A drop-collapsing test for screening surfactant-producing microorganisms

Journal of Microbiological Methods 13 (1991) 271 - 279

27 l

~) 1991 Elrevier Science Publishers B.V. 0t67-'i012/$ 3.50 MiMET 00436

A drop-collapsing test for screening surfactantproducing microorganisms D. K. Jain, D. L. Collins-Thompson, H. Lee a n d J. T. Trevors Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canad~~ (Receiw,~ 18 Deccmber 1990; revision received 8 April 1991; accepted 16 April m-,,

Summary A sensitive rapid method was devised for screening bacterial colonies that produce surfactants. Drops of cell suspensions of surfactant-producing colonies collapsed on an eil-coated surface. Drops of cell suspensions of colonies that did not produce, or produced very low concentrations of surfactants remained stable. The stability of drops was dependent on biosurfactant concentration and it correlated with surface tension but not with emulsifying activity. Microbial colonies grown in the presence of carbohydrates or hydrocar0ons could be readily screened for surfactant production by this method.

Key words: Biosurfactant; Drop collapse; Microbial screening

Introduction Surfactants are surface-active substances that alter conditions prevailing at interfaces, including air- liquid, liquid- liquid and liquid- solid interfaces. In general, suruutH ,....~ llyu,ulo'lluuJt, uyUlUpnlltC factants are amphipathic, containing ,_~.t. . . . . ,-^,-: . . .alJu . , ,---.-, ......... • groups. The hydrophobic group, usually a hydrocarbon chain, tends to be expelled by water and the polar, hydrophilic group, tends to ~emain in water. Due to their arnphipathic natme, surfactants are not uniformly distributed in the solvent, but congregate at the solvent surface. Synthetic sur factants are used for a variety of domestic and industrial purposes. Biosurfactants are important because: (i) new structural types of biosurfactants can be produced and, thus, they expand the range of available surfactants; (it) they are usually biodegradable; (iii) there are prospects of producing biosurfactants cheaper from waste products; and (iv), in some cases, they are more potent than the synthetic surfactants under extreme cJnditions of temperature, pH or salinity [1-3]. Surfactants produced by bacteria have beer: implicated in oil recovery from Correspondence to: D.K. Jain, Ontario Hydro, Biological Research Section, 800 Kipling Avenue, KD 118, Toronto, Ontario M8Z 5S4, Canada.

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o i l - s a n d mixtures [4], in sulphur-wetting during sulphur oxidation by microorganisms [5], in inhibition of human blood clotting [6] and in hydrocarbon degradation [7]. To j,..=en microorganisms that produce surfactants, they are usually grown individually in liquid media containing suitable substrates. After several days, the culture filtrates are tested for surface activity by measuring surface tension or interracial tension. This approach is labour-intensive and slow. Moreover, strain improvement through mutagenesis will be laborious and time consuming if a faster method is not applied to select for desired mutant clones. Mulligan et al. [8] developed a plate assay to screen colonies that produce surfacrants. The assay was based on the observation that the surfactant produced by Bacillus subti!is lyses sheep red blood cells [91 and decreases surface tension of liquid media [8]. However, the method could not be applied to screen for microorganisms that required hydrocarbon(s) for surfactan: pred'.-.eti9n because hydrocarbon(s) reacted with the red blood cells. Furthermore, hemolytic activity may also be associated with the presence of lytic enzymes instead of surfactant production ~y the microorganism. In this paper, we describe a sensitive and rapid method to screen for bacterial colonies producing surfactants. Solutions containing potent surfactants will be unable to form stable drops on an oily surface. Solutions without a surfactant will retain the drop configuration on the oily surface. The ability of the surfactant to effect drop collapse was correlated with its surface tension but not emulsifying activity. Materials and Methods

