APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1980, p. 511-5170099-2240/80/03-0511/07 $02.00/0

Vol. 39, No. 3

Cell Surface Measurements in Hydrocarbon and CarbohydrateFermentations

R. J. NEUFELD,* J. E. ZAJIC,t AND D. F. GERSONtBiochemical Engineering, Faculty ofEngineering Science, The University of Western Ontario, London,

Ontario, Canada N6A 5B9

Acinetobacter calcoaceticus was grown in 11-liter batch fermentations withhexadecane or sodium citrate as the sole source of carbon. Surface and interfacialtension measurements of the microbial broth indicated that surface-active com-pounds were being produced only during growth on the hydrocarbon substrate.Contact angle measurements of an aqueous drop on a smooth lawn of cells in ahexadecane bath indicated a highly hydrophobic surface of the cells in the initialstages of the hydrocarbon fermentation (1200 contact angle). At this stage, theentire cell population was bound to the hydrocarbon-aqueous interface. Thecontact angle dropped rapidly to approximately 450 after 14 h into the fermen-tation. This coincided with a shift of the cell population to the aqueous phase.Thus, the cells demonstrated more hydrophilic characteristics in the later stagesof the fermentation. Contact angles on cells grown on sodium citrate ranged from18 to 240 throughout the fermentation. The cells appear to be highly hydrophilicduring growth on a soluble substrate. From the contact angle and aqueous-hydrocarbon interfacial tension, the surface free energy of the cells was calculatedalong with the cell-aqueous and cell-hydrocarbon interfacial tension. The resultsof these measurements were useful in quantitatively evaluating the hydrophobicnature of the cell surface during growth on hydrocarbons and comparing it withthe hydrophilic nature of the cell surface during growth on a soluble substrate.

A great deal of interest has been expressed inrecent years about the mechanisms by whichmicrobial cells absorb and metabolize liquid hy-drocarbons (3, 6, 9, 11-14, 18, 19, 22, 27, 30).Utilization of hydrocarbons by microbes often isaccompanied by the production of extracellularsurface-active materials which stimulate growthdue to increased emulsification of the hydrocar-bon phase (6, 7). Noticeably lacking in the re-ported research are measurements and discus-sions of the various interfacial phenomena in-volved in the successful attack of the hydrocar-bon by the microbial cell. This has been due inpart to difficulties encountered in evaluating thecell-aqueous and cell-hydrocarbon interfacialtensions.This paper specifically deals with the interfa-

cial phenomena as they relate directly to themicrobial cell surface throughout the course ofa hydrocarbon fermentation. This involves themeasurement of the cell surface free energy andthe cell-hydrocarbon and cell-aqueous interfa-cial tensions.An interface may be defined as the boundary

t Present address: College of Science, University of Texasat El Paso, El Paso, TX 79968.

t Present address: Basel Institute for Immunology, CH-4058, Basel 5, Switzerland.

511

between two distinct phases in a heterogeneoussystem. An obvious example of a liquid-liquidinterface is the aqueous-oil interface in a hydro-carbon fermentation. Due to the immiscibility ofoil in water, this interface provides a distinctboundary to which cells with more hydrophobicsurfaces tend to be attracted. An organism wouldhave an advantage when colonizing the interfa-cial region if it had a relatively hydrophobicsurface combined with the ability to metabolizethe hydrocarbon phase. A few papers have de-scribed the importance of the available interfa-cial area to the rates of growth of various mi-crobes on hydrocarbons (6, 9, 31).The boundary between any pair of phases

possesses a surface tension (10). This surfacetension may be conceptualized in terms of aforce per unit length of boundary of the surfaceor as the energy per unit area of surface. Theunits involved are dimensionally equivalent:

newton newton m joulem m m 2 (1)

For convenience, liquid surfaces are more eas-ily handled as a surface force, whereas solidsurfaces are normally treated as a surface freeenergy even though these concepts are thermo-dynamically equivalent.

