Characterization of a Novel Pseudomonas sp. That MineralizesHigh Concentrations of Pentachlorophenol
PETRA M. RADEHAUS AND STEVEN K. SCHMIDT*Department of Environmental, Population and Organismic Biology,
University of Colorado, Boulder, Colorado 80309-0334
Received 23 March 1992/Accepted 17 June 1992
A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as
Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source ofcarbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies,stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralizea higher concentration ofPCP (160 mg liter-') than any previously reported PCP-degrading pseudomonad. Ata PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded,although an active population of Pseudononas sp. RA2 was still present in these cultures after 2 weeks. Theinhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strainRA2. The highest specific growth rate (FL = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-' butdecreased at higher or lower PCP concentrations, with the lowest , (0.05 h-1) occurring at 150 mg liter-'.Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at allPCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to belowexpected values at PCP concentrations greater than 100 mg liter-'. These results indicate that, in addition toits effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp.strain RA2. PCP and glucose were simultaneously mineralized by Pseudomonas sp. strain RA2, but glucose hadno effect on the rate of PCP mineralization. PCP, on the other hand, significantly enhanced the metabolism ofglucose by Pseudomonas sp. strain RA2.
Pentachlorophenol (PCP) is a widely used wood treatmentagent and biocide (22). The misuse, accidental spillage, andimproper disposal of this toxic compound have resulted inextensive pollution of water and soil (6, 22, 26). Under theComprehensive Environmental Response, Compensationand Liability Act, PCP was included in the list of hazardouswastes (8). The U.S. Environmental Protection Agencyconsiders 1 mg of PCP liter-1 hazardous for land disposal(10).Because of its high degree of chlorination and toxicity,
PCP is recalcitrant to microbial attack and has accumulatedin some environments. However, several studies have dem-onstrated that PCP is subject to biodegradation by a limitednumber of naturally occurring bacterial and fungal species.Pure cultures of bacteria, such as Flavobacterium (31),Pseudomonas (40), Rhodococcus (1, 11), Arthrobacter (6),Corynebactenum (30), and Mycobacterium (11) spp., andcultures of fungi, such as Phanerochaete (21) and Tricho-derma (4) spp., have the enzymatic capacities to degradePCP. Because of the toxicity of PCP, however, high concen-trations inhibit cell growth and consequently the degradationprocesses (6, 40). Therefore, many studies have been done incontinuous-flow systems with low PCP concentrations (7,26). These studies are useful and may aid in the developmentof clean-up strategies for areas with dispersed pollution (29).In contrast, some areas with point source pollution containPCP at concentrations as high as 9,000 mg/kg of soil. At suchsites, bioremediation efforts are very costly and often fail (3).As a result of this and other problems, only 7.5% of allremediation technologies used at Superfund sites in the fiscalyear 1989 were biological treatment methods (9). To make
* Corresponding author.
bioremediation a more effective technology, further researchis needed to gain a better understanding of the basic mech-anisms underlying enhancement and inhibition of microbialdegradation of high concentrations of toxic compounds.The Pseudomonas sp. strain used in this study was
isolated from a heavily contaminated soil and was selectedfor its ability to degrade relatively high concentrations ofPCP. In spite of an extensive literature search, no previouslydescribed bacterium was found that matches the character-istics of this organism. The goals of the work presented herewere to characterize the organism, show that it could min-eralize PCP, determine the range of PCP concentrations thatPseudomonas sp. strain RA2 could mineralize, test theeffects of glucose additions on PCP degradation, and, finally,to study the inhibition of PCP degradation at higher PCPconcentrations.
(This work is part of the Ph.D. thesis of Petra Radehaus tobe submitted to the Westfalische Wilhelms Universitat Mun-ster, Naturwissenschaftliche Fakultat, Munster, Germany.)
MATERIALS AND METHODS
Chemicals. Unlabeled PCP (99% pure) was obtained fromFluka AG (Buchs, Switzerland), and unlabeled D-glucosewas from Mallinckrodt, Inc. (Paris, Ky.). Uniformly 14C-labeled PCP (specific activity, 11.9 mCi mmol- ) and[U-14C]glucose (296 mCi mmol-1) were purchased fromSigma Chemical Co. (St. Louis, Mo.).
