Growth of Ralstonia eutrophaon inhibitory concentrations of phenol:diminished growth can be attributed to hydrophobic perturbation of

phenol hydroxylase activity

David Leonard1, Nicholas D. Lindley*Laboratoire Biotechnologie-Bioproce´des, UMR5504 INSA/CNRS and UR792 INRA, Centre de Bioingenierie Gilbert Durand, Institut National des

Sciences Appliquees, Complexe Scientifique de Rangueil, 31077 Toulouse cedex 04, France

Received 30 September 1998; received in revised form 10 February 1999; accepted 2 March 1999

Abstract

The effect of phenol concentration on growth and biodegradative capacity ofRalstonia eutropharegarding phenol was examined. Kineticanalysis indicated that phenol had a strong inhibitory effect on phenol metabolism and growth rate, although biomass yields remainedconstant, indicating that this phenomena was not caused by increased maintenance requirements. Measurements of specific enzyme activitiesinvolved specifically in the catabolic pathway of meta fission of phenol indicated that gene expression cannot explain the diminishedmetabolic rates at inhibitory phenol concentrations. This phenomenon is due to in vivo inhibition of enzyme activities and notably to phenolhydroxylase activity. Furthermore, other nonmetabolizable organic alcohols provoked a similar effect on both specific growth rate andphenol hydroxylase activity, indicating that inhibition was probably associated with modified membrane fluidity, partially offset by a changein the fatty acid composition of cellular lipids. © 1999 Elsevier Science Inc. All rights reserved.

Keywords: Ralstonia eutropha; Phenol inhibition; Phenol hydroxylase; Meta pathway

1. Introduction

Phenolic compounds are hazardous pollutants that aretoxic at relatively low concentrations. They can be detectedin effluents from oil refineries and both chemical and agro-chemistry industries. The effluents containing such com-pounds are usually treated in continuous activated sludgeprocesses, which are known to be sensitive to fluctuation inthe phenolic load [1]. Although phenol is not readily bio-degraded, many organisms have been shown to grow onphenol as the sole source of carbon. However, most of thesemicrobes show signs of substrate inhibition at concentra-tions as low as 0.5 mM [2]. The Haldane equation, whichrelates specific growth rate (m) to the concentration ofphenol taking into account the maximum specific growth

rate (mmax) and the substrate affinity (Ks) and inhibition (Ki)constants:

m 5 mmax3@phenol#

Ks 1 @phenol#1@phenol#2

Ki

(1)

has frequently been used to describe this inhibition [3,4],even though the biological phenomena underlying the ob-served growth kinetics has not been adequately explained.

Toxicity of aromatic compounds is frequently attributedto the disruption of membrane structure by hydrophobicinteractions with the lipid bilayer structure caused by thelipophilic nature of such compounds [5]. The partitioning oflipophilic compounds into the lipidic bilayer of membranesmight be expected to provoke significant changes in thestructure, the integrity, and the function of the membranes,thereby modifying the activities of enzymes directly asso-ciated with these membranes [6]. To some extent theseperturbations can be overcome by modifying the fatty acidsand lipopolysaccharide composition of the cell wall.Heipieper et al. [7] have shown that phenol induced an

* Corresponding author. Tel.:133-561-559-489; fax:133-561-559-400.

E-mail address:lindley@insa-tlse.fr (N.D. Lindley)1 Present address: SOREDAB, La Tremblaye, 78125 La Boissie`re

Ecole, France.

Enzyme and Microbial Technology 25 (1999) 271–277

0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.PII: S0141-0229(99)00039-3

increase in the degree of saturation of the membrane lipidsof Pseudomonas putidaand also provoked the isomerizationof the unsaturated fatty acids fromcis to trans isomers.