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512 NEUFELD, ZAJIC, AND GERSON

Although the interfacial tension of liquids hasbeen relatively easy to measure, determinationof the surface free energy of solids has beendifficult. Recent work by Zisman (32) and Neu-mann et al. (25) has provided a mechanism bywhich the surface energy of low-energy solidsurfaces may be evaluated. This is important asit relates to hydrocarbon fermentations, becauseit provides a direct measure of the cell hydro-phobicity which is necessary to understand thewetting of the microbial surface by hydrocarbonand the adhesion of microbial cells to the oil-water interface. Solid surface free energies maybe determined by using the contact angle tech-nique described in the literature (4, 23, 28, 29).This technique is based on the hypothesis ofThomas Young in 1805 that the competitionbetween the cohesive forces of a liquid to itselfand the adhesive forces between the liquid anda solid surface result in a contact angle which atequilibrium is constant and specific to the par-ticular system (2). The equation normally usedto describe this situation is:

YSV- YSL = YLV COS 0 (2)

where YLv is the liquid surface tension, ysv is thesolid surface free energy, -YSL is the interfacialtension between solid and liquid, and 0 is theequilibrium contact angle (Fig. 1).

In practice, an equilibrium contact angle isdifficult to determine, since contact angles showhysteresis (2) between an advancing maximumand a receding minimum. These angles may beachieved by advancing or receding the liquiddrop over the solid surface. The main causes ofhysteresis are heterogeneity of the surface androughness. Neumann and Good (24) demon-strated that low-energy components of a smoothheterogeneous solid surface determine the ad-vancing angle, whereas, higher-energy compo-nents determine the receding angle. Since someinteractions are to be expected between anaqueous drop and a cell surface, an advancingcontact angle would provide more useful infor-mation (29). This angle is thermodynamicallysignificant (24) and may be used in conjunctionwith the Young equation (equation 1). In thepresent study, contact angles were obtained bypermitting an aqueous drop to fall onto the cellsurface from a fixed height. This angle may beequated with the advancing contact angle.Once a contact angle has been determined,

equations 3 and 4 may be used to calculate thecell surface free energy and the interfacial ten-sion between the cell and the liquid phase (25).

( 1_7_)2

1 - 0.015 Is YILv

VAPOUR

LIQUID \--*-LAWN OF CELLS

SOLID SURFACE

FIG. 1. Contact angle of sessile drop on solid sur-face. YLV is the liquid surface tension; ysv is the solidsurface free energy; YsL is the interfacial tensionbetween solid and liquid; and 0 is the contact angle.

(0.015 Ysv - 2.00) '/ysv YLV + YLVcos (=

YLV (0.015 'y-sv YLV - 1)(4)

Neumann et al. (25) have published a Fortranprogram which incorporates the necessary selec-tion criteria in the determination of ysv and YSLfrom equations 3 and 4. Microbial cells range insurface energy from 15 mJ/m2 for the highlyhydrophobic organism, Mycobacterium butyri-cum, up to 72 mJ/m2, the approximate surfacetension of water (8).

MATERLALS AND METHODSIsolation and maintenance of cultures. The mi-

crobe studied herein has been tentatively identified asAcinetobacter calcoaceticus. This culture was isolatedfrom a chunk of asphalt found in the Thames River,London, Ontario. Isolation was accomplished on amineral salts medium (pH 7.0) with 2% (vol/vol) Essono. 9 kerosene added as the carbon source. The basicmineral salts medium consisted of 2 g of NaNO3, 1 g ofK2HPO4, 0.5 g of KH2PO4, 0.1 g of KCl, 0.5 g of MgSO4.7H20, 0.015 g of CaCl2.2H20, and 0.018 g of FeSO47H20 per liter of distilled water. All mineral salts werecertified A.C.S. grade, purchased from Fisher Scien-tific Co. The cultures were agitated in 500-ml flaskscontaining 100 ml of medium, at 200 rpm on a tem-perature-controlled (30°C) Gyrotory shaker (NewBrunswick Scientific).