Bacterial enrichment. Pseudomonas sp. strain RA2 was
isolated from a soil at the Broderick Wood Products site nearDenver, Colo. The soil at this Superfund site is heavilycontaminated with wood-preserving wastes. Soil (1 g) fromthe upper surface layer was added to 100 ml of an inorganicsalts solution containing 10 mg of PCP liter-'. After 7 days
of incubation at 22°C, 5 ml of the enrichment culture wastransferred to 100 ml of fresh medium containing 10 mg ofPCP liter-'. After 7 days of incubation at 22°C, serialdilutions were plated on a medium containing inorganicsalts, 50 mg of PCP per liter, and 15 g of purified Difco agar(Difco Laboratories, Detroit, Mich.) per liter. Isolated colo-nies appearing on the medium after 2 weeks of incubation at22°C were restreaked on fresh agar before they were trans-ferred back to liquid medium. By transferring serial subcul-tures to liquid medium with PCP concentrations increasingto 150 mg liter-', PCP degraders that could tolerate higherconcentrations of PCP were enriched. No supplementalsubstrate additions were made during the enrichment pro-cess, and thus the bacterium selected had the capability togrow on PCP without supplemental carbon sources orgrowth factors.
Bacterial identification. A variety of microbiological andbiochemical tests were used to characterize and identify thePCP-degrading bacterium. Motility was tested in soft agar (4g of agar liter-'), and flagella were stained by using themethod of Heimbrook et al. (13). Poly-,B-hydroxybutyrategranules were stained with Nile Blue A (23) and observedwith a Dialux 22 epifluorescence microscope (E. Leitz, Inc.,Rockleigh, N.J.). The ability of the organism to use differentcarbon and energy sources was tested in liquid cultures, onagar plates, and in Biolog GN MicroPlates (Biolog, Inc.,Hayward, Calif.). In addition, the cellular fatty acid compo-sition was analyzed by Microbial ID, Inc. (Newark, Del.).The absorption maxima of acetone-extracted yellow pig-ments from Pseudomonas sp. strain RA2 were determinedwith a Lambda 3B spectrophotometer (The Perkin-ElmerCorp., Norwalk, Conn.).
Culture conditions. Pseudomonas sp. strain RA2 grewpoorly on PCP agar, and thus the cells were maintained inmineral salts solution with 80 mg of PCP per liter as the solecarbon source and were subcultured every 10 days. Afterbeing in the stationary phase for 24 to 48 h, cells from thesestock cultures were used as the inoculum for all PCPdegradation experiments. In all experiments, the initial bac-terial density was 2 x 106 ml-'. All batch culture experi-ments were carried out at 21 + 2°C in unshaken 250-mlErlenmeyer flasks covered by inverted beakers and filledwith 130 ml of the mineral salts medium described below.The flasks were kept in the dark to avoid photo-decomposi-tion of PCP. For the identification tests described above,Pseudomonas sp. strain RA2 cells were used that had beengrown for 4 days at 24°C on solid Trypticase so? agarmedium containing 6 g of Trypticase soy broth liter- (BBLMicrobiology Systems, Cockeysville, Md.) and 15 g of BactoAgar (Difco) per liter of deionized water.Media and substrates. The mineral salts medium was
composed of (per liter of deionized water) 2.4 g of Na2HPO4,2.0 g of KH2PO4, 0.1 g of NH4NO3, 0.01 g of MgSO4- 7H20,0.01 g of CaCl2, and 1 ml of trace element solution (25).Media were sterilized by autoclaving at 121°C for 20 min.Solutions of CaCl2 were autoclaved separately from theother salts to avoid the formation of a precipitate. Aftercooling, the solutions were mixed under sterile conditions.The culture medium was strongly buffered (Na2HPO4-KH2PO4) at pH 6.9 to avoid inhibition of microbial activitydue to hydrochloric acid production during PCP degrada-tion. Depending on the experiment, the medium wasamended with 0.0004 to 0.75 mM (0.1 to 200 mg liter-') PCPas the sole carbon and energy source. PCP was added froma stock solution (10 g liter-1) that was prepared in 1 N NaOHand then adjusted to pH 7.5 with H3PO4. In experiments in
which the PCP concentration was 0.1 mg liter-', only theuniformly '4C-labeled PCP was added. In dual-substrateexperiments, 0.44 mM (80 mg liter-') filter-sterilized D-glu-cose was added as a supplemental carbon source.
