Phenol and Catechol Biodegradationby the Haloalkaliphile Halomonascampisalis: Influence of pH andSalinity

V I C T O R A . A L V A A N DB R E N T M . P E Y T O N *

Center for Multiphase Environmental Research,Consortium for Extremophile Research,Washington State University, Dana Hall,118 Spokane Street, Pullman, Washington 99164-2710

Removal of aromatic compounds from alkaline and/orsaline industrial wastewater is an environmental concernfor industry. In addition, aromatics may be accumulatingin soda lakes, unique natural systems, where the fate andtoxicity of these contaminants is unknown. To determinethe feasibility of aromatic compound biodegradation in salineand alkaline conditions, the effect of pH and salinity onthe biodegradation of phenol as a model aromatic wastecompound by the haloalkaliphilic bacterium Halomonascampisalis was examined. Phenol was degraded as a sourceof carbon and energy at pH 8-11 and 0-150 g/L NaCl.Metabolic intermediates catechol, cis,cis-muconate, and(+)-muconolactone were identified, thus indicating thatphenol was degraded via the â-ketoadipate metabolicpathway. Although phenol and catechol were completelydegraded in all cases, small amounts of cis,cis-muconateaccumulated proportionally to increases in pH. Therewas no noticeable influence of salinity on cis,cis-muconateaccumulation except at 0 g/L NaCl where it was completelydegraded. These results indicate that it may be feasibleto use haloalkaliphiles for the treatment of aromatics presentin saline and/or alkaline systems. This is the first reportof phenol and catechol biodegradation under combined salineand alkaline conditions.

IntroductionWith limited freshwater supplies, water-use reduction strat-egies are being implemented by industry; therefore, it isexpected that pollutant concentration and ionic strength willincrease in industrial process wastewater. In addition, it isknown that traditional pollutant biodegradation is lessefficient or does not function when salinity increases aboveseawater levels or pH differs significantly from neutral (1).This is a common and challenging problem for petrochemical,pesticide, pharmaceutical, mining, and other industries (2-4). An industrial effluent of great environmental concern ispetroleum refinery spent caustic (5), an alkaline and salinewaste stream that contains phenol as well as other aromaticcompounds. Currently, spent caustic effluents are treatedby energy-demanding wet air oxidation (6) or the uneco-nomical Fentons’s reagent treatment (7). A treatment al-ternative where aromatics could be biologically removed in

alkaline and saline conditions would benefit the (petro)-chemical industry and a wide variety of other industries. Weforesee an increasing need for the use of biological treatmentadapted to saline and alkaline environments in industrialwastewater treatment systems.

In addition to industrial systems, aromatics (and otherpollutants) may be accumulating in natural systems such asthe often overlooked soda lakes (8). Information on thebiodegradation of contaminants in these unique environ-ments is not available, and as a result, the potential foraccumulation and toxicity of contaminants in these aqueoussystems is unknown. For these environments, “acceptable”contaminant concentrations must be rationally addressedusing defensible scientific information including an under-standing of the chemical, physical, and microbiologicalfactors that influence contaminant fate.

Although high salinity and alkalinity negatively affect manymicroorganisms, vibrant ecosystems are found in naturallysaline and alkaline environments (9). Alkaliphiles are mi-croorganisms with an optimum growth rate at pH 9 or higher(9). Halophiles grow optimally between 0.5 and 2.5 M NaCl(2, 10). Haloalkaliphiles are bacteria that thrive in both salineand alkaline environments such as soda lakes including LakeMagadi (Kenya); Mono Lake, CA; and Soap Lake, WA. In sodalakes, pH ranges from 10 to 12 and salinity ranges from 35to 310 g/L NaCl depending on the site (9). To date, mostpublished research on haloalkaliphiles has focused onmicrobiological classification and genetic characterization,with limited work to discover their biotechnological potential(11). In particular, little research has characterized halo-alkaliphile capabilities for environmental applications, eventhough they have a clear potential use in the degradation ofcontaminants (2).