While such studies indicate that the cell membrane is aprobable site of growth inhibition, the nature of this inhibi-tion, particularly in species able to grow on phenol, remainsto a large extent speculative. In this study, the effect ofphenol concentration on the rate of growth and biodegrada-tion has been examined. The results show that growth in-hibition is primarily caused by the effect of phenol onsubstrate consumption rate, this being attributed to the in-hibition of phenol hydroxylase activity rather than a modi-fied expression of the genes encoding this enzyme. Further-more, similar effects were observed with other non-metabolizable solvents, suggesting that the modified in vivoenzyme activity can be attributed to hydrophobic stress andprobably involves a modified membrane structure.

2. Materials and methods

2.1. Bacterial strain and growth conditions

Ralstonia eutropha335 American Type Culture Collec-tion (ATCC 17697) was used throughout the present study.The mineral salts medium used for growth has been de-scribed previously [8]; the pH of the basal salt medium wasadjusted to 7, and the medium was sterilized by autoclavingat 121°C for 20 min. A stock solution of potassium phos-phate (1 M: pH 7) was autoclaved separately and added tothe sterile mineral salts medium at a final concentration of40 mM. Phenol was filter-sterilized through membranes(pore size of 0.2mm) and added to the medium beforeinoculation. A 3.5-l fermentor (Chemap, Volketswil, Swit-zerland) of 2-l working volume was used for batch cultures.The temperature was maintained constant at 30°C; the pH at7, with automatic addition of KOH (3 M); and the oxygenpartial pressure was maintained above 60% of air saturation(i.e. .0.132 mM dissolved oxygen in the medium), bymodifying both the stirrer speed and the volumetric air flowrate. The bioreactor was inoculated with 10% (v/v) late-exponential-phase shake flask cultures grown on phenol (5mM). The inoculum was aseptically centrifuged (50003 gfor 10 min at ambient temperature) and resuspended in freshmedium to remove any accumulated metabolites.

2.2. Measurement of fermentation parameters

Biomass was measured by cell dry weight determinationof filtered and washed culture samples after drying to con-stant weight under partial vacuum at 60°C. An averagebiomass formula of C4H6.98O1.84N0.86 (with 3.6% ash), de-termined by elemental analysis (Ecole de Chimie, Toulouse,France) was used for stoechiometric balancing.

The concentration of phenol was analyzed by high pres-sure liquid chromatography by using an Aminex (Bio-Rad,

Ivry sur Seine, France) HPX-87H column (3003 7.8 mm)and the following operating conditions: temperature, 65°C;mobile phase, 5 mM H2S04 1 7% (v/v) CH3CN; flow rate,0.8 ml/min. Detection was made at 210 nm with a variable-wavelength UV detector, and quantification was by peakintegration.

Gas flow rates were measured in both the inflowing andoutflowing gas lines, and gas composition was analyzed bygas chromatography methods with the use of samples takendirectly from the gas outlet line injected into a two-columnseparation technique (Porapak Q (Supelco, Saint-QuentinFallavier, France), followed by a 0.5 nm molecular sieve)maintained at 40°C with helium as carrier gas and catharo-metric detection as previously described [9]. The amount ofCO2 produced by the microorganism was calculated fromthe increased percentage of CO2 In the effluent gas ascompared to the air inlet concentration.

Specific rates of phenol consumption and gas metabo-lism were estimated by interpolation of the measured vari-ations in the concentrations of these compounds and inbiomass accumulation after smoothing the curves by usingthe best-fit computer program Sigma Plot® (Jandel Scien-tific, Erkrath, Germany).

2.3. Determination of enzyme activities

Approximately 50–100 mg (wet weight) freshly har-vested cells was washed twice in 50 mM Tris/HCl (pH 7.5,50 mM) at 4°C and resuspended into 5 ml of Tris/carbal-lylate buffer (pH 7.8, 9 mM Tricarballylic acid1 35 mMTris/HCl) containing MgCl2 (5 mm) and glycerol (20%v/v). The cells were disrupted By ultrasonication (4 cyclesof 20 s interspaced with 1-min periods of cooling on ice),and the resulting crude extracts were centrifuged (15 0003g for 10 min at 4°C) to obtain soluble protein extracts,which were used to assay enzyme activities. Phenol hydrox-ylase, catechol 2,3-dioxygenase (EC1.13.1.2), and 2-hy-droxymuconic semialdehyde acid dehydrogenase activitieswere measured by previously described methods [9]. Spe-cific activities of enzymes measured in cell extracts wereexpressed relative to protein content as determined by themethod of Lowry et al. [10] by using bovine serum albuminas the protein standard, and extrapolated to whole cell ac-tivities by assuming that soluble protein accounted for 50%of the dry cell weight.