Determination of bacterial growth. Microorganisms in thecultures were enumerated by direct microscopic counting ina calibrated Thoma counting chamber (0.02-mm depth;Fleischhacker KG, Schwerte, Germany). At least four filledchambers were counted per sample. In some experiments,the spread-plate technique was used for cell enumerationand for checking the purity of the cultures. Duplicate 0.1-mlportions of a series of 10-fold dilutions in mineral saltsmedium were plated on 0.2-strength TSA medium. Colonycounts were made after 7 days of incubation at 24°C. Theyield coefficient (Y), defined as the cell dry weight per totalweight of PCP degraded, was assessed by filtration of at least50 ml of early-stationary-phase cultures through dried andpreweighed 0.2-,um-pore-size polycarbonate filters (Nucle-pore Corp., Pleasanton, Calif.). After filtration, these filterswere dried at 80°C for 20 h and reweighed, and the cell dryweight was calculated. All yield measurements were done induplicate.
Analytical techniques. The mineralization of PCP andD-glucose was monitored by a technique similar to that ofSchmidt et al. (34) and Hess et al. (14). In this procedure,tracer concentrations of uniformly 14C-labeled compoundswere added to the culture media (approximately 14,000dpm/ml). The final substrate concentration was reached byadding different amounts of unlabeled compounds. To mea-sure the unmineralized radioactive carbon that was not fixedin biomass, 2.5-ml samples were removed at appropriateintervals and filtered through 0.2-,um-pore-size polycarbon-ate filters (Nuclepore Corp.). In addition, unfiltered sampleswere taken so that the percentage of carbon fixed in cellscould be calculated. One-milliliter aliquots of these sampleswere transferred to 4-ml Omni vials (Wheaton Industries,Millville, N.J.) and acidified with 3 drops of concentratedH2SO4 to force dissolved CO2 out of the samples. After theaddition of 2.5 ml of ScintiVerse II scintillation cocktail(Fisher Scientific Co., Pittsburgh, Pa.), the samples wereshaken vigorously and counted in a liquid scintillationcounter (LKB Wallac, 1209 RackBeta, Turku, Finland). Thecounting efficiency was about 97% under these conditions.In addition, the mineralization of PCP was assessed bymeasuring 14C02 evolution by a technique similar to that ofHess et al. (14). Evolved "4CO2 was captured at regularintervals in 2 ml of 0.5 N NaOH in a sidearm biometer flask.The radioactivity of the NaOH samples was determined in aliquid scintillation counter as described above.
Mineralization of PCP was also determined by measuringthe release of chloride ions with a model 96-17 B combina-tion electrode (Orion Research, Inc., Boston, Mass.). Theelectrode was calibrated by adding known concentrations ofsodium chloride standards to the mineral salts medium usedin the experiments. Uninoculated flasks were run in eachexperiment as controls for PCP volatilization.Data analysis. To obtain estimates of kinetic parameters,
PCP mineralization curves were analyzed by using nonlinearregression techniques (14) on a Macintosh Plus Computer(Apple Computer Inc., Cupertino, Calif.). The least squareof difference between data and model curves was minimizedby the method of Levenberg and Marquardt as described byDennis and Schnabel (5).