Cultures were maintained in a lyophilized state,followed by three serial transfers on the appropriatecarbon source before the commencement of the fer-mentation. The inoculum size was 5% (vol/vol) of alate-exponential-phase culture.

Fermentation conditions. The reactor used wasa Chemap 14-liter fermentor with a liquid volume of11 liters. Three sets of standard flat-blade turbineimpellers provided the agitation at 600 rpm. The aer-ation rate was 1 volume of air/volume of liquid perminute. The temperature was controlled at 30°C. Themedium utilized consisted of the basic mineral saltswith either 2% (vol/vol) n-hexadecane (HumphreyChemical Co.) or 2% (wt/vol) sodium citrate (Fishercertified A.C.S. grade) added as the carbon source.The pH during growth on sodium citrate was auto-matically controlled at pH 7.5 with the addition of 5 NHCI.

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CELL SURFACE MEASUREMENTS 513

Biomass determination. The cell broth was cen-trifuged at 12,000 x g for 20 min at 0°C. The hexadec-ane phase which was found at the liquid surface aftercentrifugation was removed in the frozen state alongwith attached cells and dried overnight at 100°C. Theaqueous medium was decanted off, and the pellet waswashed and dried overnight at 100°C. This techniqueprovided an estimate between the "bound cells" orthose adhering to the hydrocarbon phase, and "freecells" in the aqueous phase or those which may beeasily detached from the hydrocarbon phase.

Contact angle determination. The fermentationbroth was filtered through a 1.2-,um pore size mem-brane filter forming a smooth layer of cells on thesurface. The membrane filter was placed in a hexadec-ane-filled glass bath. A light was projected throughthe bath, horizontal to the cell surface, and a 1-id dropof cell-free broth was placed on the cells. The lightpassed into the objective lens ofa microscope mountedhorizontally, and the image of the drop was focusedon a screen. The contact angle was then measureddirectly.

Surface and interfacial tension measurement.A modified du Nouy surface tensiometer was used tomeasure surface and interfacial tensions. The FisherAutotensiomat measures the force required to pass aring through a sample surface. The surface tensionwas taken to be the peak of a stress-strain curve. Aparameter which will be referred to as C55' is definedas the reciprocal of the dilution required to achieve asurface tension of 55 mN/m and is similar in somerespects to a critical micelle concentration. The use ofa "relative" index of this type is necessary when study-ing the production of a chemically unidentified surfac-tant and possibly a mixture of surfactants (7).The interfacial tension was measured by dipping

the ring into the aqueous sample, layering an equalvolume of n-hexadecane on the surface, and thenmeasuring the force required to pull the ring throughthe aqueous-oil interface. All measurements weremade with fresh interfaces (less than 5 min) at 25 +20C.

RESULTSThe biomass concentrations throughout the

course of the hexadecane and sodium citratefermentations are illustrated in Fig. 2. Concen-trations of hydrocarbon bound and free cellsincluding the fraction of unbound cells to thetotal biomass population during growth on thehydrocarbon are also illustrated in Fig. 2. Duringthe first 10 h of the hexadecane fermentation,the entire cell population remained bound to thehydrocarbon phase. This could be observed inthe fermentor when the agitation and aerationwere shut off. The broth rapidly cleared due tocomplete partitioning of the cells with the float-ing hydrocarbon phase. Between 10 and 18 h, upto 85% of the cells were released from the inter-face into the aqueous phase.A higher maximum specific growth rate was

observed during growth on the hydrocarbon

1.0 r

UA 4

06 I.0is.ZOA

,,,, o10- 0Q2-o,

>.