2.4. Lipid extraction and analysis

Cell suspensions (containing the equivalent of 50 mg drycells) were centrifuged, washed with Tris/HCl (pH 7.5, 50mM). Cells were broken after freezing in liquid nitrogen andthawing to room temperature and were then subjected toultrasonic disruption for 5 min. The lipids were extractedwith chloroform:methanol (2:1) under argon for 2 h underreflux before recovering the organic phase and evaporatingunder argon. The extracted lipids were then saponified by

272 D. Leonard, N.D. Lindley / Enzyme and Microbial Technology 25 (1999) 271–277

suspending in 500ml of a solution of 20% (w/v) KOH and8% (v/v) ethanol. The suspension, placed in sealed tubes,was incubated at 110°C for 2 h before cooling and theaddition of 10% (v/v) H2SO4. The free acids were thenextracted into ether, recovered, and evaporated under nitro-gen. Esterification of the fatty acids was by methylationwith diazomethane (added until a persistent yellow colorwas observed) after resuspending in ether. After 15 minincubation at room temperature, the reaction mixture wasevaporated under nitrogen, and the methyl esters thus ob-tained were analyzed by vapor phase chromatography byusing a capillary column (OV1 WCOT (Supelco, Saint-Quentin Fallavier, France), of 25 m3 0.22 mm). Productseparation involved a temperature gradient (100–280°C at2°C/min) after injection at 250°C. Helium was used as thegas phase, and detection was by flame ionization at 300°C.all samples were diluted in petroleum ether to give a con-centration of approximately 50 ng/ml, and an injection vol-ume of 1ml was used. Pentadecanoic acid was used as aninternal standard, and the peaks were identified by retentiontimes compared to a standard solution containing equimolarquantities of a range of fatty acids methylated by the sameprocedure as used for cell lipids.

2.5. Chemicals

All chemicals were analytical grade and obtained fromeither Sigma Chimie (St Quentin Fallavier, France) or C. F.Boehringer & Soehne (Mannheim, Germany), except for2-hydroxymuconic semialdehyde, which is not availablecommercially and was therefore synthesized by the proce-dure described by Feist et al. [11].

3. Results

3.1. Effect of phenol concentration on the growth ofR.eutropha

The growth on phenol (8 mM) was not exponential(Fig. 1). As phenol concentration decreases, both the growthrate (m) and the consumption rate of phenol (qphenol) in-creased to reach maximal values ofmmax5 0.366 0.02 h21

and qphenol max5 5.76 0.2 mmol g21 h21, values similar tothose obtained at the critical dilution rate in carbon-limitedchemostat cultures [9]. During the period in which specificrates of growth and phenol consumption were approachingthe maximal rates obtained, a yellow color characteristic of2-hydroxymuconate semialdehyde (2-hms) accumulationwas observed. The 2-hms was re-assimilated toward the endof the culture when specific rates decreased because of thedepletion of phenol from the medium. Phenol was com-pletely mineralized when cell growth was finished.

The acceleration of the growth rate when phenol concen-tration decreased was characteristic of a substrate inhibitionphenomenon. To further characterize this inhibition effect, a

series of batch cultures with various initial phenol concen-trations of between 1 and 15 mM were examined. Kineticparameters were measured in the initial growth phase (i.e.the first 3 h of growth and before significant changes ineither the pH or the phenol concentration within the cul-ture), and it can be seen that phenol exerts a strong inhibi-tory effect on both the growth rate and the phenol degrada-

Fig. 1. Fermentation time course for the growth ofRalstonia eutrophaonphenol and the specific metabolic rates associated with this culture. Phenolconcentration (f), biomass concentration (Œ), specific rates of growth(solid line), phenol consumption (dotted line), and CO2 production (E).

