Journal of Microbiological Methods 32 (1998) 273280

Journal of Microbiological Methods

Application of a modied drop-collapse technique for surfactant quantitation 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 rened 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 and requires little specialized equipment. The assay is performed in a 96-microwell plate, where each well is thinly coated with oil. A 5 mL sample droplet is added to the center of a well and observed after 1 min. The droplet will either bead up, spread out slightly or collapse, depending on the amount of surfactant in the sample. The basis for this method is the type of oil used 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 (a negative response). In the quantitative method, each well is coated with 2 mL of mineral oil, and a dissecting microscope is used to measure the diameter of the droplet at 1 min. Results with both a test biosurfactant (rhamnolipid) and a test synthetic surfactant (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 du Nouy ring method; a smaller volume is required (5 mL vs. 20 mL), the effective range of measurement is greater and it does not require specialized equipment. 1998 Elsevier Science B.V. Keywords: Biosurfactant; Surfactant; Screening assay; Drop-collapse test

1. Introduction Most surfactants are produced from petroleum and require both synthesis and several purication steps, which is a costly process (Davidson and Milwidsky, 1972; Garrett, 1972). Despite this, new applications for surfactants have increased demand world-wide. One alternative to synthetic surfactants are microbially produced surfactants, also called biosurfac*Corresponding author. Tel.: 520 621 7231; fax: 520 621 1647; e-mail: rmmiller@ag.arizona.edu

tants. Like synthetic surfactants, biosurfactants reduce the surface and interfacial tensions of aqueous media. Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications, including remediation of organics and metals, enhanced transport of bacteria, enhanced oil recovery, as cosmetic additives and in biological control (Desai and Banat, 1997; Herman 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 potential applications has increased interest in micro-

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

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bially produced surfactants; however, little is currently known concerning the ecology or distribution of biosurfactant-producing organisms in the environment. Biosurfactants are natural, biodegradable materials and may be less toxic than synthetic surfactants (Margaritis and Creese, 1978; Van Dyke et al., 1991). In addition, because of their unique structures, biosurfactants may have a greater range of properties that can be exploited commercially (Cooper and Zajic, 1980). Interest in microbially produced surfactants has led to a need for the further development of rapid and efcient qualitative and quantitative methods for screening and analyzing biosurfactant-producing organisms. Several methods exist to measure surfactant concentrations in a liquid medium. These methods measure the surface force between a liquid and air (surface tension) or the force between two liquids (interfacial tension), which is then correlated to surfactant concentration. Such methods include capillary height, drop-weight and drop-volume, ring, bubble pressure, pendant drop, sessile drop, hanging plate, surface potential, as well as methods based on the spreading of oils, and the method of ripples (Harkins and Alexander, 1959). Currently, the most widely used method for the measurement of surface and interfacial tension is the du Nouy ring method, which measures the force required to pull a platinum wire ring through the liquidair or liquidliquid interface. The reasons for the wide use of this method are its accuracy, ease of use and the fact that it provides a fairly rapid measurement of surface and interfacial tension; however, it does require the purchase of specialized equipment (Harkins and Alexander, 1959). Other limitations of this method include the volume of sample required for analysis and the restricted range of concentrations that can be analyzed without dilution. The objective of this research was to rene a qualitative drop-collapse technique described previously by Jain et al. (1991) so that it could be easily used to both qualitatively screen surfactant-producing microorganisms and quantify surfactant concentration. Qualitative and quantitative applications were then conducted to assess the efcacy of this method. The qualitative test was evaluated by screening a range of diverse biosurfactant-producing microorganisms isolated from soils. The quantitative

test was used to generate standard surfactant concentration curves for two surfactants, one synthetic (sodium dodecyl sulfate) and the other microbial (rhamnolipid), and was also used to determine the concentration of rhamnolipid in efuent fractions that were collected during a column study where rhamnolipid was applied to remove soil-bound cadmium. In all cases, drop-collapse results were compared to results obtained with the du Nouy ring method.

2. Materials and methods

2.1. Chemicals
Two well-dened anionic surfactants were used as standards in the study. The rst was a rhamnolipid biosurfactant produced by Pseudomonas aeruginosa IGB83. The rhamnolipid produced by this microorganism is a mixture of monorhamnolipid and dirhamnolipid, with an average molecular weight of 577 g / mol (Torrens et al., 1998). Production and purication of rhamnolipid have been described previously (Miller and Zhang, 1997; Zhang and Miller, 1992, 1995). The second surfactant was sodium dodecyl sulfate (SDS), a synthetic surfactant obtained from Sigma (Arlington, IL, USA), with a molecular weight of 288 g / mol. For surfactant quantitation experiments, standard curves were prepared from stock solutions of surfactant in puried water (Barnstead, Nanopure water, Dubuque, IA, USA) or in 7 mM KNO 3 , depending on the experiment. All surfactant solutions were adjusted to pH 7.07.2 with 1 M NaOH. Several coating oils were tested in this study, including stylet oil (white mineral oil), mineral oil, hexadecane, kerosene, 10W-30 Castrol, 10W-40 Pennzoil and silicone oil. These were all obtained from Aldrich (Milwaukee, WI, USA), except for Pennzoil (Oil City, PA, USA) and Castrol (Swindon, UK).