J

TIME (h)

FIG. 2. Growth ofA. calcoaceticus on hexadecane( ) and sodium citrate (-). Upper curve repre-sents the fraction of free (unbound) cells to the totalcell population during growth on hexadecane. Totalbiomass (0) is compared with the concentration ofcells bound to the hydrocarbon (E) and cells fiee inthe aqueous phase (A).

than on the soluble substrate (0.40 h-1 comparedwith 0.23 h-'); however, the maximum biomassyields were similar. The exponential phase ofgrowth ended earlier during growth in the solu-ble substrate than on hexadecane (10 h com-pared with 13 h).The surface and interfacial tension of the

whole broth remained fairly constant duringgrowth on sodium citrate (Fig. 3). The surfacetension was approximately 70 mN/m, slightlybelow the surface tension of water (72.8 mN/m).The surface tension of the hexadecane brothdropped from 52 to 26.5 mN/m between 10 and14 h into the fermentation. A corresponding dropin the aqueous-hydrocarbon interfacial tensionfrom 22.5 to 3 mN/m was observed. This alsocoincides with the shift in the cells from thehydrocarbon to the aqueous phase. The inter-facial tension between the cell-free broth andhexadecane dropped from 31 to 10 mN/m be-tween 10 and 18 h into the fermentation. Anincrease in the relative surfactant concentration(C55-1) coincided with the drop in surface ten-sion. This is in sharp contrast to the cells grow-ing on sodium citrate, where the C55 1 was lessthan 1 throughout the entire fermentation, in-dicating that little or no biosurfactant was beingproduced during growth on sodium citrate.The three-phase contact angle at the cell-

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514 NEUFELD, ZAJIC, AND GERSON

0

Z 60

z

z 50 - 0 0 \

40_w~~~~~

30-30-"-A-- -

10

5 10 15 20 25 30

TIME (h)

FIG. 3. Surface and interfacial tension measure-ments and relative surfactant concentration ofwholemicrobial broth during growth on hexadecane( ) and sodium citrate (-----). Symbols: 0, surfacetension ofwhole broth; A, interfacial tension betweenhexadecane and whole broth; El relative surfactantconcentration-C55 1, relative units.

120-

$~100 Y

W 8 0z

, 60

z0

5 10 15 20 25 30TIME (hours)

FIG. 4. Contact angle of cell-free broth on cells inhexadecane. Bars represent one standard deviationfrom the mean. Each value represents the mean of 10measurements. Measurements on cells originatingfrom the hexadecane fermentation ( ); angles oncells originating from the sodium citrate fermentation

aqueous-hydrocarbon junction is plotted in Fig.4. The cells grown on hexadecane are clearlyhighly hydrophobic initially, with contact anglesof 120°; however, the angle drops rapidly to

between 40 and 460 after 14 h. An additionaldecrease in angle was noted toward the end ofthe fermentation. The angles were similar,whether distilled water or cell-free broth wasused as the aqueous phase. As the cell-free brothmore closely resembles the aqueous phase whichthe cell encounters in the fermentor, subsequentdata were calculated using cell-free broth.Contact angles on cells metabolizing sodium

citrate were in striking contrast to those grownon hexadecane. The mean angle throughout theentire fermentation was 20.70 with a standarddeviation of 2.10. The angles tended to dropslightly as the fermentation progressed; how-ever, the cells are clearly more hydrophilic thanthose growing on the hydrocarbon substrate.

Plots of ysv (cell surface free energy), ys, (cell-hydrocarbon interfacial tension) and YS2 (cell-aqueous interfacial tension) are presented in Fig.5. The surface free energy of the cells growingon hexadecane is 42 mj/M2 initially, rising to58.5 mj/M2 at 10 h, the point at which the cellsmove to the aqueous phase. The initial lowsurface free energy confirms the hydrophobicnature of the cell surface in the initial stages ofthe hydrocarbon fermentation.The interfacial free energies between the cells

and the two liquid phases undergo dramaticchanges between 6 and 10 h into the fermenta-tion. Before 8 h, ys2 > ysi; at 8 h, ys2 = yss; and

E E-zE E

%Dz

c00

o0z z

-4

<i u

-I

*z,6 "a,1' A1I.