Phenol is a toxic industrial compound whose presence inthe environment poses significant risks to aquatic biota andis lethal to fish at relatively low concentrations of 5-25 mg/L(12). Phenol has traditionally been removed from industrialeffluents by physicochemical methods (e.g., ozone treat-ment), but these treatments can be complex and expensive(13). In this study, phenol was chosen as the model compoundsince it is a toxic aromatic compound of environmentalinterest and biodegradation information is readily availablefor nonhaloalkaliphilic microorganisms. With phenol, limitedreports show biodegradation in either saline or alkalineconditions but not both. Woolard and Irvine (14) showedthat halophilic mixed cultures degraded phenol (with 99%phenol removal efficiency) in 140 g/L NaCl wastewater atneutral pH. Peyton et al. (15) reported biodegradation rateparameters and growth yields for halophilic mixed culturesthat were able to degrade up to 300 mg/L phenol in 100 g/LNaCl solutions. Hinteregger and Streichsbier (16) showedthat an unidentified Halomonas sp. degraded phenol insolutions up to 140 g/L NaCl at pH 7. In alkaline conditions,Kamekar et al. (17) reported phenol degradation at pH 10 bybacteria isolated from a 2.6 g/L NaCl soda lake in India.

It is known that aerobic nonhaloalkaphilic bacteria canmetabolize phenol to catechol. Catechol is further degradedby two known pathways. In the meta pathway, catechol istransformed to 2-hydroxymuconic semialdehyde (2 HMS)and further converted to acetaldehyde and pyruvate (18, 19).In the â-ketoadipate pathway, catechol is metabolized tocis,cis-muconate followed by (+)-muconolactone, which isfurther transformed into â-ketoadipate, succinate, and acetyl-CoA (20, 21). This latter pathway is widespread amongterrestrial microorganisms because it allows the use, assubstrates, of aromatic compounds produced by plants (22).

* Corresponding author phone: (509)335-4002; fax: (509)335-4806;e-mail: bmp@wsu.edu.

Environ. Sci. Technol. 2003, 37, 4397-4402

10.1021/es0341844 CCC: $25.00 2003 American Chemical Society VOL. 37, NO. 19, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4397Published on Web 09/06/2003

To our knowledge, the biodegradation of phenol by halo-alkaliphilic bacteria has not been previously reported. Wereport here that a haloalkaliphilic bacterium, Halomonascampisalis, is able to degrade phenol at pH values rangingfrom 8 to 11 and NaCl concentrations ranging from 0 to 150g/L. We also identify the degradative metabolic pathway andthe effect of different pH values and NaCl concentrations onthe distribution of metabolites.

Materials and MethodsMicroorganism and Culture Conditions. A pure culture ofH. campisalis, a moderately haloalkaliphilic bacteriumisolated near Soap Lake in central Washington (23), was grownon a basal mineral medium containing (in g/L) 0.25 KH2PO4,1.0 NH4Cl, 2.0 Na2B4O7, 0.0125 FeCl3, 0.06 CaCl2, and 0.05MgCl2. The medium was supplemented with a specifiedamount of added NaCl, 1 mL of trace element solution (15),and 10 mg of yeast extract per 100 mg of phenol. The mediumwas autoclaved and cooled to room temperature prior to pHadjustment with sterile filtered (0.45 µm) NaOH, catechol,and phenol. To provide inocula, glycerol-preserved samples(-80 °C) of H. campisalis previously grown in 50 mg/L phenolwere used to initiate each experiment. Aliquots of 10% (v/v)were transferred into fresh medium after 90% of the phenolhad been degraded. During late log-growth phase of the thirdtransfer, aliquots of 10% (v/v) were collected, centrifuged(20 °C, 2000g), washed, and resuspended in fresh medium.A cell-free control and three replicate flasks were used forthe experiment where cultures were grown in 500-mLErlenmeyer flasks (200 mL of liquid volume) fitted with foamstoppers and shaken at 140 rpm in an environmental chambermaintained at 30 °C, with limited exposure to light. AnEppendorf pipet (Westbury, NY) with a sterile tip was usedto remove samples for substrate, metabolites, and proteinanalysis at predetermined time intervals. Experiments werestopped 6 h after the substrate was exhausted. A cellcomposition of CH1.8O0.5N0.2 was used for carbon balancecalculations (24).