Fig. 2. Effect of phenol concentration on initial rates of growth (Œ) andphenol consumption (f) in batch cultures ofR. eutrophaon phenol. Theinset shows the linear relationship between these two specific metabolicrates.

273D. Leonard, N.D. Lindley / Enzyme and Microbial Technology 25 (1999) 271–277

tion rate (Fig. 2). No growth was obtained at phenolconcentrations higher than 12 mM. It is important to notethat the inhibitory effect of phenol on growth is directlycorrelated, in a linear manner, to the diminished rate ofphenol consumption (Fig. 2, inset). This linear relationshipbetween growth rate and substrate consumption rate, asobserved in chemostat experiments in which growth ratewas varied by feeding rate, while retaining extremely lowresidual phenol concentrations [9], thereby overcoming sub-strate inhibition phenomena, indicates that biomass yieldswere constant at all phenol concentrations (0.67 g biomass/gphenol; value measured experimentally). As would be ex-pected, CO2 production was also closely correlated to phe-nol consumption (Fig. 1) and indicates that no significantcarbon was accumulating as overflow metabolites. Further-more, this constant biomass yield indicates that mainte-nance energy requirements do not increase as a function ofthe phenol concentration, in contrast to the situation ob-served for this strain with benzoate [12]. These resultssuggest that phenol inhibition is directly related to a dimin-ished capacity to catabolize phenol. The absence of metab-olite accumulation other than 2-hms at relatively high ratesof phenol degradation for which 2-hms dehydrogenase hasbeen shown to be rate limiting [9] suggests that the initialreactions of phenol catabolism are the principle sites ofaction for this inhibition. In light of this, the specific activityof key enzymes was examined to determine whether thisphenomenon could be attributed to gene expression phe-nomena or to perturbed biochemical activity of the en-zymes.

3.2. Effect of phenol concentration on the level ofcatabolic pathway enzyme expression

The activities of the first three catabolic enzymes (phenolhydroxylase; catechol 2,3 dioxygenase; 2-hms dehydroge-nase) and the two enzymes used for the assimilation ofacetaldehyde [the acetaldehyde dehydrogenase and theacetyl-coenzyme A (CoA) synthetase] were assayed fromcells sampled throughout the batch culture described above

(Fig. 1). Activities of 2-hms dehydrogenase, acetaldehydedehydrogenase, and acetyl-CoA synthetase were constantwhatever the phenol concentration, whereas activities ofphenol hydroxylase and catechol-2,3-dioxygenase decreaseslightly as phenol depletion took place (Table 1). Similarmodifications in enzyme activity were seen when phenolaccumulated in chemostat washout experiments; phenol ac-cumulates and provokes a diminished growth rate (Fig. 3).These variations were similar to those seen in response tothe modified growth rate in phenol-limited chemostat cul-tures [9]. It would appear, therefore, that these variations inspecific enzyme activities can be attributed to growth rate-dependent modifications of gene expression rather than to adirect response to the phenol concentration. Furthermore,the observed modifications in enzyme activity would tend toincrease the concentration of catabolic enzymes under con-ditions in which diminished rates of phenol degradation andgrowth inhibition were observed. In light of these results, itwould appear that the inhibitory effect of phenol is mostprobably caused by a biochemical phenomenon modifyingthe in vivo enzyme activity.