PENTACHLOROPHENOL MINERALIZATION BY A PSEUDOMONAS SP. 2881
TABLE 1. Characteristics of Pseudomonas sp. strain RA2
Test Bacterial reaction
MorphologyGram stainFlagellaOxidaseCatalaseFluorescent pigment (on TSA)Growth at 8°CGrowth at 37°CStarch hydrolysisVoges-ProskauerMethyl redGelatin hydrolysisBlood agarEosin methylene blue agarMannitol salt agarTriple sugar iron agarLipase test (Tween 80)Poly-3-hydroxybutyrate productionGrowth on:Bromo succinic acidCitrateDextrinD-FructoseD-Galactonic acidD-Galacturonic acidGentiobioseD-GlucoseGlycogenGlycyl-L-glutamic acidp-Hydroxybenzoate,-Hydroxybutyric acidL-LeucineMaltoseMethyl pyruvatep-NitroanilinePentachlorophenolPhenolL-ProlineSebacic acidSuccinic acidSucroseUric acid
Rod
Polar, one++
,-HemolysisNo precipitate, purple colonies, no acid production
No growth, unchanged red butt and slantNo carbohydrate fermentation, no reduction of sodium thiosulfate
+
+
++++++
+
+
+
RESULTSCharacterization of the PCP-degrading bacterium. The
PCP degrader is a gram-negative, oxidase- and catalase-positive, aerobic rod-shaped bacterium. It has one polarflagellum and accumulates poly-3-hydroxybutyrate. Cells ofthis bacterium were motile in soft agar and wet mounts. Cellsgrown in liquid culture with PCP were approximately 2 to 3,um in length. The organism grew slowly on solid mediumsuch as nutrient agar or TSA. Visible colonies developed in4 days. In addition, the organism grew better on diluted agarmedium than on full-strength medium. Higher colony countswere obtained on 0.2-strength TSA (6 g of Trypticase soybroth liter-') than on normal-strength TSA (30 g of Trypti-case soy broth liter-'). Typical colonies on TSA weretransparent yellow and adhered to the agar surface. Acetoneextracts of the yellow cell pigment had absorption maxima at450 and 475 nm. Further cell characteristics, primarilyresults of studies on the utilization of different carbonsources, are presented in Table 1. Some reactions on theBiolog GN MicroPlates were ambiguous and therefore arenot included in Table 1. As a result of the listed taxonomiccharacteristics and the fatty acid profile, the PCP degrader
has been tentatively identified as a Pseudomonas sp. (24).This organism will henceforth be called Pseudomonas sp.strain RA2.
Mineralization of PCP. Under batch culture conditions,Pseudomonas sp. strain RA2 cells were capable of com-pletely mineralizing PCP (Fig. 1). Figure 1 shows the disap-pearance of 40 mg of PCP per liter, the growth of Pseudo-monas sp. strain RA2, and the release of 26 mg of chlorideliter-1 during PCP mineralization. The molecular weight ofPCP consists of 66.6% chlorine, and therefore the data inFig. 1 indicate stoichiometric release of chloride during PCPmineralization. Correspondence of chloride release and de-pletion of radiolabeled PCP indicate that the dechlorinationreactions and PCP uptake happened simultaneously. Somecells of Pseudomonas sp. strain RA2 became unusually long(up to 15-fold normal cell length) during the exponentialgrowth phase, but only coccoid cells were observed in thestationary phase. Final cell dry weights were roughly pro-portional to initial PCP concentrations (Fig. 2). In theradiotracer studies, approximately 14 to 17% of 14C-labeledPCP was incorporated in cellular material at all PCP concen-trations.
FIG. 1. Mineralization of 40 mg of PCP liter-1 as sole source ofcarbon and energy by Pseudomonas sp. strain RA2 and simulta-neous cell growth and chloride ion production.
Replicate tests were done to measure the release of 14C02durin~mineralization of PCP at a concentration of 40 mgliter- . On average, 70% of the labeled carbon was recov-ered as 14Co2, 14% was assimilated into cells, and 3%remained in solution at the end of the 6-day experiment.Approximately 13% of the initial 14C was not recovered,probably because of the loss of 14C02 during sampling.
Inhibition of PCP degradation. Several experiments weredone to determine the range of PCP concentrations thatPseudomonas sp. strain RA2 could mineralize. In batchcultures, Pseudomonas sp. strain RA2 was capable of com-pletely mineralizing PCP at a concentration of 160 mg liter-'but was unable to mineralize PCP at a concentration of 200mg liter-1. The viable cell number in the culture with 200 mg
14
12 [I-
ua
I-
Co
Lo
D
1-1
3
10
8
6
4
2L20 40 60 80 100 120 140 160
Pentachlorophenol (mg U1)
FIG. 2. Effect of initial PCP concentrations on the formation ofbiomass (dry weight) and final cell densities in cultures of Pseudo-monas sp. strain RA2.