Analytical Techniques. Samples for phenol and itsmetabolites were centrifuged and analyzed by reversed-phasehigh-pressure liquid chromatography (HPLC) in a Hewlett-Packard 1100 series HPLC (Palo Alto, CA). The HPLCconditions for the determination of phenol, catechol, 2-HMS,muconolactone, and cis,cis-muconate were as follows: Zor-bak C18, 4.5 mm i.d × 150 mm column (Agilent Technologies,Palo Alto, CA); 80% mobile phase A (10 mM KH2PO4 bufferadjusted to pH 2.5); and 20% methanol. A UV detector wasused for most metabolites (phenol, 270 nm; (+)-mucono-lactone and cis,cis-muconate, 260 nm; 2-HMS, 375 nm), anda fluorescence detector was used for catechol. The detectionlimit for phenol and catechol was 0.05 mg/L and for (+)-muconolactone and cis,cis-muconate was 0.1 mg/L.

Samples were taken for optical density measurements at620 nm (8453 spectrophotometer Hewlett-Packard, Palo Alto,CA) and were correlated to protein measured using bovineserum albumin (BSA) as the standard according to Bradford(25). Cell samples for protein measurement were centrifuged,washed with fresh medium, and disrupted in a cold bath (4°C) in a sonicator with 3 pulses of 1 min each.

Headspace oxygen and carbon dioxide samples wereanalyzed with a Hewlett-Packard 5890 series gas chromato-graph (Agilent Technologies, Palo Alto, CA); with oven anddetector temperature of 250 °C and a 30 m × 0.5 mm i.d. GSMolesieve capillary column (J&W Scientific, Folson, CA).

Organic carbon (OC) and inorganic carbon (IC) wereanalyzed on a Shimadzu 5000 total organic carbon (TOC)analyzer (Kyoto, Japan) equipped with a nondispersiveinfrared detector. The OC concentration was obtained byseparately determining the IC concentration and the totalcarbon (TC) concentration in the sample and then subtracting

the IC concentration from the TC concentration (TOC ) TC- IC). The measurement of IC was obtained by reacting thesample with a strong acid (phosphoric acid) at ambienttemperature. The TC concentration in the sample wasobtained using high-temperature combustion (680 °C) ofthe sample with a platinum catalyst.

Phenol, catechol, and cis,cis-muconate were ACS reagentgrade from Sigma Chemical Co. (Milwaukee, WI). (+)-Muconolactone (commercially unavailable) was graciouslydonated by Dr. Nicholas Ornston, Yale University. Methanol(HPLC grade) was obtained from J. T. Baker (Phillipsburg,NJ). Phenol and catechol carbon balances were conductedin 500-mL serum bottles sealed by rubber septa, whichallowed liquid and headspace sampling without gas exchange.

Results and DiscussionPhenol Degradation at Various NaCl Concentrations. AtpH 9.5, H. campisalis degraded 130 mg/L phenol as a carbonand energy source at 0, 25, 50, 100, and 150 g/L NaCl addedto the basal medium (Figure 1). At all NaCl concentrationstested, catechol was produced and was subsequently de-graded. In contrast to catechol, cis,cis-muconate accumulatedin solution. (+)-Muconolactone was not detected in this seriesof experiments (data not shown). In general, salinity hadonly some influence on catechol, except at 150 g/L NaClwhere catechol minimally accumulated. Salinity had onlylimited effect on cis,cis-muconate accumulation (Figure 1).Only at 0 g/L NaCl was cis,cis-muconate completely degraded.Separate tests with cis,cis-muconate as a sole carbon andenergy source for H. campisalis at pH 9.5, 100 g/L NaCl,

FIGURE 1. Phenol (A), catechol (B), cis,cis-muconate (C), and protein(D) profiles during biodegradation at 0, 25, 50, 100, and 150 g/L NaClin pH 9.5 solutions. (+)-Muconolactone was not detected in anysample of this series (data not shown). Results are the average oftriplicates (error bars are the corresponding standard deviation).