5 10 15 20 25 30

TIME (h)

FIG. 5. Cell surface free energy and cell-aqueousand cell-hydrocarbon interfacial tensions duringgrowth on hexadecane ( ) and sodium citrate(-----). Symbols: 0, cell surface free energy; A, cell-hexadecane interfacial tension; E, cell-aqueous in-terfacial tension.

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0---,"

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VOL. 39, 1980

after this point ys2 < -ysi. Thus, at 10 h, a sub-stantial difference exists between -ysi and yS2. Atsubsequent time intervals the interfacial freeenergy between the cells and the aqueous phaseapproaches zero. The difference between ysi and_YS2 is the free surface energy difference (AG)resulting when a particle is moved from phase 2to phase 1 (1):

AG2 b1 = 47rR2 (ysI - YS2) (5)

where R is the cell radius. Processes pccur in thedirection in which the free energy change isnegative. If equation 5 is negative, the cells willtend toward phase 1 or the hydrocarbon phase.Similarly, if equation 5 is positive (where ysi>YS2), then the cells will have their lowest energyin the aqueous phase.According to Albertsson (1), a spherical par-

ticle in a two-phase liquid system can occupyone of five positions (Fig. 6), depending on theabsolute value of the partition factor (PF) andthe relative sizes of ysi and Ys2.

PF = YSI - YS2 (6)Y12

where Y12 is the interfacial tension between liq-uid phases 1 and 2. Based on the results of Fig.

|YSI -YS2

|Ysl s2 |I

IYSI_yS2 > 1Y12

IS-YS2< 1

Y12

YSI -S2< 1

Y12

YS1>YS2 o

YSi <YS2

YSI >YS2

0

-0--

YS1-YS2<1

Y12FIG. 6. Possiblepositions ofa sphericalparticle in

a two-phase liquid system at different mutual rela-tions between -y2, ysi, and ys2. The upper phase isphase 1, and the lower is phase 2 (from Albertsson[1]). y12 is the interfacial tension between phases 1

and 2; ysi is the interfacial tension between the par-ticle and phase 1; and ys2 is the interfacial tensionbetween the particle andphase 2.

CELL SURFACE MEASUREMENTS 515

5 10 15 20 25 30TIME Ihours)

FIG. 7. (ysI - yS2)/(y12) as a function of timethroughout the course ofa hydrocarbon fermnentation.See legend to Fig. 6 for a description of symbols.

5, it appears that the cells should have a tend-ency to find their most stable position in phase1, or the hydrocarbon phase up until 8 h intothe fermentation, where the reverse is true after8 h. Whether the cells are wholly in the upperor lower phase or bound to the interface (Fig.6c, d, and e) will depend on the PF. This param-eter is plotted in Fig. 7, where it may be seenthat the PF is less than 1 at all times. Thiswould suggest that the cells are bound to theinterface throughout the fermentation, a seem-ing contradiction. However, commencing at 6 h,there is a tendency for the PF to rapidly ap-proach 1 but level off after 14 h at 0.72. The cellpopulations tend to follow the thermodynamicexpectations. Variability within the populationat a given time may be due to turbulence in thefermentor, and the resultant rapid turnover ofthe hydrocarbon-aqueous interface. In addition,centrifugal forces present during centrifugationwill tend to pull some cells from the interface,thus reducing the total measured bound popu-lation. Intimate contact between the cells andthe hydrocarbon may be expected especially inthe initial stages ofthe fermentation consideringthe hydrophobic nature of the cell surface.