Table 1Specific activities of the enzymes of the phenol degradation pathway during batch cultures described (Fig. 1) and washout experiment (Fig. 3)

Specific activities (mmol/min mg21 of proteins)

Time (h) Phenol(mM)

Phenolhydroxylase

Catechol-2-3-dioxygenase

Aldehydedehydrogenase

Acetaldehydedehydrogenase

Acetyl-coAsynthase

Batch5.75 6.1 0.22 1.086 0.05 ND 0.0476 0.01 0.156 0.0199.6 3.3 0.20 1.116 0.025 0.446 0.04 0.0556 0.003 0.146 0.012

11.2 0.26 0.17 0.766 0.023 0.476 0.09 0.0436 0.004 0.146 0.013Chemostat washout

1.25 0.15 NDa 0.306 0.02 0.196 0.05 ND ND1.6 2.7 ND 0.556 0.03 0.316 0.04 ND ND3.3 8.7 ND 0.686 0.03 0.346 0.06 ND ND

a ND 5 no data.

Fig. 3. Washout phase of a phenol-grown chemostat culture ofR. eutrophacaused by an increase in the dilution rate from 0.29 h21 at time 0 to 0.33h21. Phenol outlet concentration (f), biomass concentration (Œ), 2-hmsconcentration (l), and specific growth rate (E).

274 D. Leonard, N.D. Lindley / Enzyme and Microbial Technology 25 (1999) 271–277

3.5. Effect of phenol concentration on the in vitro activityof catabolic enzymes

The first three enzymes (phenol hydroxylase, catechol-2,3-dioxygenase, and 2-hms dehydrogenase) involved inphenol catabolism were inhibited to varying extents whenthe in vitro phenol concentration was modified (Fig. 4).When 10 mM phenol was added to the assay mixtures,catechol-2,3-dioxygenase maintained 85% and 2-hms dehy-drogenase 45% of maximum activity, while phenol hydrox-ylase retained only 25% of its maximal activity. Phenolhydroxylase, the first enzyme of the pathway, is the mostinhibited by phenol concentration. The Haldane equation(1) models the inhibitory effect of phenol on this enzymeand predicts an apparentKs of 10 mM and an apparentKi of3.6 mM. This result, however, has to take into account thatphenol hydroxlase activity is assayed in whole cells, andtherefore, any effect of catechol-2,3-dioxygenase (this canbe excluded in view of the specific data concerning thisactivity) or the respiratory chain activities could introduceexperimental artifacts. Before it can be concluded that phe-nol hydroxylase is the most probable site of phenol-relatedinhibition, the possible effects on respiratory activity needto be examined.

3.6. Effect of phenol on cell respiration

The effect of phenol concentration on the respiratoryactivity of R. eutrophawas evaluated by measuring oxygenconsumption of whole cells harvested from fructose-growncultures. Washed cells, resuspended in fresh, air-saturatedmedium containing fructose (5 mM) and variable phenolconcentrations were examined for their respiratory con-sumption of oxygen. The time of the test (approximately 15min) was too short for induction of the phenol catabolic

enzymes shown to be absent at significant levels in fructose-grown cells. Respiratory activity on fructose was inhibitedby phenol. This inhibitory effect was less important thanthat observed on the phenol hydroxylase activity (seeabove): at a phenol concentration of 10 mM, 50% decreasein respiration was observed as compared to 75% inhibitionof phenol hydroxylase activity. This indicates that respira-tory activity on fructose is less inhibited than phenol hy-droxylase activity, but extrapolation of this result to phenol-grown cells is somewhat hazardous in light of the branchednature of the respiratory chain [13]. The use of KCN atvarious concentrations confirmed the presence of three dif-ferent oxygenases with differing sensitivity to KCN in fruc-tose-grown cells, as postulated by Komen et al. [13], whoused a similar approach. For phenol-grown cells, metabolicflux calculations have shown that approximately 40% ofoxygen consumption observed in cells catabolizing phenolcan be attributed to respiratory consumption, the remainderbeing directly associated with phenol hydroxylase and cat-echol-2,3-dioxygenase activities. Total inhibition of this re-spiratory activity was obtained by addition of 0.2 mM KCN,enabling phenol hydroxylase to be assessed for its sensitiv-ity to phenol in the absence of potential artifacts caused byrespiratory activity. Under these conditions, it is clear thatphenol hydroxylase is even more sensitive than estimated,with a Ki value of approximately 2 mM.

