Application of a Modified Drop-collapse Technique for Surfactant

Journalof

MethodsMicrobiological

Journal of Microbiological Methods 32 (1998) 273–280

Application of a modified drop-collapse technique for surfactantquantitation and screening of biosurfactant-producing

microorganisms*Adria A. Bodour, Raina M. Miller-Maier

Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ 85721, USA

Received 8 December 1997; received in revised form 27 February 1998; accepted 1 March 1998

Abstract

A drop-collapse method has been refined for use as both a qualitative assay to screen for surfactant-producing microbes,and as a quantitative assay to determine surfactant concentration. The assay is rapid, easy to perform, reproducible andrequires little specialized equipment. The assay is performed in a 96-microwell plate, where each well is thinly coated withoil. A 5 mL sample droplet is added to the center of a well and observed after 1 min. The droplet will either bead up, spreadout slightly or collapse, depending on the amount of surfactant in the sample. The basis for this method is the type of oilused to coat each well. In the qualitative method, each well is coated with 1.8 mL of Pennzoil and either the drop collapses,indicating the presence of surfactant (a positive result), or the drop remains beaded, indicating the absence of surfactant (anegative response). In the quantitative method, each well is coated with 2 mL of mineral oil, and a dissecting microscope isused to measure the diameter of the droplet at 1 min. Results with both a test biosurfactant (rhamnolipid) and a test syntheticsurfactant (sodium dodecyl sulfate) indicate a direct linear correlation between droplet diameter and surfactant concentration.The drop-collapse method has several advantages over commonly used methods that measure surface tension, such as the duNouy ring method; a smaller volume is required (5 mL vs. 20 mL), the effective range of measurement is greater and it doesnot require specialized equipment. 1998 Elsevier Science B.V.

Keywords: Biosurfactant; Surfactant; Screening assay; Drop-collapse test

1. Introduction tants. Like synthetic surfactants, biosurfactants re-duce the surface and interfacial tensions of aqueous

Most surfactants are produced from petroleum and media. Biosurfactants are a unique class of com-require both synthesis and several purification steps, pounds that have been shown to have a variety ofwhich is a costly process (Davidson and Milwidsky, potential applications, including remediation of or-1972; Garrett, 1972). Despite this, new applications ganics and metals, enhanced transport of bacteria,for surfactants have increased demand world-wide. enhanced oil recovery, as cosmetic additives and inOne alternative to synthetic surfactants are microbiological control (Desai and Banat, 1997; Hermanbially produced surfactants, also called biosurfac- et al., 1995; Miller, 1995; Miller and Zhang, 1997;

Stanghellini and Miller, 1997; Van Dyke et al., 1993;* Zhang and Miller, 1995). This wide range of po-Corresponding author. Tel.: 520 621 7231; fax: 520 621 1647;e-mail: [email protected] tential applications has increased interest in micro-

0167-7012/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PI I : S0167-7012( 98 )00031-1

274 A.A. Bodour, R.M. Miller-Maier / Journal of Microbiological Methods 32 (1998) 273 –280

bially produced surfactants; however, little is cur- test was used to generate standard surfactant con-rently known concerning the ecology or distribution centration curves for two surfactants, one syntheticof biosurfactant-producing organisms in the environ- (sodium dodecyl sulfate) and the other microbialment. Biosurfactants are natural, biodegradable ma- (rhamnolipid), and was also used to determine theterials and may be less toxic than synthetic surfac- concentration of rhamnolipid in effluent fractionstants (Margaritis and Creese, 1978; Van Dyke et al., that were collected during a column study where1991). In addition, because of their unique structures, rhamnolipid was applied to remove soil-bound cad-biosurfactants may have a greater range of properties mium. In all cases, drop-collapse results were com-that can be exploited commercially (Cooper and pared to results obtained with the du Nouy ringZajic, 1980). method.