Referring to Fig. 5, it may be seen that A.calcoaceticus, growing with sodium citrate asthe sole source of carbon, maintains a substan-tially higher surface free energy and cell-hydro-carbon interfacial tension than cells growing onthe hydrocarbon substrate. The mean surfacefree energy of the cells metabolizing the solublesubstrate was 71.7 ± 2.0 mJ/m2, and the meancell-hydrocarbon interfacial tension was 29.3 +3.2 mJ/m2. Even though cells were not exposed

YSI"S2 C.--

YSlzYS2 ---O---

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516 NEUFELD, ZAJIC, AND GERSON

to hydrocarbon during the sodium citrate fer-mentation, a cell-hydrocarbon interfacial ten-sion was calculated for comparative purposes.The cell surface was highly hydrophilic through-out the course of the fermentation as indicatedby the low cell-aqueous interfacial tension (lessthan 0.1 mJ/m2).

DISCUSSIONStudies of contact angles on solid surfaces are

numerous in the literature along with descrip-tions of the difficulties associated with measur-ing and interpreting contact angle data. Never-theless, based on these results and those byother workers (29), it appears that contact anglesare a useful tool in studying cellular interactionsat interfaces.Most studies on the nature of surfaces and the

types of molecular forces acting across interfaceshave concentrated on the contact angle ofliquidson solids in air. Few have examined the wetta-bility of a solid in a three-phase system involvingthe solid phase and two immiscible liquids (5).In this study, the wetting liquid is polar in anonpolar medium. This technique also permitsthe cells to be examined without the possibledifficulties encountered with dried or semidriedcell surface preparations.A number of interesting observations in the

literature have been related to the relative hy-drophobic-hydrophilic nature of the microbialcell surface. Mudd and Mudd (20, 21) in 1924and Reed and Rice (26) in 1931 observed thatnon-acid-fast bacteria, when in the aqueousphase of a water-oil emulsion, adhere to theinterface or, if mobile, are able to return to theaqueous phase after an encounter with the in-terface. Acid-fast bacteria, on the other hand,are immediately transferred to the hydrocarbonphase after entering the interface. It was postu-lated that this difference in behavior was due tothe presence of nonpolar fatty (hydrophobic)substances on the surface of the acid-fast orga-nisms which were more easily "wetted" by thenonpolar liquid as opposed to the polar (hydro-philic) nature of the non-acid-fast bacteria. Theextent of partitioning of the cells was related tothe degree of acid-fastness of the species tested.

Marshall and Cruickshank (16) observed thatcells of Flexibacter CW7 which are long, taperedfilaments always orient with the pointed enddirected to an oil-water interface. This behaviorhas been observed with other organisms as well(15, 16). It was postulated that while the major-ity of the cell surface was hydrophilic, the por-tion directed toward the interface may be hydro-phobic.Mudd and Mudd (20) observed that bacteria

accumulate at the oil-water interface much more

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rapidly and to a greater extent in systems ofhigh interfacial tension, whereas the oppositesituation existed in systems of low interfacialtension. Mimura et al. (17) reported that syn-thetic surfactants weakened the affinity of Can-dida petrophilum to oil. This observation wasconfirmed in the present study, where a shift inthe cells from hydrocarbon to the aqueous phasecoincided with a sharp drop in the aqueoussurface tension and aqueous-hydrocarbon inter-facial tension. Since Acinetobacter spp. are non-acid-fast, it is not likely that the cells wouldreadily pass into the hydrocarbon phase; how-ever, the initial highly hydrophobic surfacewould suggest that the stable equilibrium posi-tion of cells would be on the nonpolar side of theinterface. As the surface tension and aqueous-hydrocarbon interfacial tension decrease, therewould be a tendency for the aqueous phase todisplace the hydrocarbon from the cell surface.Assuming that the surface areas of the microbialcells remain relatively constant, the partitioningof cells in a two-phase liquid system is dependenton -ys, and ys2 (1). The partition coefficient of amicrobial cell is difficult to evaluate due to thedifficulty in establishing the stable equilibriumposition of the cell. If the cells adhere to theinterface, certain criteria should be used to es-tablish a partition coefficient. When cells havebeen observed growing in the hydrocarbonphase, it is difficult to determine clearly if thecells are in fact in the hydrocarbon phase orfirmly attached to the aqueous-oil interface. Per-haps the criteria of Albertsson (Fig. 6) may beuseful in establishing the true location of thecell.A few studies have indicated that the propor-