All chemicals used were of reagent grade. Virgin heavy atmospheric gas reference oil refined from a light Canadian crude oil was obtained from Esso Research Centre, Sarnia, Ontario, Canada. The microorganisms used in this study were: Pseudomonas aeruginosa UG2 which produces emulsifying agent(s) when grown in a carbohydrate medium [10], Bacillus sutures ATCC. -~aa~"' " "" -wm~n""-'- also produces bLIIt .... clt~t,,Ldlll-a . . . . . . . . . I.n. ,1 , . a , h..h.,a r~l. L , , _ , , , . * U ,,~,, , ~ L ~ medium t-,,, Acinetobacter calcoaceticus ATCC 31012 which produces surface-active compounds in a hydrocarbon supplemented medium but not in a carbohydrate medium [11]; and Escherichia coli Nx r (resistant to nalidixic acid). P. aeruginosa UG2 was isolated in our laboratory [10]. B. subtilis A.TCC 21331 and A. calcoaceticus ATCC 31012 were purchased from the American "Dpe Culture Collection, Maryland, USA. E. coli Nx r was supplied by G. Palmateer, Ontario Ministry of the Environment, London, Ontario, Canada. The polystyrene plates (11 × 8 cm) used for drop collapse testing were covering lids of Microplates supplied by Biolog, Hayward, California, USA. The plate contained circles of 8 mm in internal diameter and ~- 0.25 mm in height as shown in Fig. 2. The circles helped in the formation of drops of defined size and also prevented drops from moving on oily surfaces. The plate was evenly coated with 100/xl Esso reference oil. Solutions of cetyltrimethyl ammonium bromide (CTAB), a cationic surfactant, and sodium dodecyl sulphate (SDS), an anionic surfactant, were prepared at varying concentrations in distilled water. The drop-collapsing ability was studied at room temperature (21-22°C) by pipetting 135/zl of surfactant solution inside each circle.

273 To examine microbial colonies for surface activity, the organisms were grown on tryptic soy agar (TSA, Difco) plates at 30 °C. After 24 h, a loopful of cells from a bacterial colony was suspended in 150/~1 distilled water and 135/A of the cell suspension was pipetted inside the circle on tne plate. After 1- 2 min, the drops were examined visually. Those that retained the drop configuration were considered stable while those that collapsed or spread were unstable. The effect of culture filtrates on drop stability was also examined. In these e~,perimerits, the organisms were grown in liquid media supplemented with glucose, sodium acetate or hexadecane as C sources. A loopful of cells from a TSA slant was transferred to 50 ml of yeast N base (YNB, Difco) without amino acids and ammonium sulphate, but supplemented with 2 g.1-1 NaNOs and 2% (w/v or v/v) filter-sterilized C substrates in 250-ml Erlenmeyer flasks. The flasks were incubated at 30 °C for 5 days, with gyratory shaking at 200 rpm. Liquid cultures containing hexadecane were kept overnight at 4 °C for hexadecane to solidify. Liquid cultures free of hexadecane were pipetted and transferred to another flask. Bacterial cells were removed by centfifugation and filtration through 0.45-/~m Millipore filters. There was no difference in surfactant activity of the culture filtrates before and after solidification of hexadecane, or after filtration. Emulsifying activity of the culture filtrate was estimated as described previously [10]. Briefly, a U.5-ml sample of test solution was added to 0.1 rnl of a hexadecane: 2-methylnaphthalene mixture (1 : 1, v/v) and 4.4 ml of 20 mM Tris buffer, pH 7, containing 10 mM MgSO 4. Samples were mixed by vortexing for l0 s and held stationary for 30 min. The turbidity of the mixture was measured spectrophotometrically at 540 rim. 1 U of e~ulsifying activity was defined as the amount of emulsifier giving an absorbance of I. The cr;~ical micelle concentration (F~r~cdilution) values of culture filtrates were calculated as described by Guerra-Santos et al. [12]. The Fcmcdilution value is an indirect measurement of the concentration of surfactants in a given solution; increasing Fcmc dilution values indicate increasing concentrations of surfactants. The eultnr~

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mat (Fisher Scientific, Toronto, Canada), increased. The dilution factor at which the surface tension increased was recorded as the Fcm~ dilution value. Hemolytic activity of bacterial strains was determined by inoculating sheep blood agar plates as described [8]. The plates were incubated at 30 °C for 4 8 - 72 b. Hemolytic activity of bacterial cell suspensions, SDS solutions, or culture filtrates was examined by placing 3 0 - 75 #l of aqueous solutions into a well (6 ram diameter) in blood agar. The well was bored using the wide end of a Pasteur pipette. After 6 h of incubation at 30 °C, plates were examined for clearing zones around the wells. All experiments were repeated two times. In all instances, similar trends were observed. Results The surface tension and drop-collapsing ability of SDS and CTAB measured at various concentrations are shown in Fig. 1. In the presence of low concentrations of these detergents, the drops remained stable on the plate, i.e., they did not collapse. However, drops containing SDS at concentrations > 0.05°-/o (w/v) and a surface tension