3.7. Effect of alcohols on phenol-hydroxylase activity

Because the major site for phenol-associated inhibitionof both growth and phenol consumption appears to be thephenol hydroxylase activity, the question arises whether thisis a typical substrate-inhibition phenomenon or a more gen-eral effect caused by the chemical properties of phenol.Alcohols are known to interact with the lipid bilayer ofcells: the insertion of these lipophilics agents into the cel-lular membrane provokes a change in the degree of freedomor fluidity of the membrane [6,14]. Indeed when cell lipidswere analyzed, the fatty acid profile during growth on phe-nol was seen to be somewhat different than that seen oneither fructose or acetate. During growth on phenol, themembrane lipids ofR. eutrophacontain predominantlypalmitic acid (C 16:0), palmitoleic acid (C 16:1), and oleicacid (C 18:1) in equal amounts, whereas considerably lowerlevels of palmitic acid and higher levels of oleic acid areseen on both fructose and acetate (Table 2). These modifi-

Fig. 4. Effect of phenol concentration on the in vitro specific activity ofvarious enzyme activities in cells harvested from rapidly growing batchcultures ofR. eutrophaon phenol. Phenol hydroxylase as measured di-rectly (F) or independently of respiratory activity (E), catechol-2,3-dioxy-genase (f), and 2-hms dehydrogenase (Œ).

Table 2Change in fatty acids composition ofRalstonia eutropha

Substrate % of fatty acids

C 16:0 C 16:1 C 18:1

Fructose 21 37.6 40.4Acetate 22.9 30.8 45.9Phenol 31.8 35.4 31.3

275D. Leonard, N.D. Lindley / Enzyme and Microbial Technology 25 (1999) 271–277

cations of the degree of fatty acid saturation are typical ofthose seen in microorganisms when membrane fluidity isperturbed by the accumulation of hydrophobic fermentationproducts. Unlike what has been described forP. putida[7,15] no trans isomer of the unsaturated acids (palmitoleicacid or oleic acid) occurred during growth on phenol, eventhough our analytical method did enable good separation ofa commercial mixture of palmitoleic acid isomers (cis andtrans) esterified by the same techniques as used for cell lipids.

To examine whether this phenomenon was due to mod-ified membrane fluidity, three other non-metabolizable al-cohols were selected according to their hydrophobicity, ormore precisely their partition coefficient between an organic(octanol) phase and an aqueous phase, i.e. Log (PO/W), andexamined for their effect on cell growth on fructose. Theinhibitory effect of the chosen alcohols [butanol 2 (LogP 50.78), t-amyl alcohol (LogP 5 1.40), phenol (LogP 51.57), and 4-methyl-2-pentanol (LogP 5 1.80)] on growthof R. eutrophacorrelated both to an increase of the Log Pand the concentration (Fig. 5). It would appear, therefore,that hydrophobic compounds other than phenol are able tobring about inhibition of cell growth.

After these preliminary tests, the effect of thet-Amylalcohol, which has a partition coefficient similar to that ofphenol, on phenol hydroxylase activity was examined with cellsuspensions containing both KCN (0.2 mM, see above) and thesubstrate phenol at a non-inhibitory concentration (0.2 mM).The t-Amyl alcohol inhibited phenol hydroxylase activitywith a Ki of 40 mM (coherent with the effect on specificgrowth rate), suggesting that at least some of the inhibitionphenomena can be attributed to hydrophobic effects.

4. Discussion

Phenol can be used as a sole source of carbon and energyby R. eutropha. This strain has a maximal growth rate (mmax

5 0.36/h), in batch culture, lower than other phenol degrad-ers, such asP. putida(mmax 5 0.48/h [16]) andR. eutrophaJMP134 (mmax 5 0.4/h [17]). This reduced growth capacityon phenol has been attributed to the low specific activities ofthe 2-hms dehydrogenase activity [9] as revealed by theaccumulation of 2-hms, a yellow intermediate of the metapathway at high growth rates.