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showed no growth (data not shown). Our results are inagreement with previous reports showing that many bacteriathat normally grow on phenol and catechol could not growon cis,cis-muconate alone (26) since it is highly polar andnot able to permeate the cell membrane (27). Our data suggestthat, at this low NaCl concentration, the H. campisalis cellmembrane may undergo changes that allow cis,cis-muconateto reenter the cell, thus allowing its complete degradation.A potentially related phenomenon was observed by Vreeland(28), who showed that the cell membrane of Halomonaselongata, a close relative of H. campisalis, was less compactand internally coherent and was less tightly bound to thecytoplasmatic membrane at lower salinity. At the same time,the overall cell wall lipid composition showed a decrease inthe amount of negatively charged lipids, thus decreasing itshydrophilicity (29). In addition, when H. elongata was grownin low salinity conditions, the cell wall presented observablefracture faces that were not present in high-salt-grown cells(29). A similar phenomenon may be occurring with the cellmembrane of H. campisalis that could allow cis,cis-muconateto reenter the cell (only at the lowest NaCl concentrationtested) such that complete degradation can occur. To ourknowledge, this is the first report of a bacterium that candegrade cis,cis-muconate in low salinity but is not able touse this same metabolite in medium and high salinityconditions.

While a key metabolite of the â-ketoadipate pathway(cis,cis-muconate) was detected in all tests, a key metabolitein the meta pathway (2-hydroxymuconic semialdehyde,2-HMS) was not detected in any sample. Furthermore,enzymatic tests at pH 8 and 0 g/L NaCl for were positive forcatechol 1,2-dioxygenase and negative for catechol 2,3-dioxygenase (data not shown). Therefore, it appears thatunder the range of conditions tested; only the â-ketoadipatepathway is used by H. campisalis for phenol biodegradation.Two Halomonas sp. have been reported to use the â-keto-adipate pathway for aromatics biodegradation in salineconditions and neutral pH (16, 30). Use of the â-ketoadipatepathway is reported to be more efficient in carbon conversionto cell mass (growth yield) than the meta pathway. The metapathway utilizes phenol at a higher rate but results in lowercell yields (31).

Phenol Degradation at pH 8-11. H. campisalis degradedphenol as the sole source of carbon and energy in 100 g/LNaCl at pH ranging from 8 to 11 (Figure 2). Catechol appearedtransiently and was later degraded. In contrast to salinityexperiments, with increased pH, significant accumulationof cis,cis-muconate was detected (Figure 2). The increasingaccumulation of cis,cis-muconate with increasing pH is ofenvironmental and industrial significance. These data un-derscore the fact that, in extreme environments, we cannotassume that contaminant biodegradation end points will beidentical to nonextreme counterparts. This result has im-plications on the fate of nonpoint source contaminants inextreme environments such as soda lakes.

From an industrial perspective, the microbial productionand accumulation of cis,cis-muconic acid in alkaline solutionsmay be of interest to industries where this metabolite is avaluable raw material. cis,cis-Muconate can be converted toadipic acid, a commodity chemical for nylon production,which is useful for new functional resins, pharmaceuticals,and agrochemicals (32, 33). In addition to cis,cis-muconateaccumulation, (+)-muconolactone, which follows cis,cis-muconate in the â-ketoadipate pathway (20, 21), was detectedat pH 8 and pH 9 but not at pH 10 and pH 11 (Figure 2).These results indicate that in our experiments its accumula-tion appears to be pH-dependent. Sistrom and Stanier (34)reported that, in experiments with cell-free extracts ofPseudomonas fluorescens, the transformation of cis,cis-

FIGURE 2. Phenol (A), catechol (B), cis,cis-muconate (C), (+)-muconolactone (D), and protein (E) profiles during biodegradationat pH 8, 9, 9.5, 10, and 11 in 100 g/L NaCl solutions. Results are theaverage of triplicates (error bars are the corresponding standarddeviation).

FIGURE 3. cis,cis-Muconate accumulation as a product of phenolbiodegradation by H. campisalis at different pH values in 100 g/LNaCl solutions (results fitted using the least-squares techniquefrom Sigma Plot, R 2 ) 0.88, standard deviation errors bar fromtriplicates).