1-
a-to
0
0
a2
120
100
80
60
40
20
I---4
2aca3
0 2 4 6 8 10
Time (days)
FIG. 3. Mineralization of 150 mg of PCP liter-' as sole source ofcarbon and energy by Pseudomonas sp. strain RA2 and simulta-neous cell growth.
of PCP liter-' slowly decreased from 2 x 106 cells per ml toapproximately 104 cells per ml over a period of 16 days. Themost profound effect of increasing PCP concentration wason the number of cells produced per unit of PCP mineralized(Fig. 2). In contrast, high concentrations of PCP had only amoderate effect on cell dry weight (Fig. 2) and Y. At all PCPconcentrations tested, Y was approximately 0.1.The concentration of PCP also affected the lag phase
before the onset of mineralization by Pseudomonas sp.strain RA2. The higher the concentration of PCP, the longerthe lag phase lasted before the start of measurable PCPmineralization (data not shown). The lag period before PCPdegradation at high concentrations was shown to be a lagphase in cell growth (Fig. 3). This period of adaptation wasnot observed at PCP concentrations of 40 mg liter-' or less(Fig. 1).
In addition to its effects on the lag phase, high concentra-tions of PCP also adversely affected the growth rate ofPseudomonas sp. strain RA2. The highest specific growthrate (,u) was 0.09 h-1 at 40 mg of PCP liter-'. The growthrate decreased in proportion to increasing PCP concentra-tion, and the lowest growth rate of 0.05 h- was observed at150 mg of PCP liter-' (Fig. 4). The inhibition constant (K,)for PCP mineralization was obtained by using linear regres-sion to fit a straight line (r = 0.98) to the descending portionof the curve shown in Fig. 4. The Ki obtained was 155 mg ofPCP liter-'. To distinguish Ki from Ks, Ki is defined as thehighest concentration of PCP at which ,u equals ,umax/2 (32).An attempt was made to fit the differential form of theHaldane equation (17, 32) to the growth rate response curve(Fig. 4), but there were too few datum points in the curve toobtain an accurate estimate of Ki by using this method.
Simultaneous use of glucose and PCP. In experimentstesting the effects of glucose on PCP mineralization byPseudomonas sp. strain RA2, PCP and glucose were simul-taneously degraded (Fig. 5). Almost 90% of the glucose and95% of the PCP were mineralized by the cells in thisdual-substrate experiment; 40% of the mineralized glucoseand 12% of the mineralized PCP were incorporated into cellbiomass. Cell density reached approximately 4 x 108 cellsml-' in this experiment.
PENTACHLOROPHENOL MINERALIZATION BY A PSEUDOMONAS SP. 2883
0.11
0:
0.10 -
0.09 -
0.08 .
0.07 -
0.06
0.05
100.
80
0._
80V
0.04 1
0.03 10 20 40 60 80 100 120 140 160
Pentachlorophenol (mg L-1)
FIG. 4. Effect of PCP concentrations on the specific growth rate(,u) of Pseudomonas sp. strain RA2 (standard deviations are shownas bars).
Glucose had notion (data not sh(growth rate (R,) wIn contrast, PCPmineralization bygrowth rate of Pglucose (0.083 h-glucose alone (0.growth rate on P(
The PCP-minerwas isolated fronidentified as Pseut
100
a*0
as
80
0
0
80
60
40
20
0
FIG. 5. Simultanmg of glucose liter-
60
40
20
0 1 2 3 4 5 6 7 8 9
Time (days)
FIG. 6. Effect of 80 mg of PCP liter-' as a su?plemental carbonsource on the utilization of 80 mg of glucose liter- by Pseudomonassp. strain RA2.