In this study the substrate inhibition phenomenon hasbeen characterized and seen to be within the same range(0–12 mM) as seen for other bacteria. This similarity insubstrate inhibition kinetics is coherent with a non-specificmechanism of phenol inhibition. To characterize this moreprecisely, phenol inhibition onR. eutrophagrowth wasexamined in relation to a number of physiological charac-teristics indicative of metabolic activity.

The decreased growth rate at inhibitory substrate con-centrations was directly correlated to the diminished phenolconsumption rate. Whatever the phenol concentration, bio-mass yield was constant (0.67 g/g), and therefore, growthinhibition in R. eutrophais not attributable to increasedmaintenance phenomena, as postulated previously for thisspecies [18], but confirming the observations of Yang andHumphrey [4] with P. putida. This constant growth effi-ciency is not caused by a significant modification of geneexpression. Indeed, as growth slows in washout culturesbecause of the accumulation of phenol, some enzyme activities(e.g. 2-hms dehydrogenase and catechol-2,3-dioxygenase,but not phenol hydroxylase) increase slightly, coherent withthe activity profiles already seen in phenol-limited steadystate chemostats [9]. This increase in enzyme activity wouldoffset the inhibitory phenomenon of phenol on these cyto-solic enzymes, assuming that free phenol occurs in the cells.More likely, the phenol is localized in the lipidic part of themembrane, where its accumulation provokes a change inmembrane fluidity, which will certainly exert an inhibitoryeffect on enzymes localized within the cell membrane [19].To some extent, this is compensated for by a change incellular lipids, as seen for other microorganisms subject tohydrophobic stress [14,20]. This modified membrane integ-rity would certainly explain the diminished respiration ratesobserved for fructose-grown cells in the presence of phenol.Keweloh et al. [21] examining similar phenomena for phe-nol in Escherichia coli explained this by the increasedpermeability of the cell membrane to protons, although thisis unlikely to play an important role here because it wouldlogically lead to an increased maintenance coefficient,thereby modifying the biomass yield.

Phenol hydroxylase is, however, the major site for phe-nol inhibition, and this effect is also seen with other non-metabolizable, hydrophobic compounds, suggesting thatthis enzyme is highly sensitive to hydrophobic stress. Thesubcellular localization of phenol hydroxylase, the enzymemost effected by phenol, is not yet known, but it is temptingto speculate that the most logical site would be the cellmembrane, thereby avoiding penetration of phenol into thecytosol. Because the rest of cell metabolism is presumably

Fig. 5. Effect of various organic alcohols on the specific growth rate (m) ofR. eutrophaon fructose. Phenol (F),t-Amyl-alcohol (Œ), butanol (itrif]t),and 4-methyl-2-pentanol (E).

276 D. Leonard, N.D. Lindley / Enzyme and Microbial Technology 25 (1999) 271–277

able to function more rapidly under conditions in whichphenol hydroxylase is controlling the flux into the metapathway, one possibility to improve the growth of this strainat otherwise inhibitory phenol concentrations might be toamplify the expression of the genes encoding the multicom-ponent phenol hydroxylase activity because these geneshave recently been cloned and sequenced from anotherR.eutrophastrain [22].

References

[1] Allsop PJ, Chisti Y, Moo-Young M, Sullivan GR. Dynamics ofphenol degradation byPseudomonas putida. Biotechnol Bioeng1993;41:572–80.

[2] Muller R. Bacterial degradation of xenobiotics. In: Fry JC, Gadd GM,Herbert RA, Jones CW, Watson-Craik IA, editors. Microbial control ofpollution. Cambridge, UK: Cambridge University Press, 1992. p. 35–58.

[3] Sokol W. Oxidation of an inhibitory substrate by washed cells (oxi-dation of phenol byPseudomonas putida). Biotechnol Bioeng 1987;30:921–7.