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muconate to (+)-muconolactone is a pH-dependent, freelyreversible reaction catalyzed by the muconate lactonizingenzyme according to the reaction shown in eq 1:

In addition, Ornston and Stanier (35) reported that amuconate lactonizing enzyme, isolated from Pseudomonasputida, reduced its activity approximately 70% when pH wasincreased from 7 to 9. Therefore, in both Ornston and Stanier’sresults (35) and our results (Figure 2), higher pH solutionsgave more cis,cis-muconate accumulation and yielded less(+)-muconolactone. As shown in Figure 3, a log-linearrelationship is observed between pH and cis,cis-muconateeffluxed by H. campisalis. Our results indicate that catecholis transformed into cis,cis-muconate, some of which iseffluxed from the cell. Once outside, cis,cis-muconate cannotpermeate the membrane to re-enter the cell and thusaccumulates in the medium. At pH values used here, cis,cis-muconate is present in solution as an anion (pKa, 4.6,experimentally determined), which hinders permeabilityacross the bacterial membrane.

Cell Yields. Cell yields were found to vary with salinityand pH (Figure 4). The highest value (0.39 mg of cell protein/mg of phenol) was found at pH 9.5 and 100 g/L NaCl, whichcoincides with reported optimum growth conditions for H.campisalis (23). The range of yields observed (0.2-0.39 mgof cell protein/mg of phenol) is similar to previously reportedcell yields for mixed halophilic cultures also grown on phenol(36).

Carbon Balance for Phenol and Catechol Biodegrada-tion. A carbon balance at pH 8, 100 g/L NaCl, and 70 mg/Lphenol was performed. When phenol was completely de-graded (below the detection limit of 0.05 mg/L) and theculture had reached the late log phase of cell growth, the fateof phenol carbon in terms of carbon dioxide, cell carbon,metabolites, and residual phenol (Figure 5) gave a 93.8%carbon recovery. Most of the phenol carbon was recoveredas CO2 (55.5%) while only 5.5% of the carbon accumulatedas dead-end metabolites (cis,cis-muconate + unknown).These results are similar to reports for polynuclear aromatichydrocarbons (PAHs) and phenol biodegradation at lowsalinities and circumneutral pH (37, 38). On the basis of ourresults, assuming that cis,cis-muconate is the only metabolitethat accumulates significantly and using the chemicalequation balancing technique proposed by VanBriesen and

Rittman (39), an equation that summarizes phenol biodeg-radation at pH 8 and 100 g/L NaCl is proposed in eq 2:

Other researchers (40, 41) have reported similar equationsfor mesophilic phenol biodegradation; however, completemineralization of phenol was assumed. Metabolites otherthan cis,cis-muconate were not identified, but 5.5% of initialcarbon, designated as unknown, is hypothesized to be thepolymeric product of the abiotic transformation of catechol(42). A brownish color was observed that became darker withincreased pH. In addition, a very fine black precipitate wasobserved, which has been reported during abiotic catecholtransformation at low salinity in both neutral (43) and alkalineconditions (44).

Catechol Biodegradation. Figure 6 shows that H. camp-isalis can degrade 16 mg/L catechol (10.5 mg carbon/L) atpH 8 and 100 g/L NaCl with formation of cis,cis-muconateand concomitant formation of CO2. Uninoculated abioticexperiments at pH 8-11 (Figure 7) show that, althoughcatechol concentrations decreased, no CO2 or cis,cis-mu-conate were detected, and the corresponding organic carbon

FIGURE 4. Observed yields for phenol biodegradation experiments at (A) different salinities and (B) different pH values. Values representthe average calculated cell yield for each measurement where phenol concentration was <50% of the initial value. Error bars indicatethe 95% confidence interval.

cis,cis-muconate2- + H+ 798muconate lactonizing enzyme

(+)-muconolactone1- (1)

FIGURE 5. Carbon mass profiles for H. campisalis growing on phenolat pH 8 in 100 g/L NaCl medium. Carbon recovery refers to the sumof carbon contribution from inorganic carbon, carbon dioxide, cellcarbon, and organic carbon while total carbon was measured byTOC analyzer.

C6H6O + 5.33O2 + 0.35NH3 f 1.76CH1.8O0.5N0.2 +0.06C6H6O4 + 3.89CO2 + 1.77H2O (2)

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concentration did not change, thus indicating that catecholunderwent abiotic transformation. It can be seen that at pH8-11, abiotic catechol transformation rates were substantiallyfaster at higher pH. These data indicate that, for contaminantdegradation pathways that pass through catechol, abioticcatechol transformation (with the likely polymeric productaccumulation) may be a factor to consider in contaminantdegradation in alkaline industrial wastewater and naturalalkaline environments.