Interest in microbially produced surfactants hasled to a need for the further development of rapidand efficient qualitative and quantitative methods for 2. Materials and methodsscreening and analyzing biosurfactant-producing or-ganisms. Several methods exist to measure surfactant 2.1. Chemicalsconcentrations in a liquid medium. These methodsmeasure the surface force between a liquid and air Two well-defined anionic surfactants were used as(surface tension) or the force between two liquids standards in the study. The first was a rhamnolipid(interfacial tension), which is then correlated to biosurfactant produced by Pseudomonas aeruginosasurfactant concentration. Such methods include IGB83. The rhamnolipid produced by this micro-capillary height, drop-weight and drop-volume, ring, organism is a mixture of monorhamnolipid andbubble pressure, pendant drop, sessile drop, hanging dirhamnolipid, with an average molecular weight ofplate, surface potential, as well as methods based on 577 g/mol (Torrens et al., 1998). Production andthe spreading of oils, and the method of ripples purification of rhamnolipid have been described(Harkins and Alexander, 1959). Currently, the most previously (Miller and Zhang, 1997; Zhang andwidely used method for the measurement of surface Miller, 1992, 1995). The second surfactant wasand interfacial tension is the du Nouy ring method, sodium dodecyl sulfate (SDS), a synthetic surfactantwhich measures the force required to pull a platinum obtained from Sigma (Arlington, IL, USA), with awire ring through the liquid–air or liquid–liquid molecular weight of 288 g/mol. For surfactantinterface. The reasons for the wide use of this quantitation experiments, standard curves were pre-method are its accuracy, ease of use and the fact that pared from stock solutions of surfactant in purifiedit provides a fairly rapid measurement of surface and water (Barnstead, Nanopure water, Dubuque, IA,interfacial tension; however, it does require the USA) or in 7 mM KNO , depending on the experi-3

purchase of specialized equipment (Harkins and ment. All surfactant solutions were adjusted to pHAlexander, 1959). Other limitations of this method 7.0–7.2 with 1 M NaOH.include the volume of sample required for analysis Several coating oils were tested in this study,and the restricted range of concentrations that can be including stylet oil (white mineral oil), mineral oil,analyzed without dilution. hexadecane, kerosene, 10W-30 Castrol, 10W-40

The objective of this research was to refine a Pennzoil and silicone oil. These were all obtainedqualitative drop-collapse technique described previ- from Aldrich (Milwaukee, WI, USA), except forously by Jain et al. (1991) so that it could be easily Pennzoil (Oil City, PA, USA) and Castrol (Swin-used to both qualitatively screen surfactant-produc- don, UK).ing microorganisms and quantify surfactant concen-tration. Qualitative and quantitative applications 2.2. Du Nouy methodologywere then conducted to assess the efficacy of thismethod. The qualitative test was evaluated by A Surface Tensiomat, Model 21 (Fisher Scientific,screening a range of diverse biosurfactant-producing Pittsburgh, PA, USA) was used to measure themicroorganisms isolated from soils. The quantitative surface tension of standard surfactant solutions. A 20

A.A. Bodour, R.M. Miller-Maier / Journal of Microbiological Methods 32 (1998) 273 –280 275

mL volume of each surfactant standard solution was surfactant concentrations in unknown samples. Sam-put into a clean glass 50 mL beaker and placed onto ples were replicated five times and each experimentthe tensiometer platform. A platinum wire ring was was repeated three times.submerged into the solution and then slowly pulledthrough the liquid–air interface, to measure the 2.4. Sample preparationsurface tension (dyn/cm). Between each measure-ment, the platinum wire ring was rinsed three times To ensure reproducible results, sample preparationwith water, three times with acetone and was al- for the drop collapse method and for the du Nouylowed to dry. ring method must be consistent. Parameters that

affect surface and interfacial tension include pH,2.3. Drop-collapse methodology ionic strength, temperature and the composition of

the medium (Champion et al., 1995; Miller andThe drop-collapse technique was performed in the Zhang, 1997; Zhang and Miller, 1992). In this study,