2.2. Du Nouy methodology

A Surface Tensiomat, Model 21 (Fisher Scientic, Pittsburgh, PA, USA) was used to measure the surface tension of standard surfactant solutions. A 20

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mL volume of each surfactant standard solution was put into a clean glass 50 mL beaker and placed onto the tensiometer platform. A platinum wire ring was submerged into the solution and then slowly pulled through the liquidair interface, to measure the surface tension (dyn / cm). Between each measurement, the platinum wire ring was rinsed three times with water, three times with acetone and was allowed to dry.

surfactant concentrations in unknown samples. Samples were replicated ve times and each experiment was repeated three times.

2.4. Sample preparation

To ensure reproducible results, sample preparation for the drop collapse method and for the du Nouy ring method must be consistent. Parameters that affect surface and interfacial tension include pH, ionic strength, temperature and the composition of the medium (Champion et al., 1995; Miller and Zhang, 1997; Zhang and Miller, 1992). In this study, samples and standards for any given test were prepared in identical buffer solutions and all samples were equilibrated in a 258C water bath for 30 min prior to measurement.

2.3. Drop-collapse methodology

The drop-collapse technique was performed in the polystyrene lid of a 96-microwell (12.738.5 cm) plate (VWR, Cerritos, CA, USA or Biolog, Hayward, CA, USA). The lids have 96 circular wells (i.d., 8 mm). Before use, each lid was rinsed three times each with hot water, ethanol and distilled water, and dried. After preparation, each well was coated with a thin layer of oil. Various oils were tested. It was found that the type of oil used dictates whether the assay is qualitative or quantitative. For the qualitative test, each well was coated with 1.8 mL of 10W-40 Pennzoil, which was spread as a thin coating over the bottom of the well. The coated wells were equilibrated for 24 h to ensure a uniform oil coating. For the quantitative test, 2.0 mL of mineral oil were used to coat each well and equilibration was for 12 h. For both tests, a 5 mL aliquot of sample was delivered into the center of the well using a 25 mL glass syringe (Hamilton, Reno, NV, USA) by holding the syringe at an angle of 458. The syringe was rinsed three times between each sample addition with water and then with acetone. For the qualitative test, the drop results were determined visually after 1 min. If the drop remained beaded, the result was scored as negative. If the drop collapsed, the result was scored as positive. For the quantitative test, a standard curve was prepared for each surfactant by adding drops containing varied surfactant concentrations to each well. At 1 min, the diameter of each drop was measured using a dissection microscope (153 magnication) with a calibrated micrometer. Droplets were examined at a standard time (1 min) to ensure consistent results. Standard curves were prepared by plotting the surfactant concentration versus the drop diameter and these were used to determine

2.5. Application of the qualitative test-screening for biosurfactant-producing isolates

A number of potential biosurfactant-producing bacteria were isolated from a range of Arizona soils to evaluate use of the qualitative drop-collapse method as a screening tool. The following four soils were used: (1) an uncontaminated sandy soil (Vinton) with a low organic matter content (0.1%); (2) an uncontaminated sandy loam soil (Mt. Lemmon) with a high organic matter content (4.59%); (3) a soil that had a history of contamination with waste oil and (4) a soil that was contaminated with dross containing high levels of cadmium and lead (organic matter content50.55%). Each soil (5 g) was placed in a 250 mL ask containing 50 mL of tap water and was incubated at room temperature with gyratory shaking at 200 rpm for three weeks. On days 3, 7, 14 and 21, samples from each ask were serially diluted and plated onto R 2 A agar (Becton Dickinson, Cockeysville, MD, USA) and incubated for seven days. Isolated colonies were then inoculated into 5 mL of mineral salts medium (0.4% Na 2 HPO 4 , 0.15% KH 2 PO 4 , 0.1% NH 4 Cl, 0.02% MgSO 4 ?7H 2 O, 0.0005% iron ammonium citrate, 0.001% CaCl, pH 7.2; surface tension, 64 dyn / cm) containing 2% (w / v) glucose as the sole carbon and energy source. This medium was chosen to specically select for rhamnolipid-producing microorganisms, but other media could be substituted. The cultures were incubated

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with shaking (200 rpm) for veseven days at room temperature. The cell suspensions were tested for the presence of surfactant using the qualitative dropcollapse method. All isolates that tested positive and some that tested negative in the drop-collapse test were then tested using the du Nouy ring method. The isolates were grown as above, except that the volume used was 25 mL. Cell suspensions were then centrifuged at 15,0003g for 10 min and the cell-free supernatant was measured for surface activity using the du Nouy ring method.