effect on the kinetics of PCP mineraliza- similar in some respects to the PCP-degrading Flavobacte-own). Kinetic analyses indicated that the num sp. described by Saber and Crawford (31). However,as not affected by the addition of glucose. several characteristics of Pseudomonas sp. strain RA2 dis-significantly increased the rate of glucose tinguish it from members of the genus Flavobactenum.Pseudomonas sp. strain RA2 (Fig. 6). The Pseudomonas sp. strain RA2 is motile, has a functional polarVeudomonas sp. strain RA2 on PCP and flagellum, and stores poly-3-hydroxybutyrate. These traits1) was higher than the growth rate on are not found in the genus Flavobacterium (15). Previous
.061 h-1). In the same experiment, the reports of PCP degradation by Pseudomonas spp. are rare'P alone was 0.075 h-1. (16, 27, 39, 40), and, unfortunately, some of these reports
contain incomplete or ambiguous descriptions of the organ-isms involved. Thus, a rigorous comparison of Pseudomo-
DISCUSSION nas sp. strain RA2 with previously described PCP-degrading
-alizing bacterium described in this study pseudomonads is not possible. Prior to the present study, then PCP-contaminated soil and tentatively most thoroughly described PCP-degrading pseudomonaddomonas sp. strain RA2. This bacterium is
was the Pseudomonas cepacia strain isolated by Kams et al.(16). This organism differs from Pseudomonas sp. strainRA2 in several major respects, the most important being thatP. cepacia has multiple flagella and Pseudomonas sp. strainRA2 has a single flagellum (Table 1). In addition, P. cepaciacould only significantly degrade PCP in the presence ofsupplemental carbon sources (16, 27). In contrast, Pseudo-monas sp. strain RA2 metabolized PCP as its sole source ofcarbon and energy, and glucose had no effect on PCPmineralization by Pseudomonas sp. strain RA2.The depletion of radiolabeled PCP accompanied by CO2
evolution, stoichiometric release of chloride, and propor-tional increase in cell biomass indicates that Pseudomonas
Glucose sp. strain RA2 can completely mineralize PCP (Fig. 1 and 2).PCP \ < These results also suggest that PCP is mineralized without
the release of measurable amounts of toxic metabolites ordead-end products. In contrast, some previously describedmicroorganisms could not completely degrade PCP (4) oraccumulated less-biodegradable products such as pentachlo-roanisole (11).
_______,______._____, ______,____ PCP is an extremely toxic compound, and, in this study,Pseudomonas sp. strain RA2 could not grow at a PCP145concentration of 200 mg liter-1. Even at this growth-inhib-
Time (days) iting concentration, however, a significant number of Pseu-ieous utilization of 80 mg of PCP liter-' and 80 domonas sp. strain RA2 cells survived for several weeks.-' by Pseudomonas sp. strain RA2. The fact that Pseudomonas sp. strain RA2 could survive
even at a PCP concentration of 200 mg liter-' is encouragingbecause it may allow for the future selection of mutantscapable of mineralizing very high concentrations of PCP.Some bacteria other than pseudomonads have been found tobe more resistant to high concentrations of PCP (6, 31, 37).However, Pseudomonas sp. strain RA2 can mineralizehigher concentrations of PCP than any previously describedPseudomonas sp. (16, 27, 39, 40).
Increasing concentrations of PCP adversely affected bothgrowth rates (Fig. 4) and the number of cells produced perunit of PCP but not the biomass produced per unit of PCP(Fig. 2). A similar effect of PCP on specific growth rates waspreviously described by Stanlake and Finn (37) for anArthrobacter sp. The observation of unusually long cellsduring exponential growth also indicates that Pseudomonassp. strain RA2 has the potential to grow without dividing.These results are not consistent with the assumption thatPCP acts only as an uncoupler of oxidative phosphorylation(41) because an uncoupler should reduce biomass as well ascell number. The adverse effect of PCP on bacterial prolif-eration may be partially due to its direct influence on cellmembranes. In addition to its effects on electrical conduc-tivity, PCP has been shown to alter the molecular organiza-tion of phospholipid membranes (36, 38).PCP concentration had a significant effect on the acclima-
tion phase for PCP mineralization. With increasing substrateconcentration, the acclimation period, i.e., the time intervalduring which biodegradation was not detected, increased.Similar observations have been made for phenol degradationin sewage cultures (42). In the present study, the delay inbiodegradation was linked to a delay in growth. The onset oflogarithmic growth was delayed at high PCP concentrationsas compared with the onset of growth at lower concentra-tions. A prolongation of the growth-related lag phase at highPCP concentrations has been previously reported in otherPseudomonas spp. (27, 39) and in cultures of non-PCP-degrading rumen bacteria (43). It may be that high PCPconcentrations initially reduce the number of active Pseudo-monas sp. strain RA2 cells; however, the direct countingmethod used in this study cannot be used to differentiateactive from nonactive cells. A second explanation is that theacclimation is due to proliferation of enzymes specific fordealing with high concentrations of PCP. In an experiment inwhich the total inoculum density was greatly increased (datanot shown), the lag in PCP mineralization was almostcompletely eliminated, thus supporting the first of the aboveexplanations. It may be that a higher population density canwithstand a higher PCP concentration because the concen-tration of PCP per cell is lower (20).
