 65 to 70% (13 to 14 mg/l) being degradedafter 63 days. There was a lag period of approximately 14 days before coronene degradation began. Similarly, protein concentrations decreasedslightly during the incubation period. Most PAH-contaminatedsites contain a variety of PAH compounds, ranging in size from twoto seven-benzenerings. Under these conditions, it is possible that the ability of the isolates to degradehigh molecular weight PAHs is affectedby the presenceof the low molecular weight PAH compounds. Degradation of high molecular weight PAHs in the presence of low molecular weight PAHs was investigated by performing high cell density degradation experiments using a substratemixture containing three- to seven-ring PAH compounds(data for VUN 10,003 shown in Fig. 2).
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Time (days) Figure 2. Time course degradation experiment using high cell numbers of Pseudon~~~as cepacia VUN 10.003 and a mixture of PAHs. PAHs were addedto flasks at a concentration of 50 mg/l each, the exception being coronenewhich was supplied at a concentration of 20 mg/l. The degradation ratesof fluorene (0). phenanthrene(U). fluoranthene (v), pyrene (+). benz[a]anthracene(%), benzo[a]pyrene(0). dibenz[a,h]anthracene(Cl). and coronene(A) are expressedas ug PAH degradedper mg protein.
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All three strains were capable of degrading high and low molecular weight PAHs concurrently. Degradation of the low molecular weight PAHs (three-ring compounds) was fast and extensive; in the caseof VUN 10,002, over 90% of fluorene and phenanthrenewas degradedin 7 days. The degradation lag period for the five- and seven-ring compoundsusing VUN 10,003 was reduced to approximately 7 to 10 days, compared to the 14 to 21 day lag period observed in experiments containing single substrates (Fig. 1). Also, the specific degradation rate of the high molecular weight PAHs was greater than that observed in the single substrate experiments. After 42 days, the amount of five- and seven-ring PAHs degradedper mg of protein (27 ,ug DBA, 25 pg B(a)P, and 10 pg COR) in the mixed PAH incubations (Fig. 2) was much greater than the amountsdegraded(6.7 pg DBA, 7.2 pg B(a)P and 7.5 pg COR) when the PAHs were supplied as single substrates(Fig. 1). DISCUSSION A large amount of literature exists on the microbial degradation of low molecular weight PAHs as sole carbon and energy sources. These studies have yielded fundamental information about the biodegradability of these compounds. However, little is known about the biodegradation of high molecular weight PAHs or the fate of these compounds in PAH mixtures. Presumably, this is due to the recalcitrant nature of thesecompounds. In this study we have used high cell density cultures to verify the ability of P. cepacia strains VUN 10,001, VUN 10,002 and VUN 10,003, to degrade dibenz[a,h]anthracene as a sole carbon and energy source. This work was extended to investigate the degradation of other high molecular weight compoundsby these strains. Significant decreasesin the concentration of benzo[a]pyrene, dibenz[a,h]anthraceneand coronenewere observedafter 63 days incubation. There have been no reports on the isolation of a bacterial species capable of degrading benzo[a]pyrene, dibenz[a,h]anthraceneor coronene when supplied as sole carbon and energy sources. The bacterial degradation of five-ring PAH compoundshas only been observed for benzo[a]pyrene (Grosser et al., 1991; Heitkamp and Cerniglia, 1989; Shiaris, 1989). In one study (Grosser et al., 1991), a pyrene-degrading isolate from a heterogeneous microbial community was able to degrade 0.5 mg/l benzo[a]pyrene in a liquid medium containing significant quantities of yeast extract, peptoneand starch, suggestingco-metabolic degradation of the PAH. Fatty acid profile analysis failed to identify the isolate, although it was tentatively identified as Mycobacterium sp, basedon acid-fast and Gram staining, and colony morphology. An earlier study (Heitkamp and Cerniglia, 1989) observed the degradation of benzo[a]pyrene by a microbial community enriched from a sediment sample, but an isolate capable of degrading the PAH in pure culture could not be obtained. Similar results were also observed for the degradation of benzo[a]pyrene by microbial communities in muddy sediments from Boston Harbor (Shiaris, 1989). This is the first report to demonstratethe microbial degradation of the seven-ring PAH, coronene.by a pure culture. High molecular weight PAH compoundsare usually found in the environment as mixtures at PAH-contaminated sites, e.g. contaminated soils from manufactured gas plant and wood preserving facilities contain a mixture of PAH compoundsranging in size from two- to sevenring structures, as well as a wide variety of phenolic and N-. 