tion of hydrophobic components of cells in-creases during growth on hydrocarbons. Mimuraet al. (17) found that hydrocarbon-assimilatingyeasts had a stronger affinity for the hydrocar-bon than non-assimilating yeasts. Lipophiliccompounds with an affinity for oil were ex-tracted from the hydrocarbon-grown yeasts.These compounds proved to be good emulsifiers.Kaeppeli and Fiechter (12) observed that Can-dida tropicalis demonstrated a 25% lower ad-sorption capacity for hydrocarbons when grow-ing on glucose than when metabolizing hydro-carbons. This was attributed to a lipophilic li-popolysaccharide moiety present at the cell sur-face. Hug et al. (11) reported that cells of Can-dida tropicalis required an adaptation phaseafter a substrate change from glucose to hexa-decane during which time the lipid content ofthe cell doubled. Kaeppeli et al. (13) found thatalkane-grown cells of C. tropicalis contained asurface-localized polysaccharide which was iden-tified as a mannan containing approximately 4%

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VOL. 39, 1980

covalently linked fatty acids. Cells grown onglucose lacked the polysaccharide-fatty acidcomplex. It was demonstrated that this complexwas involved in the binding affinity of the cellsto the hydrocarbon.This study provides a quantitative measure of

the hydrophobic nature of a cell growing onhydrocarbons. This is in marked contrast to themore hydrophilic nature of those same cellswhen grown on a soluble substrate. It is hopedthat future studies on the hydrophobicity of thecell surface will provide useful infornation toaid in the understanding of microbial growth onliquid hydrocarbons and other water-insolublesubstrates.

ACKNOWLEDGMENTThis work was supported by the Natural Sciences and

Engineering Research Council of Canada.

LITERATURE CITED

1. Albertason, P. 1971. Partition of cell particles and mac-romolecules, 2nd ed., p. 58-72. Wiley-Interscience, NewYork.

2. Blake, T. D., and J. M. Haynes. 1973. Contact-anglehysteresis. Prog. Surf. Membr. Sci. 6:125-138.

3. Einsele, A., H. Schneider, and A. Fiechter. 1975. Char-acterization of microemulsions in a hydrocarbon fer-mentation by electron microscopy. J. Ferment. Technol.53:241-243.

4. El-Shimi, A., and E. D. Goddard. 1974. Wettability ofsome low energy surfaces. I. Air/liquid/solid interface.J. Colloid Interface Sci. 48:242-248.

5. El-Shimi, A., and E. D. Goddard. 1974. Wettability ofsome low energy surfaces. II. Oils on solids submergedin water. J. Colloid Interface Sci. 48:249-255.

6. Erickson, L. E., and T. Nakahara. 1975. Growth incultures with two liquid phases: hydrocarbon uptakeand transport. Process Biochem. 10:9-13.

7. Gerson, D. F., and J. E. Zajic. 1978. Surfactant produc-tion from hydrocarbons by Corynebacterium lepus, sp.nov. and Pseudomonas asphaltenicus, sp. nov. Dev.Ind. Microbiol. 19:577-599.

8. Gerson, D. F., and J. E. Zajic. 1979. The biophysics ofcellular adhesion, p. 29-57. In K. Venkatsubramanian(ed.), Immobilized microbial cells, ACS symposium se-ries 106. American Chemical Society, Washington, D.C.

9. Gutierrez, J. R., and L. E. Erickson. 1977. Hydrocarbonuptake in hydrocarbon fermentations. Biotechnol.Bioeng. 19:1331-1349.