2.6. Application of the quantitative test-column experiment

The efcacy of the qualitative drop-collapse method was determined using samples collected from a column experiment that was performed to evaluate the effect of rhamnolipid on the removal of soilbound cadmium. The results of these experiments are described in detail elsewhere (Torrens et al., 1998). In brief, the column was packed with Vinton soil, saturated with Ca(NO 3 ) 2 , and then contaminated with cadmium, as Cd(NO 3 ) 2 . After cadmium loading, the soil was washed for a set number of pore volumes with an electrolyte solution (7 mM KNO 3 ) until the cadmium concentration in the efuent was approximately zero. Then, rhamnolipid (5770 mg / L) in 7 mM KNO 3 was applied to the column for 25 pore volumes. The column efuent fractions were collected and refrigerated. Each sample was then measured for biosurfactant concentration using both the du Nouy ring method and the drop-collapse method. For the drop-collapse method, each sample was diluted 1:250 with 7 mM KNO 3 . For the du Nouy ring method, each sample was diluted 1:500 with 7 mM KNO 3 . In all cases, samples were equilibrated for 30 min in a 258C water bath prior to measurement.

shown in Fig. 1 (well A, top row). The bead forms because the polar water molecules are repelled from the hydrophobic surface. In contrast, if the water droplet contains surfactant, the force or interfacial tension between the water drop and the hydrophobic surface is reduced, which results in the spreading of the water drop over the hydrophobic surface (Fig. 1, well B, top row). Of the different oils tested for their efcacy in the drop-collapse test, Pennzoil 10W-40 was found to give the best qualitative indication of the presence of surfactant. Pennzoil was considered the most effective oil because either the water drop remained beaded in the absence of surfactant or it collapsed completely. Thus, the results of the test were easy to determine visually (Fig. 1, top row). The amount of surfactant required to cause dropcollapse is dependent on the ability of the surfactant to reduce surface and interfacial tension. The more potent the surfactant, the smaller the quantity that can be detected. In terms of surface tension, the minimum amount of surfactant required to cause drop-collapse on 10W-40 Pennzoil was the amount that caused a reduction in the surface tension of water from 72 dyn / cm (water alone) to 43 dyn / cm. To put this into perspective, it takes approximately 10 mg / L of rhamnolipid to reduce the surface tension to 43 dyn / cm.

3.2. Comparison of surfactant quantitation by the drop-collapse and the du Nouy ring methods
While most of the oils tested were qualitative in nature, it was found that mineral oil allowed for quantitation of surfactant in the sample droplet. In this case, as the surfactant concentration increased, the diameter of the sample drop increased (Fig. 1, bottom row). Quantitative results for two surfactants, rhamnolipid and SDS, are presented as standard curves in Fig. 2. A linear correlation was found between the rhamnolipid concentration and the drop diameter, in the range of 0 to 100 mg / L, with an r 2 of 0.997 (Fig. 2A). For SDS (Fig. 2B), concentrations between 0 and 2400 mg / L were linearly correlated with drop diameter (r 2 50.989). The du Nouy ring method was used for comparison to the drop-collapse method. Results showing the surface tension of standard rhamnolipid and SDS solutions are shown in Fig. 3. As expected, the

3. Results and discussion

3.1. Qualitative drop-collapse test

A drop of water applied to a hydrophobic surface in the absence of surfactants will form a bead, as

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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 concentration until a plateau was reached at the critical micellar concentration (CMC) of the surfactant. CMC values were determined from these graphs to be 1845 mg / L for SDS and 27 mg / L for rhamnolipid. Thus, the effective measurement range for rhamnolipid using the du Nouy ring method was 0 to 27 mg / L and for SDS, it was from 0 to 1845 mg / L. These results indicate that the effective measurement range for the drop-collapse method is greater than that for the du Nouy ring method. A larger range of measurement is advantageous when samples contain high surfactant concentrations because less sample dilution is required, thereby minimizing error introduced by sample dilution.