0- and S-heterocyclic compounds (Foght et al., 1989; Mueller et al., 1989a; 1991). To be effective in site decontamination, microorganisms must possessthe ability to degrade all PAHs present in complex mixtures, but this is not always observed e.g. In a previous study conducted with a PAH mixture (Park et al., 1990), it was found that the two- and three-ring PAH compounds were degraded rapidly, however, the high molecular weight PAHs (four-, five- and six-ring PAHs) were recalcitrant to microbial attack. Also. there is little information available about 581
the degradation kinetics of PAH mixtures (Cemiglia, 1992), which may differ from that observed using single PAH compounds. All three strains isolated in our study were able to degrade all the PAHs used in this work simultaneously when present as a substratemixture. Furthermore, improved degradation of the five- and seven-ring PAH compounds occurred in the presence of lower molecular weight PAHs. This was reflected by a decrease in the degradation lag period of around 50% and an increase in the degradation rate per mg of protein over a 42 day incubation period. The amounts degraded per mg of protein in the PAH mixture were 280% greater for benzo[a]pyrene and dibenz[a,h]anthracene, and 33% greater for coronene, comparedto single substrateexperiments. The improved degradation of the high molecular weight PAHs was probably a result of increasedmetabolic activity due to the presenceof the more easily degraded, low molecular weight PAHs. In an earlier study, the sequential removal of PAHs by a bacterial community was observed during the biotransformation of a PAH mixture (Mueller et al., 1989b). The authors found that the recalcitrant PAHs (fluoranthrene and pyrene) were utilised only after degradation of the more labile compounds. It is interesting that in our work the concurrent degradation of all the PAHs occurred after a short lag period of 10 days, suggesting that for our isolates the catabolic pathways for these compoundsare not inhibited by the presenceof a different PAH compound. ACKNOWLEDGEMENTS We thank Dr Brent Davey (Australian Defence Industry Environmental Services) for the supply of the contaminated soil. The research was funded by a Victorian Education Foundation scholarship and a Centre for Bioprocessingand Food Technology grant to support PhD studies. REFERENCES Andrews,A. W., L. H. ThibaultandW. Lijinsky. 1978. Mutation Res. 51:311-318. Barratt, P. and P. Harold. 1991. p. 336-346. In M. C. R. Davies (ed.), Land Reclamation, An End to Dereliction? Elsevier Applied Science, New York. Cemiglia, C. E. 1992. Biodegradation3:351-368. Cemiglia, C. E. and S. K. Yang. 1984. Appl. Environ. Microbial. 47:119-124. Erickson, D. C., R. C. Loehr and E. F. Neuhauser. 1993. Water Res. 27:911-919. Foght, J. M., P. M. Fedorak and D. W. S. Westlake. 1989. Can. J. Microbial. 36:169-175. Freeman, D. J. and F. C. R. Cattell. 1990. Environ. Sci. Technol. 24:1581-1585. Fujikawa, K., F. L. Fort, K. Samejimaand Y. Sakamoto.1993. Mutation Res. 290:175-182. Grifoll, M., S. A. Selifonov, C. V. Gatlin and P. J. Chapman. 1995. Appl. Environ. Microbial. 61:3711-3723. Grosser, R. J., D. Warshawskyand J. R. Vestal. 1991. Appl. Environ. Microbial. 57:3462-3469. Guerin, W. F and G. E. Jones. 1988. Appl. Environ. Microbial. 54:929-936. Heitkamp, M. A. and C. E. Cerniglia. 1989. Appl. Environ. Microbial. 55:1969-1973. Juhasz, A. L., G. A. Stanley and M. L. Britz. 1995. Third International Symposium; In Situ and OnSite Bioreclamation, San Diego. Kastner, M., M. Breuer-Jammaliand B. Mahro. 1994. Appl. Microbial. Biotechnol. 41:267-273. Lowry, 0. H., N. J. Rosebrough,A. L. Farr and R. J. Randall. 1951. J. Biol. Chem 193:265-275. Mersch-Sundermann,V., S. Mochayedi and S. Kevekordes. 1992. Mutation Res. 278: l-9. Mueller, J. G., P.J Chapman, and P. H. Pritchard. 1989a.Environ. Sci. Technol. 23:1197-1201. Mueller, J. G., P. J. Chapmanand P. H. Pritchard. 1989b. Appl. Environ. Microbial. 55:3085-3090. Mueller, J. G., S. E. Lantz, B. 0. Blattmann and P. J. Chapman. 1991. Environ. Sci. Technol. 25: 1045-1055. Park, K. S., R. C. Sims and R. DuPont. 1990. J. Environ. Eng. (ASCE) 116:632&O. Shiaris, M.P. 1989. Appl. Environ. Microbial. 55:1391-1399. Sims, J. L., R. C. Sims and J. E. Matthews. 1990. Haz. WasteHaz. Mat. 7: 117-148.
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