polystyrene lid of a 96-microwell (12.738.5 cm) samples and standards for any given test wereplate (VWR, Cerritos, CA, USA or Biolog, Hay- prepared in identical buffer solutions and all samplesward, CA, USA). The lids have 96 circular wells were equilibrated in a 258C water bath for 30 min(i.d., 8 mm). Before use, each lid was rinsed three prior to measurement.times each with hot water, ethanol and distilledwater, and dried. After preparation, each well was 2.5. Application of the qualitative test-screeningcoated with a thin layer of oil. Various oils were for biosurfactant-producing isolatestested. It was found that the type of oil used dictateswhether the assay is qualitative or quantitative. For A number of potential biosurfactant-producingthe qualitative test, each well was coated with 1.8 mL bacteria were isolated from a range of Arizona soilsof 10W-40 Pennzoil, which was spread as a thin to evaluate use of the qualitative drop-collapsecoating over the bottom of the well. The coated wells method as a screening tool. The following four soilswere equilibrated for 24 h to ensure a uniform oil were used: (1) an uncontaminated sandy soil (Vin-coating. For the quantitative test, 2.0 mL of mineral ton) with a low organic matter content (0.1%); (2) anoil were used to coat each well and equilibration was uncontaminated sandy loam soil (Mt. Lemmon) withfor 1–2 h. a high organic matter content (4.59%); (3) a soil that

For both tests, a 5 mL aliquot of sample was had a history of contamination with waste oil and (4)delivered into the center of the well using a 25 mL a soil that was contaminated with dross containingglass syringe (Hamilton, Reno, NV, USA) by holding high levels of cadmium and lead (organic matterthe syringe at an angle of 458. The syringe was content50.55%). Each soil (5 g) was placed in a 250rinsed three times between each sample addition with mL flask containing 50 mL of tap water and waswater and then with acetone. For the qualitative test, incubated at room temperature with gyratory shakingthe drop results were determined visually after 1 at 200 rpm for three weeks. On days 3, 7, 14 and 21,min. If the drop remained beaded, the result was samples from each flask were serially diluted andscored as negative. If the drop collapsed, the result plated onto R A agar (Becton Dickinson, Coc-2

was scored as positive. For the quantitative test, a keysville, MD, USA) and incubated for seven days.standard curve was prepared for each surfactant by Isolated colonies were then inoculated into 5 mL ofadding drops containing varied surfactant concen- mineral salts medium (0.4% Na HPO , 0.15%2 4

trations to each well. At 1 min, the diameter of each KH PO , 0.1% NH Cl, 0.02% MgSO ?7H O,2 4 4 4 2

drop was measured using a dissection microscope 0.0005% iron ammonium citrate, 0.001% CaCl, pH(153 magnification) with a calibrated micrometer. 7.2; surface tension, 64 dyn/cm) containing 2% (w/Droplets were examined at a standard time (1 min) to v) glucose as the sole carbon and energy source. Thisensure consistent results. Standard curves were pre- medium was chosen to specifically select for rham-pared by plotting the surfactant concentration versus nolipid-producing microorganisms, but other mediathe drop diameter and these were used to determine could be substituted. The cultures were incubated


with shaking (200 rpm) for five–seven days at room shown in Fig. 1 (well A, top row). The bead formstemperature. The cell suspensions were tested for the because the polar water molecules are repelled frompresence of surfactant using the qualitative drop- the hydrophobic surface. In contrast, if the watercollapse method. droplet contains surfactant, the force or interfacial

All isolates that tested positive and some that tension between the water drop and the hydrophobictested negative in the drop-collapse test were then surface is reduced, which results in the spreading oftested using the du Nouy ring method. The isolates the water drop over the hydrophobic surface (Fig. 1,were grown as above, except that the volume used well B, top row). Of the different oils tested for theirwas 25 mL. Cell suspensions were then centrifuged efficacy in the drop-collapse test, Pennzoil 10W-40at 15,0003g for 10 min and the cell-free supernatant was found to give the best qualitative indication ofwas measured for surface activity using the du Nouy the presence of surfactant. Pennzoil was consideredring method. the most effective oil because either the water drop

remained beaded in the absence of surfactant or it2.6. Application of the quantitative test-column collapsed completely. Thus, the results of the testexperiment were easy to determine visually (Fig. 1, top row).