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< 59.3 dyn. cm-1 collapsed on an oil-coated plate (Fig. la). Similarly, drops containing CTAB at concentratons > 0.007°7o (w/v) and a surface tension < 66.9 dyn.cm-I collapsed on the reference-oil-coated plate. The stability of drops in the presence of bacterial cells was examined. Drops of cell suspensions of P. aeruginosa UG2 (grown on a TSA plate; Fig. 2b) and B. subtilis ATCC 21331 (not shown) were unstable and collapsed on plates as for drops of 0.2070 (w/v) SDS (Fig. 2a). In contrast, drops of cell suspensions of TSA grown A. calcor;ceticus ATCC 31012 and E. coli Nx r were s,~able (not shown), indicating the absence of significant surfactant produced by these strains. Extracellular surfactant production by P. aeruginosa UG2, A. calcoaceticus ATCC 31012 and E. coli Nx r was examined by growing these straitls in liquid med;~ cow,raining various C sources. After 5 days of growth, culture filtrates were used for determhm...n of ~,,r~'~,-, tension, F~mc dilution value, the highest dilution that exhibited dropc,.~Uapsing ability, and emulsifying activity (Table 1). The culture filtrate of P. aeruginosa UG2 grown on glucose (Fig. 2f) or sodium acetate (not shown) did not form stable drops. The culture filtrate of A. calcoaceticus ATCC 31012 grown oai glucose formed stable drops (Fig. 2d). However, filtrates from hexadecane-grown culture of A. calcoaceticus ATCC 31012 readily exhibited drop collapse. The c, dture filtrate of F. coil Nx r formed a stable drop (Fig. 2e), indicating the absence of surfactant production.

275

Fig. 2. Effect of surfactants on drop stability: (a) 0.2% (w/v) sodium dodecjrl sulphate solution; (b) cell suspension of P aeruginosu UG2 grown on a TSA plate; (~1ultrapure water; (3) culture filtrate of A. calcoacericus ATCC 31012 grown on glucose; (e) culture filtrate of E. coli Nxr grown on glucose; (f) culture filtrate of I? YX,-+KI.W UG2 grown on glucose.

The tendency to promote drop collapse was correlated with surface activity. For example, I? aeruginosa UG2 produisd higher amounts of extracellular surfactant when grown on glucose than on sodium acetate. This was indicated by a higher FCm, dilution value, lower surface tension and higher dilution of the glucose-grown cuiturc filtrate that exhibited drop collapse (Table 1). Similarly, A. calcoaceticus ATCC 31012 produced higher amount of surfrictant on hexadecane than on g!~cose as iiidicated by lower surface tension and drop-collapsing tendency of the filtrate from the hexadecane-grown culture (Table 1). E. coli Nxr did not significantly decrease the surface tension of the filtrate and, therefore, did not show drop colla The emulsifying activity of culture filtrates of aeruginosa ?JG%,A. ~~~~o~~&~~~~~s XCC 31012 and E. coli Nxr was examined. The culture filtrate of F! aeruginosa UG2

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S U R F A C E ACTIVITY O F B I O S U R F A C T A N T S P R O D U C E D IN L I Q U I D CULTURE F I L T R A T E S a

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grown on glucose or on sodium acetate, and that of A. calcoaceticus ATCC 31012 grown on hexadecane exhibited similar emulsifying activity (Table 1). A. catcoaceticus ATCC 31012 culture filtrate from cells grown on glucose and E. coti N×r culture filtrate did not exhibit significant emulsifying activity (Table 1). Hemol~ic activity of cell suspensions ofP. aeruginosa UG2, B. subti!is ATCC 21331~ A. calcoaceticus ATCC 31012 and E. coil Nx r was examined by suspending colonies, previously grown on TSA plates, in distilled water and pipetting into wells in blood agar. After 6 h of incubation at 30 °C, zone clearing was not detected. The wells were also tested with 3 0 - 75 tzl of culture filtrates of P. aeruginosa UG2 grown on glucose which contained significant amounts of surfactant activity (Table 1) but again lysis of red blood cells was not detected probably due to diffusion restriction of the UG2 surfactant through the agar. Ho~vevet, wells containing 0.2-0.5 % (w/v) SDS exhibited clear zones in blood agar plates within 6 h of incubation. When P. aeruginosa UG2, B. subtilis ATCC 21331, A, calcoaceticus ATCC 31012 and E. co6 Nx r were grown at 30°C on blood agar plates, no clearing of zones was observed around colonies up to 24 h of growth. However, cell suspensions of P. aeruginosa UG2 and B. subtilis ATCC 21331 grown in blood agar exhibited drop collapse, indicating surface activity. Cell suspensions of A. caleoaceticus ATCC 31012 and E. coil Nx r formed stable drops, suggesting the absence of surface activity in the suspensions. Clear zones were observed after 4 8 - 72 h of growth on blood agar plates around P. aeruginosa UG2 and B. subtilis ATCC 21331 colonies but not around A. ealeoaceticus ATCC 31012 and E. coil Nx r colonies.