Several experiments which assessed the effects of glucoseon PCP mineralization in Pseudomonas sp. strain RA2showed that relatively high concentrations of PCP andglucose could be simultaneously mineralized by this organ-ism (Fig. 5). These findings are consistent with previouswork that demonstrated simultaneous mineralization of glu-cose and a variety of aromatic compounds in a number ofPseudomonas spp. (12, 28, 33, 34). In addition, P. cepaciaATCC 1100 has been shown to cometabolize PCP in thepresence of glucose, whereas in the absence of glucose, P.cepacia could not degrade significant quantities of PCP (27).
It was hoped that glucose would increase the growth rateof Pseudomonas sp. strain RA2, as has been noted withother xenobiotic-degrading bacteria (33, 34), or amelioratethe toxic effects of PCP (20, 35). Contrary to expectations,however, glucose had no effect on the rate of PCP mineral-ization. This was in spite of the fact that the cell density was
increased from 5 x 107 cells ml-' in the absence of glucoseto 4 x 108 cells ml-' when glucose was added.An unexpected finding of this study was that PCP signifi-
cantly enhanced the mineralization of glucose by Pseudomo-nas sp. strain RA2 (Fig. 6). One explanation for this findingis that more glucose-metabolizing cells were produced in thepresence of PCP than in its absence. Data to test thishypothesis were not obtained in the present study. PCP didsignificantly increase the growth rate of Pseudomonas sp.strain RA2 from 0.06 h-1 in the absence of PCP to 0.08 h-1when PCP was present. Another likely explanation for thestimulatory effect of PCP on glucose metabolism is that PCPis an uncoupler of oxidative phosphorylation. Uncouplerscan increase the overall metabolic rate of an organism (18),which would in turn increase the rate of glucose metabolism.Bauer and Capone (2) noted that the addition of PCP tooxygenated sediment slurries caused significant increases inthe percentage of glucose respired.The results of the dual-substrate experiments also indicate
that Pseudomonas sp. strain RA2 has a preference for PCPover glucose as a growth substrate. Pseudomonas sp. strainRA2 grew faster on 80 mg of PCP liter-' (,u = 0.08 h-1) thanon 80 mg of glucose liter-' (p, = 0.06 h-1) despite the factthat the percent carbon content of glucose is much higherthan that of PCP. These results are probably attributable toselective pressures in the soil from which Pseudomonas sp.strain RA2 was isolated. This soil was highly contaminatedwith PCP and creosote and probably was not receivingsignificant inputs of glucose from natural sources. Thus,selective pressure in this soil would have favored organismsthat specialize on PCP or components of creosote. Anotherreason for the preference of this organism for PCP is thatselective pressure for PCP mineralization was always main-tained in laboratory cultures of Pseudomonas sp. strainRA2. Unlike many PCP-degrading bacteria used by previousworkers, the Pseudomonas sp. strain RA2 cell line used inall PCP degradation experiments was never grown on richlaboratory media. Preference for a xenobiotic chemical overa naturally occurring compound has been previously re-ported by LaPat-Polasko et al. (19), who found that Pseu-domonas sp. strain LP preferred methylene chloride overacetate in their experimental system.
In conclusion, Pseudomonas sp. strain RA2 was shown tomineralize relatively high concentrations of PCP and glucosehad no effect on the kinetics of PCP mineralization in thisorganism. Future work with this organism will be directed atselecting strains of Pseudomonas sp. strain RA2 that canmineralize even higher concentrations of PCP, with the hopethat they can be used in bioremediation efforts at sites thatare heavily contaminated with PCP.
ACKNOWLEDGMENTSWe thank H. J. Rehm and J. Silverstein for valuable discussions,
M. Schwierskott for technical assistance, and P. Brooks, M. Fisk,E. Kelly, and R. Mullen for reviewing a draft of the manuscript. Weare indebted to R. Schimmel, A. M. Colpitts, and K. Griffiths forproviding soil samples and to J. Hanken and W. Bowman for the useof equipment.
This work was supported by grants from the Colorado WaterResources Research Institute, the National Science Foundation,and the Council on Research and Creative Work, University ofColorado, Boulder.
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