10. Hiemenz, P. C. 1977. Principles of colloid and surfacechemistry. Marcel Dekker Inc., New York.

11. Hug, H., H. W. Blanch, and A. Fiechter. 1974. Thefunctional role of lipids in hydrocarbon assimilation.Biotechnol. Bioeng. 16:965-985.

12. Kaeppeli, O., and A. Fiechter. 1976. The mode of inter-action between the substrate and cell surface of thehydrocarbon-utilizing yeast Candida tropicalis. Bio-technol. Bioeng. 18:967-974.

13. Kaeppeli, O., M. Muller, and A. Fiechter. 1978. Chem-ical and structural alterations at the cell surface of

CELL SURFACE MEASUREMENTS 517

Candida tropicalis, induced by hydrocarbon substrate.J. Bacteriol. 133:952-958.

14. Mallee, F. M., and H. W. Blanch. 1977. Mechanisticmodel for microbial growth on hydrocarbons. Biotech-nol. Bioeng. 19:1793-1816.

15. Marshall, K. C. 1973. Mechanism of adhesion of marinebacteria to surfaces, p. 625-632. In R. F. Acker, B. F.Brown, J. R. Depalma, and W. P. Iverson (ed.), Proc.3rd Irt. Cong. Mar. Corrosion Fouling. NorthwesternUniversity Press, Evanston, Ill.

16. Marshall, K. C., and R. H. Cruickshank. 1973. Cellsurface hydrophobicity and the orientation of certainbacteria at interfaces. Arch. Mikrobiol. 91:29-40.

17. Mimura, A., S. Watanabe, and I. Takeda. 1971. Bio-chemical engineering analysis of hydrocarbon fermen-tation. III. Analysis of emulsification phenomena. J.Ferment. Technol. 49:255-271.

18. Miura, Y. 1978. Mechanism of liquid hydrocarbon uptakeby microorganisms and growth kinetics. Adv. Biochem.Eng. 9:31-56.

19. Miura, Y., M. Okazaki, S. Hamada, S. Murakawa,and R. Yugen. 1977. Assimilation of liquid hydrocar-bon by microorganisms. I. Mechanism of hydrocarbonuptake. Biotechnol. Bioeng. 19:701-714.

20. Mudd, S., and E. B. H. Mudd. 1924. Certain interfacialtension relations and the behavior of bacteria in films.J. Exp. Med. 40:647-660.

21. Mudd, S., and E. B. H. Mudd. 1924. The penetration ofbacteria through capillary spaces. IV. A kinetic mech-anism in interfaces. J. Exp. Med. 40:633-646.

22. Nakahara, T., L. E. Erickson, and J. R. Gutierrez.1977. Characteristics of hydrocarbon uptake in cultureswith two liquid phases. Biotechnol. Bioeng. 19:9-25.

23. Neumann, A. W. 1974. Contact angles and their temper-ature dependence: thermodynamic status, measure-ment, interpretation and application. Adv. Colloid In-terface Sci. 4:105-191.

24. Neumann, A. W., and R. J. Good. 1972. Thermody-namics of contact angles. I. Heterogeneous solid sur-faces. J. Colloid Interface Sci. 8:341-358.

25. Neumann, A. W., R. J. Good, C. J. Hope, and M.Sejpal. 1974. An equation of state approach to deter-mine surface tensions of low-energy solids from contactangles. J. Colloid Interface Sci. 49:291-304.

26. Reed, G. B., and C. E. Rice. 1931. The behaviour of acid-fast bacteria in oil and water systems. J. Bacteriol. 22:239-247.

27. Scott, C. C. L., and W. L. Finnerty. 1976. Characteri-zation of intracytoplasmic hydrocarbon inclusions fromthe hydrocarbon-oxidizing Acinetobacter species H01-N. J. Bacteriol. 127:481-489.

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