Discussion Microorganisms with surface activity have been screened by measuring their cell surface hydrophobicity [13], contact angle [14], surface tension and interfacial tension [15], red blood ceil iysis [8] and wetting of water-repelliiig material such as cotton and glass [16]. We describe here a method ~or rapid determination of bacLcrial c~tonics with surface activity. The method is based on the ability of surfactams to destabilize liquid droplets on an oily surface, and this ability is correlated with its surface tension. The drop collapsing is related also to the spreading tension. The spreading tension of surfactant solution-on-oil is related to oil surface tension, surface-tension of sorfactant solution and oil-surfactant solution interfacial tension according to the following equation [17]: ' Y s p = "Yk--'Ysurf--)'k/surf,where %p = spreading tension, 7~ = oil surface tension, "/surf = surface tension of surfactant solution, and ')'k/surf = oil-surfactant solution interfacial tension. Since only one type of oil was used, the oil surface tension was constant. The more negative the surface tension and interracial tension due to increasing concentration of surfactant, the greater the degree of spreading tension of surfactant solution-on-oil, and this corresponds to increasing tendency for drops of surfactant solution to collapse on oily surface. Drops with higher spreading tension or lower surface tension will collapse on oily surfaces. In contrast, drops with lower spreading tension or higher surface tension will not spread on an oily surface. Previously, a method based on red blood cell lysis to screen surfactant-producing

278 colonies was developed by Mulligan et al. [8]. Hemolytic zones around colonies were related to the ability of microbes to produce surfactants. However, the method had three limitations. F~rs,, the method was not very specific because clearing zones around the colonies could also indicate the presence of lyric enzymes ~roduced by the colony Therefore, further screening in liquid media for surfactant production was required. Second, microorganisms which required hydrocarbons for biosurfactant production could not be screened [8]. Therefore, the method could be applied only to screen for biosurfactant production on nonhydrocarbon substrates. Third, the absence of hemolytic activity could be due to diffusion restriction of the surfactant through the ag~r. Our method is very specific since only organisms which produce significant surfaceactive compounds will cause collapse of aqueous drops on oily surfaces. Furthermore, colonies growing on hydrocarbon media and producing biosurfactant will also give positive responses. For example, our study confirmed that A. calcoaceticus ATCC 21012 requires hexadecane for biosurfactant production. The biosurfactant lowered surface tension of the medium and did not form a stable drop on the oily surface. The drop-collapsing techmque is more sensitive than the red bloo¢l ceii iysis technique [8] for screening biosurfactant producing colonies. After 24 h of growth on blood agar plate, hemol3¢ic zon~¢ could not be detected around colonies of P aeruginosa UG2 and B. subtiiis ATCC 21331. However, cell suspensions of these colonies caused collapse of drops on oil-coated surfaces, indicating that even smaller quantities of biosurfactants produced by the colonies can be detected by the drop-collapsing technique. The drop-collapsing activity did not correlate with emulsifying activity. An emulsion is formed wnen one liquid phase is dispersed as microscopic droplets in another liquid continuous phase. A. calcoaceticus ATCC 31012 is known to produce biosurfactant with high emulsifying activity but is relatively poor at lowering surface tension [11]. Although A. calcoaceticus ATCC 31012 culture filtrate had emulsifying activity as high as P. aeruginosa UG2 culture filtrates, a much h'_:gherconcentration of A. cal~

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Acknowledgements This research was supported by a grant from Ontario Ministry of the Environment. The views and ideas expres,~d in this paper are those of the authors and do not necessarily reflect the views and policies of the Ministry of the Environment, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. We thank D. Hamilton for help with graphic work, G. Palmateer for supplying the E. coil strain and S. Guiiey for technical assistance.