3.3. Application of the qualitative drop-collapse method

Bacterial colonies were isolated from the four soils used in this study and screened for surfactant production using both the qualitative drop-collapse and the du Nouy methods. Both methods gave similar results, showing that surfactant-producers were pres-

ent in each soil, although there were temporal shifts in the proportion of surfactant-producers isolated. In general, each soil sampling yielded 1525 different isolates. Each of these isolates was screened for surfactant production. For each soil, the day seven, sampling yielded the highest number of surfactantproducers and the two contaminated soils yielded the highest proportion of surfactant-producers, with up to 31% of the isolates tested being positive for surfactant production on day seven. The group of isolates obtained on days 3, 14 and 21 generally contained only one to two surfactant-producers and, in some cases, none. It is not known at this time what might cause the observed temporal shift in the numbers of surfactant-producers isolated. The du Nouy ring method showed surface tension reduction in all positive isolates (2742 dyn / cm) and little to no reduction in surface tension for negative isolates (5068 dyn / cm). This comparison conrmed that the drop-collapse method provides comparable results to the du Nouy ring method. The advantage of the drop-collapse method for screening purposes is that the test volume required is much smaller (5 mL) than the volume required for the du Nouy ring test (20 mL). Thus, the cultures can be

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Fig. 2. The quantitative drop-collapse method. The gure shows the results obtained with two different surfactants: (A) P. aeruginosa IGB83 with a CMC of 27 mg / L and (B) SDS with a CMC of 1845 mg / L. Each point represents the mean and standard deviation of ve replicates from experiments that were carried out in triplicate.

Fig. 3. Du Nouy ring method shows the same two surfactants (A) P. aeruginosa IGB83 with a CMC of 27 mg / L and (B) SDS with a CMC of 1845 mg / L. Each point represents the mean and standard deviation of triplicate samples.

grown up in smaller test tubes, requiring less media and less room for incubation.

3.4. Application of the quantitative drop-collapse method

The efcacy of the drop-collapse method for surfactant quantitation was tested by determining the concentration of surfactant within column fraction efuents. Typical results from such an experiment are shown in Fig. 4. Both the du Nouy ring and drop-collapse methods showed similar overall results; however, the drop-collapse results have smaller associated standard deviations. One reason for this may be that a smaller dilution was required for the drop-collapse samples (1:250) than for the du Nouy

ring method (1:500). The difference in dilution requirement is because the drop-collapse method has a larger range of measurement, three to four times the CMC, than the du Nouy ring method, up to the CMC. The larger range of measurement for dropcollapse means that less sample dilution is required, thereby minimizing dilution error. We also found in this study that the drop-collapse method was less operator-dependent. Several operators in our laboratory used both methods and consistently showed less variability using the drop-collapse method.

4. Summary The use of a drop-collapse technique for the screening of surfactant-producing microorganisms has been reported previously (Jain et al., 1991). In

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range that can be measured is greater in the dropcollapse method; (3) the drop-collapse test was just as easy to perform and more reproducible than the du Nouy ring method (Fig. 4) and (4) the drop-collapse method uses only a common dissecting microscope and calibrated micrometer, while the du Nouy ring method requires a surface tensiometer, a piece of equipment that is not routinely found in most microbiology laboratories because of its cost.

Acknowledgements This research was supported by Grant P42 ES04940 from the National Institute of Environmental Health Sciences, NIH. Our thanks to Debora Gage-Fasse for technical help in evaluating the dropcollapse method and to Dr. G. Soberon-Chavez, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, for providing P. aeruginosa IGB83. We would also like to thank Bruce Russell for his expertise in photographing the qualitative and quantitative results in Fig. 1.

Fig. 4. Breakthrough curve for a Vinton soil column treated with 10 mM biosurfactant IGB83. The drop-collapse method ( j ) was compared to the du Nouy ring method ( s ) for the determination of determine biosurfactant concentrations. Separate standard curves were used for this experiment, with standards prepared in 7 mM KNO 3 .

addition, other rapid screening methods of biosurfactant-producing microorganisms, such as axisymmetric drop shape analysis by prole (ADSA-P) or colorimetric methods, have been reported (Desai and Banat, 1997; Hansen et al., 1993; Shulga et al., 1993; Siegmund and Wagner, 1991; Van der Vert et al., 1991). In this study, the drop-collapse technique has been rened and further developed into a quantitative assay for use both in screening surfactant-producing isolates and in determining surfactant concentration. The type of oil used to coat the test wells dictates the application of the method. Specically, Pennzoilcoated wells can be used to rapidly screen for surfactant-producing organisms, while mineral oilcoated wells can be used to quantitate surfactant concentration. A comparison of the traditional du Nouy ring method to the drop-collapse method revealed that the drop-collapse test has several advantages over the surface tension measurement: (1) the drop-collapse method requires only 5 mL of sample, in comparison to a minimum volume of 20 mL required by the du Nouy ring method; (2) the effective concentration

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