The haloalkaliphilic bacterium H. campisalis degradesphenol and catechol in alkaline (pH values of 8-11) andsaline environments (0-150 g/L NaCl) using the â-keto-adipate pathway. The valuable metabolite cis,cis-muconateincreasingly accumulated in the medium with increasing pH,thus presenting industrial and environmental implications.From an industrial point of view, the production of cis,cis-muconate is economically attractive since this metabolite isan intermediate in the production adipic acid and othervaluable chemicals. From an environmental perspective, thefate of contaminants in extreme environments, such as salineand alkaline lakes, may not be the same as the much moreextensively studied nonextreme environments. A carbonbalance for phenol biodegradation at pH 8 and 100 g/L NaClshows that most of the carbon is converted to CO2 and thatonly a small fraction accumulated as an unknown, likely tobe the polymeric product of abiotic catechol transformation.Experiments show that the rate of formation of this polymerof catechol increases with increasing pH; therefore, it maybe necessary to examine further the potential accumulationof metabolites in extremophile waste treatment systems.Clearly, there is a great potential for the use of haloalkaliphilesand potentially other extremophiles to expand the use ofbiological treatment of industrial wastewater. Furthermore,in extreme environments the ultimate fate of nonpoint sourcecontaminants cannot be assumed to be identical to non-

extreme conditions. Further study of contaminant biodeg-radation and fate in extreme conditions is necessary topromote understanding applicable to both natural andindustrial systems.

AcknowledgmentsThis project was supported by Washington State Universitythrough the Chemical Engineering Department, the Centerfor Multiphase Environmental Research, and the State ofWashington Water Research Center Grant 01HQGR0107. Weare also grateful to the National Science Foundation,Integrated Graduate Education and Research Training (IG-ERT) program and Microbial Observatories Program for theirsupport.

Literature Cited(1) Kargi, F.; Dincer, A. J. Environ. Eng. 1999, 125, 966-971.(2) Margesin, R.; Schinner, F. Appl. Microbiol. Biotechnol. 2001, 56,

650-663.(3) Kubo, M.; Hiroe, J.; Murakami, M.; Fukami, H.; Tachiri, T. J.

Biosci. Bioeng. 2001, 91, 222-224.(4) Prisciandaro, M.; Borghi, A.; Veglio, F. Ind. Eng. Chem. Res. 2002,

41, 5034-5041.(5) Rajganesh, B.; Sublette K. Biotechnol. Prog. 1995, 11, 228-230.(6) Carlos, T.; Maugans, C. Hydrocarbon Process. 2002, 89-92.(7) Sheu, S.; Weng, H. Water Res. 2001, 8, 2017-2021.(8) Peyton, B.; Yonge, D. Biodegradation of Non-Point Source

Pollutants in Soap Lake, Washington; Technical Report WRR-11; State of Washington Water Research Center: Pullman, WA,2002.

(9) Horikoshi, K. Microbiol. Mol. Biol. Rev. 1999, 63, 735-750.(10) Ventosa, A.; Nieto, J.; Oren, A. Microbiol. Mol. Biol. Rev. 1998,

62, 504-544.(11) Ventosa, A.; Nieto, J. World J. Microbiol. Biotechnol. 1995, 11,

85-94.(12) Saha, C.; Bhunia, F.; Kaviraj, A. Bull. Environ. Contam. Toxicol.

1999, 63, 195-202.(13) Lin, H.; Chuang, S. Technol. Environ. Chem. 1994, 44, 243-258.

FIGURE 6. Carbon concentration profiles for H. campisalis growingon catechol at pH 8 in 100 g/L NaCl medium. Catechol biodegradation,cis,cis-muconate accumulation, inorganic carbon, and cell carbonare also shown. In addition, the control for abiotic catecholtransformation is shown.

FIGURE 7. Abiotic catechol degradation at different pH values in100 g/L NaCl medium. Open symbols refer to catechol measured byHPLC. Closed symbols refer to organic carbon measured by TOCanalyzer.