[4] Yang RD, Humphrey AE. Dynamic and steady state studies of phenolbiodegradation in pure and mixed cultures. Biotechnol Bioeng 1975;17:1211–35.

[5] Sikkema J, de Bont JAM, Poolman B. Interactions of cyclic hydro-carbons with biological membranes. J Biol Chem 1994;269:8022–8.

[6] Sikkema J, de Bont JAM, Poolman B. Mechanism of membranetoxicity of hydrocarbons. Microbiol Rev 1995;59:201–22.

[7] Heipieper HJ, Diefenbach R, Keweloh H. Conversion of cis unsatur-ated fatty acids to trans, a possible mechanism for the protection ofphenol-degradingPseudomonas putidaP8 from substrate toxicity.Appl Environ Microbiol 1992;58:1847–52.

[8] Ampe F, Lindley ND. Acetate utilization is inhibited by benzoate inRalstonia eutropha: Evidence for transcriptional control of the ex-pression ofacoEcoding for acetyl-CoA synthetase. J Bacteriol 1995:177:5826–33.

[9] Leonard D, Lindley ND. Carbon and energy flux constraints incontinuous cultures ofRalstonia eutrophagrown on phenol. Micro-biology 1998;144:241–8.

[10] Lowry OH, Rosebrough NJ, Farret AL, Randall RJ. Protein mea-surement with the Folin phenol reagent. J Biol Chem 1951;193:265–75.

[11] Feist CF, Hegeman GD. Phenol and benzoate metabolism byPseudo-monas putida: Regulation of tangentiel pathways. J Bacteriol 1969;100:869–77.

[12] Ampe F, Lindley, ND. Flux limitations in the ortho pathway ofbenzoate degradation ofRalstonia eutropha: metabolite overflow andinduction of themetapathway at high substrate concentrations. Mi-crobiology 1996;142:1807–17.

[13] Komen R, Zannoni D, Schmidt K. The electron transport system ofRalstonia eutrophaH16. II. Respiratory activities and effect of spe-cific inhibitors. Arch Microbiol 1991;155:436–43.

[14] Ingrahm LO. Adaptation of membrane lipids to alcohols. J Bacteriol1976;125:670–8.

[15] Weber FJ, Isken S, de Bont JAM. Cis/trans isomerization of fattyacids as a defence mechanism ofPseudomonas putidastrains to toxicconcentrations of toluene. Microbiology 1994;140:2013–17.

[16] Hill GA, Robinson CR. Substrate inhibition kinetics: Phenol degra-dation by Pseudomonas putida.Biotechnol Bioeng 1975;121:272–85.

[17] Muller RH, Babel W. Determination of the Ks values during thegrowth of Ralstonia eutrophaon phenol, 2,4-dichlorophenoxyaceticacid and fructose. Acta Biotechnol 1995;15:347–53.

[18] Muller RH, Simon D, Grobe HJ, Babel W. Substrate inhibition understationary growth conditions –nutristat experiments withRalstoniaeutrophaJMP 134 during growth on phenol and 2,4-dichlorophe-noxyacetate. Appl Microbiol Biotechnol 1997;48:648–55.

[19] Weber FJ, de Bont JAM. Adaptation mechanisms to the toxic effectsof organic solvents on membranes. Biochim Biophys Acta 1996;1286:225–45.

[20] Heipieper HJ, Keweloeh H, Rehm HJ. Influence of phenols on growthand membrane permeability of free and immobilizedEscherichiacoli. Appl Environ Microbiol 1991;57:1213–17.

[21] Keweloh H, Weyrauch G, Rehm HJ. Phenol-induced membraneschanges in free and immobilizedEscherichia coli. Appl MicrobiolBiotechnol 1990;33:66–71.

[22] Hino S, Watanabe K, Takahashi N. Phenol hydroxylase cloned fromRalstonia eutrophastrain E2 exhibits novel kinetic properties. Mi-crobiology 1998;144:1765–72.

277D. Leonard, N.D. Lindley / Enzyme and Microbial Technology 25 (1999) 271–277