279

References I Parkinson, M. (1985) Bio-surfact~t~ts. ~iql~cla~l. ~civ. 3, 65-83. 2 Zajic, J. E., Guignard, M. and Gers~0, 13-1~.11977)bPr~pc,lies and biod-.gradatior, of bi,~e~,.,~i¢i~r fr-_,~ Corynebacterium hydrocarbocta.~ttts. I~ic~Cbh~l. l~ioeng. 19, 1303- t320. 3 Kretschmer, A., Bock, H. and Wa,~aer, P. (l~b8:~)Chemical and physical character ization of late, facial;active iipids from Rhodococcuv er_vtBe~tolis ~rc~vn on n-alkanes. Appl. Environ. Microbio!o 43. 864- 870. 4 Davis, J. Bo (1967) Petroleum Microlaiol0~, I~ls~vier, New York, 604 pp. 5 Jones, G. E. and Starkey, R. L. (l~bl) Stz~-f~c~.~ive s~ibstances produ,~ed by Thiobacillus thiooxidans. J. Bacterioi. 82, 788-789. 6 Arima, K., Kakinuma, A. and "P~rntjt,3, ~'. (1~68) Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: is~,latiola, qll~r~cr~rizatioa and its inhibition of fibrin clot formation. Bicchem. Biophys. Res. Cemrraup. ~1, 4~'--,~9~,. 7 Suzuki, T., Tanaka, K. and Kinoshil~, S. (1~3~9)Thee:~tre,cellular accumulation of trehalose and glucose by bacteria grown on n-alkanes. Agri¢. fiiC~l, tZhera. 33, 190-195. 8 Mulligan, C, N., Cooper, D. G. a~l bletaf~[tt, R. ,I, ~(19g~,)Selection of microbes producing biosurfactants in media without hydrocarbons. J. l::etr~'~, q~cql~n~,l.62, 311-314. 9 Bernheimer, A. W. ~~ ' .n:.,igad,L. ~.~,!97C; ,~ z : . ~ n d properties of a cytolytic agent produced by Bacillus subtilis. J. Gen. Microbiol. 61~ 361~.~b~3. 10 Berg, G., Seech, A.G., Lee, H. aod Treaters, J. 1~. (1990) Identification and characterization of a soil bacterium with extracellular enntalsifyi~ ~tivit~. J. Environ. Sci. Health A25, 753-764. 11 Rosenberg, E. and Kaplan, N. (19~b)Shcl~,a¢live properties of Acinelobacter exopolysacchar~des. In: Bacterial Outer Membranes a~ I~lod~l~'ster0~ (haot~ye, M., ed.), pp. 3! !- 338, John Wiley & Sons, New York. 12 Guerra-Santos, L., K/ippelli, O. arid Fiech~vk A. (1984)pseuclomonas aeruginosa bi6surfactant production in continuous culture with glueos~ ~ carl~qfa source. Appl, Environ. Microbiol. 48, 301-305. 13 Neu, T. R. and Poralla, K. (1990) l~ul~if¥~g ~ager~tsfrom bacteria isola,~ed during screening for cells with hydrophobic surfaces. App|, Ylicr¢~iC~l. l]ivteehnol. 32, 521-525. 14 Backman, P. A. and DeVay,J. E. (1~7l) ~,q~l~esvrl t he raode of action and biogenesis of the phy;.~to×in syringomycin. Physiol. Plant Path01. K ~ I ~ 3 . 15 Zajic, J.E. and Panchal, C.J. (1976) 13~0-,¢rnoIsifiers. CRC Crit. Rev. Microbiol. 5, 39-66. 16 Matsuyama, T., Sogawa, M. and ~/ano, 1, (19g'/~ Direct colony ~hin-layer chrgmatography and rapid ch~,~acterization of Serratia marc¢~cens t~ot.ar~t~~lefective in production of we~ting agents. Appl. Eavi ron. Microbioi. 53, 1186-1188. ! 7 Panchal, C. J., Zajic, J. E. and Gers0~, 1:3,~: (1#7':3)Mt~ltiple-phase emulsions using microbial emulsifiers. J. Colloid lnterL Sci. 68, 2~tl ~ 30).