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(14) Woolard, C.; Irvine, R. Water Res. 1995, 29, 1159-1168.(15) Peyton, B.; Wilson, T.; Yonge, D. Water Res. 2002, 36, 811-4820.(16) Hinteregger, C.; Streichsbier, F. Biotechnol. Lett. 1997, 19 (11),

1099-1102.(17) Kamekar, P.; Sarnaik, S.; Kelkar, S. J. Appl. Microbiol Symp. Suppl.

1999, 8, 128S-133S.(18) Wang, S.; Loh, K. Enzyme Microb. Technol. 1999, 25, 177-184.(19) Kang, M.; Park, J. J. Chem. Technol. Biotechnol. 1997, 69, 226-

230.(20) Stanier, R.; Ornston, L. In Advances in Microbial Physiology, 1st

ed; Rose, A. H., Tempest, D. W., Eds.; Academic Press: London,1973; Vol 9, Chapter 4.

(21) Bayly, R. C.; Wigmore, G. J. J. Bacteriol. 1973, 113, 1112-1120.(22) Parke, D.; D’Argenio, D.; Ornston, L. J. Bacteriol. 2000, 182,

257-263.(23) Mormile, M.; Romine, M.; Garcia, T.; Ventosa, A.; Bailey, T.;

Peyton, B. Syst. Appl. Microbiol. 1999, 22, 551-558.(24) Stephanopoulos, N.; Aristidou, A.; Nielsen, J. Metabolic Engi-

neering: Principles and Methodologies, 1st ed.; Academic Press:San Diego, 1998; p 119.

(25) Bradford, M. Anal. Biochem. 1976, 72, 248-254.(26) Ornston, N.; Parke, D. In Current Topics in Cellular Regulation;

Horecker, B., Stadtman, E., Eds.; Academic Press: New York,1977; Vol 12, Chapter 6.

(27) Ornston, N.; Stanier, R. J. Biol. Chem. 1966, 211, 3776-3786.(28) Vreeland, R. In The Prokaryotes, 2nd ed.; Ballows, A., Ed.; Spinger

Verlag: New York, 1992; Vol. I, Chapter 3.(29) Hart, D.; Vreeland, R. J. Bacteriol. 1988, 170, 132-135.(30) Rosenberg, A. Arch. Microbiol. 1983, 136, 117-123.

(31) Muller K. Acta Biotechnol. 2002, 3-4, 221-234.(32) Choi, J.; Lee, E.; Cho, M.; Choi, C. J. Ferment. Bioeng. 1997, 1,

70-76.(33) Niu, W.; Draths, K.; Frost, J. Biotechnol. Prog. 2002, 18, 201-

211.(34) Sistrom, W. R.; Stanier, R. Y. J. Biol. Chem. 1954, 210, 821-836.(35) Ornston, N.; Stanier, R. J. Biol. Chem. 1966, 211, 3776-3786.(36) Peyton, B.; Wilson, T.; Yonge, D. Water Res. 2002, 36, 4811-

4820.(37) Orupold, K.; Masirin, A.; Tenno, T. Chemosphere. 2001, 44, 1273-

1280.(38) Bouchez, M.; Blanchet, D.; Vandecasteele, P. Appl. Microbiol.

Biotechnol. 1996, 45, 556-561.(39) VanBriesen, J. M., Rittman, B. E. Biotechnol. Bioeng. 2000, 67,

35-52.(40) Maskow, T., Babel. W. Thermochim. Acta 1998, 309, 97-103.(41) Yuan, Z.; VanBriesen, J. M. Biotechnol. Bioeng. 2002, 80, 100-

113.(42) Borraccino, R.; Kharoune, M.; Giot, R.; Agatos, S.; Nyns, E.;

Naveau, H.; Pauss, A. Water Res. 2001, 15, 3729-3737.(43) Reardon, K.; Mosteller, D.; Rogers, B. J. Biotechnol. Bioeng. 2000,

69, 385-400.(44) Balla, J.; Kiss, T.; Jameson, R. Inorg. Chem. 1992, 31, 58-62.

Received for review February 27, 2003. Revised manuscriptreceived June 27, 2003. Accepted July 15, 2003.

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