The amount of surfactant required to cause drop-The efficacy of the qualitative drop-collapse meth- collapse is dependent on the ability of the surfactant

od was determined using samples collected from a to reduce surface and interfacial tension. The morecolumn experiment that was performed to evaluate potent the surfactant, the smaller the quantity thatthe effect of rhamnolipid on the removal of soil- can be detected. In terms of surface tension, thebound cadmium. The results of these experiments are minimum amount of surfactant required to causedescribed in detail elsewhere (Torrens et al., 1998). drop-collapse on 10W-40 Pennzoil was the amountIn brief, the column was packed with Vinton soil, that caused a reduction in the surface tension ofsaturated with Ca(NO ) , and then contaminated water from 72 dyn/cm (water alone) to 43 dyn/cm.3 2

with cadmium, as Cd(NO ) . After cadmium load- To put this into perspective, it takes approximately3 2

ing, the soil was washed for a set number of pore 10 mg/L of rhamnolipid to reduce the surfacevolumes with an electrolyte solution (7 mM KNO ) tension to 43 dyn/cm.3

until the cadmium concentration in the effluent wasapproximately zero. Then, rhamnolipid (5770 mg/L) 3.2. Comparison of surfactant quantitation by thein 7 mM KNO was applied to the column for 25 drop-collapse and the du Nouy ring methods3

pore volumes. The column effluent fractions werecollected and refrigerated. Each sample was then While most of the oils tested were qualitative inmeasured for biosurfactant concentration using both nature, it was found that mineral oil allowed forthe du Nouy ring method and the drop-collapse quantitation of surfactant in the sample droplet. Inmethod. For the drop-collapse method, each sample this case, as the surfactant concentration increased,was diluted 1:250 with 7 mM KNO . For the du the diameter of the sample drop increased (Fig. 1,3

Nouy ring method, each sample was diluted 1:500 bottom row). Quantitative results for two surfactants,with 7 mM KNO . In all cases, samples were rhamnolipid and SDS, are presented as standard3

equilibrated for 30 min in a 258C water bath prior to curves in Fig. 2. A linear correlation was foundmeasurement. between the rhamnolipid concentration and the drop

2diameter, in the range of 0 to 100 mg/L, with an rof 0.997 (Fig. 2A). For SDS (Fig. 2B), concen-

3. Results and discussion trations between 0 and 2400 mg/L were linearly2correlated with drop diameter (r 50.989).

3.1. Qualitative drop-collapse test The du Nouy ring method was used for com-parison to the drop-collapse method. Results show-

A drop of water applied to a hydrophobic surface ing the surface tension of standard rhamnolipid andin the absence of surfactants will form a bead, as SDS solutions are shown in Fig. 3. As expected, the


Fig. 1. Qualitative drop-collapse method: (A) Water control (no surfactant), (B) 1000 mg/L rhamnolipid. Quantitative drop-collapse method:(A) Water control, (B) 25 mg/L rhamnolipid, (C) 50 mg/L rhamnolipid, (D) 75 mg/L rhamnolipid and (E) 100 mg/L rhamnolipid.

surface tension decreased with increasing surfactant ent in each soil, although there were temporal shiftsconcentration until a plateau was reached at the in the proportion of surfactant-producers isolated. Incritical micellar concentration (CMC) of the surfac- general, each soil sampling yielded 15–25 differenttant. CMC values were determined from these graphs isolates. Each of these isolates was screened forto be 1845 mg/L for SDS and 27 mg/L for surfactant production. For each soil, the day seven,rhamnolipid. Thus, the effective measurement range sampling yielded the highest number of surfactant-for rhamnolipid using the du Nouy ring method was producers and the two contaminated soils yielded the0 to 27 mg/L and for SDS, it was from 0 to 1845 highest proportion of surfactant-producers, with upmg/L. to 31% of the isolates tested being positive for

These results indicate that the effective measure- surfactant production on day seven. The group ofment range for the drop-collapse method is greater isolates obtained on days 3, 14 and 21 generallythan that for the du Nouy ring method. A larger contained only one to two surfactant-producers and,range of measurement is advantageous when samples in some cases, none. It is not known at this timecontain high surfactant concentrations because less what might cause the observed temporal shift in thesample dilution is required, thereby minimizing error numbers of surfactant-producers isolated.introduced by sample dilution. The du Nouy ring method showed surface tension

reduction in all positive isolates (27–42 dyn/cm) and3.3. Application of the qualitative drop-collapse little to no reduction in surface tension for negativemethod isolates (50–68 dyn/cm). This comparison con-

firmed that the drop-collapse method provides com-Bacterial colonies were isolated from the four soils parable results to the du Nouy ring method. The

used in this study and screened for surfactant pro- advantage of the drop-collapse method for screeningduction using both the qualitative drop-collapse and purposes is that the test volume required is muchthe du Nouy methods. Both methods gave similar smaller (5 mL) than the volume required for the duresults, showing that surfactant-producers were pres- Nouy ring test (20 mL). Thus, the cultures can be


Fig. 3. Du Nouy ring method shows the same two surfactants (A)Fig. 2. The quantitative drop-collapse method. The figure shows

P. aeruginosa IGB83 with a CMC of 27 mg/L and (B) SDS withthe results obtained with two different surfactants: (A) P. aerugin-

a CMC of 1845 mg/L. Each point represents the mean andosa IGB83 with a CMC of 27 mg/L and (B) SDS with a CMC of

standard deviation of triplicate samples.1845 mg/L. Each point represents the mean and standard devia-tion of five replicates from experiments that were carried out intriplicate.

ring method (1:500). The difference in dilutionrequirement is because the drop-collapse method has

grown up in smaller test tubes, requiring less media a larger range of measurement, three to four timesand less room for incubation. the CMC, than the du Nouy ring method, up to the

CMC. The larger range of measurement for drop-3.4. Application of the quantitative drop-collapse collapse means that less sample dilution is required,method thereby minimizing dilution error. We also found in

this study that the drop-collapse method was lessThe efficacy of the drop-collapse method for operator-dependent. Several operators in our labora-

surfactant quantitation was tested by determining the tory used both methods and consistently showed lessconcentration of surfactant within column fraction variability using the drop-collapse method.effluents. Typical results from such an experimentare shown in Fig. 4. Both the du Nouy ring anddrop-collapse methods showed similar overall re- 4. Summarysults; however, the drop-collapse results have smallerassociated standard deviations. One reason for this The use of a drop-collapse technique for themay be that a smaller dilution was required for the screening of surfactant-producing microorganismsdrop-collapse samples (1:250) than for the du Nouy has been reported previously (Jain et al., 1991). In


range that can be measured is greater in the drop-collapse method; (3) the drop-collapse test was justas easy to perform and more reproducible than the duNouy ring method (Fig. 4) and (4) the drop-collapsemethod uses only a common dissecting microscopeand calibrated micrometer, while the du Nouy ringmethod requires a surface tensiometer, a piece ofequipment that is not routinely found in mostmicrobiology laboratories because of its cost.

Acknowledgements

This research was supported by Grant P42ES04940 from the National Institute of Environmen-tal Health Sciences, NIH. Our thanks to DeboraGage-Fasse for technical help in evaluating the drop-collapse method and to Dr. G. Soberon-Chavez,Fig. 4. Breakthrough curve for a Vinton soil column treated with

10 mM biosurfactant IGB83. The drop-collapse method (j) was Instituto de Biotecnologia, Universidad Nacionalcompared to the du Nouy ring method (s) for the determination Autonoma de Mexico, for providing P. aeruginosaof determine biosurfactant concentrations. Separate standard IGB83. We would also like to thank Bruce Russellcurves were used for this experiment, with standards prepared in 7

for his expertise in photographing the qualitative andmM KNO .3quantitative results in Fig. 1.

addition, other rapid screening methods of biosurfac-tant-producing microorganisms, such as axisymmet-

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Application of a Modified Drop-collapse Technique for Surfactant