Journal of Microbiological Methods 70 (2007) 503 – 510

www.elsevier.com/locate/jmicmeth

The application of a high throughput analysis method for the screening of

potential biosurfactants from natural sources
Chien-Yen Chen a,b , Simon C. Baker c,⁎, Richard C. Darton a
a
Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK
b
Department of Earth and Environmental Sciences, National Chung Cheng University, 168 University Road, Min-hsiung, Chia-yi 621, Taiwan
c
School of Life Sciences, Oxford Brookes University, Gypsy Lane, Oxford OX3 0BP, UK
Received 26 April 2007; received in revised form 6 June 2007; accepted 6 June 2007
Available online 23 June 2007

Abstract

The presence of biosurfactants in growth media can be evaluated by a variety of methods, none of which are suitable for high throughput
studies. The method described here is based on the effect of meniscus shape on the image of a grid viewed through the wells of a 96-well plate.
The efficacy of the method was demonstrated by the selection of a bacterium (producing a biosurfactant able to reduce the surface tension of pure
water from 72 to 28.75 mN m− 1) from a culture collection isolated from aviation fuel-contaminated land. The assay was found to be more
sensitive, rapid and easy to perform than other published methods. It does not need specialised equipment or chemicals and excludes the bias
which results from the surfactant properties of medium used for bacterial growth.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Biosurfactant; Emulsification test; Surface tension; High throughput screening

1. Introduction (Rodrigues et al., 2006). The best known biosurfactant, surfactin,

Biosurfactants are a diverse group of surface-active biomo-
lecules produced by many living organisms (Maier, 2003; Banat,
1995). These amphiphilic compounds contain a hydrophobic
and a hydrophilic moiety, and have the ability to reduce
interfacial tension between different fluid phases. Their uses and
potential commercial applications have been reported in several
fields, including surfactant-assisted flooding for enhanced oil
recovery in the oil industry, emulsifiers in the food industry, and
moisturizers in the cosmetic industry (Cameotra and Makkar,
2004; Desai and Banat, 1997; Banat et al., 2000).
There has also been interest in the biological activity of these
biosurfactants, which has helped to revive an interest in them as
potential sources of new drugs. For example mannosylerythritol
lipid and viscosin have been shown to display antibiotic effects
and a number of other potential biomedical applications
⁎ Corresponding author. Tel.: +44 1865 484057; fax: +44 1865 483242.
E-mail address: simon.baker@brookes.ac.uk (S.C. Baker).
0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2007.06.006
is a lipopeptide antibiotic produced by Bacillus subtilis. It exhibits
an antiviral and antibacterial action (Maget-Dana and Ptak, 1995;
Vollenbroich et al., 1997) and could be the prototype of a new
generation of antitumor drug because of its excellent interfacial
properties (Peypoux et al., 1999).
The development of rapid and reliable methods for screening
and selection of microbes from thousands of potentially active
organisms and the subsequent evaluation of surface activity holds
the key to the discovery of new biosurfactants. An emulsification
test, followed by the measurement of surface tension reduction
and various physical and chemical properties, have been used for
evaluating potential biosurfactant-producing microorganisms by
many researchers. The most suitable and popular test is that
applied by Cooper and Goldenberg (1987). This method assays
biosurfactant activity by measuring the height and stability of an
emulsion layer formed by vigorously shaking the culture broth
with an equal volume of kerosene or hexadecane. While this
method is simple and easy to perform, it does not give quantitative
data, and so a number of other methods have been developed.
These include the placement and analysis of media droplets on
slides, a drop-collapsing test, an axisymmetric drop shape
504 C.-Y. Chen et al. / Journal of Microbiological Methods 70 (2007) 503–510

analysis by profile, several colorimetric methods, a salt 2. Materials and methods

aggregation test, an assessment of bacterial adhesion to
hydrocarbon compounds and a replica plate test. The various 2.1. Bacterial strains
methods for initial evaluation of biosurfactant can be ranked,
according to how they satisfy three requirements of a screening A culture collection of 36 strains had been previously
test (Table 1): isolated from a site contaminated by aviation fuel (Nicklin-
Faull, University of London, Birbeck College, unpublished
(1) The ability to identify potential organisms. data). Bacteria were initially identified by the API system
(2) The ability to assess quantitatively how effective the supplied by Bio-Merieux. The API 20E test kit was used, and
surfactant is. the manufacturer's instructions were followed by incubating
(3) The ability to screen many candidates quickly. bacteria at 30 °C. Examination of the strips was conducted after
24 h. The identification of strains was carried out according to
The performance of these methods according to these criteria the API identification manual.
is shown in Table 1.
The objective of the work reported in this paper is to develop 2.2. Medium for biosurfactant production
a method for rapid screening of organisms deemed likely
candidates for the production of biosurfactant. For the purpose An M9 minimal salt medium was used as described by Miller
we have selected a method and apparatus for measuring surface (1972), except that the following were added to final concentra-
configuration described previously by Vaux and Cottingham tions of: Na2EDTA, 140 mM; ZnSO4·7H2O, 7.6 mM; MnSO4·4-
(2001). In this technique liquid samples are confined in wells of H2O, 25 mM; CuSO4·5H2O, 3.2 mM; (NH4)6Mo7O24·4H2O,
a multiwell microtitre plate, and a light beam is passed through 0.9 mM; CoCl2·6H2O, 6.7 mM. Glucose that had been filter-
the sample, offset from the centre of the well. The intensity of sterilised (Millipore membrane PVDF, 0.22 μm filter) was added
the reflected and transmitted light beams is dependent upon the to M9 minimal salt medium to give a concentration of 0.2% (w/w).
angle of incidence with the liquid surface, which varies with the This glucose was the only carbon source for bacterial growth.
surface tension of the liquid. Thus measurement of the
transmitted intensity can be used as an indicator of the presence 2.3. Culture conditions
of biosurfactant.
Stocks of 36 bacteria were thawed, transferred to Petri dishes
with nutrient agar and grown overnight at 30 °C. One single
Table 1 colony of every plate was inoculated to a 25 mL universal tube
Summary of simple and rapid methods currently used for evaluating with 10 mL M9 minimal medium and cultured overnight at
biosurfactant 30 °C, at 200 rpm on a rotary shaker. Cells were removed from
Analytical technique Qualitative Quantitative Analysis the broth by centrifugation at 13,000 rpm for 30 min and the
analysis analysis speed supernatant of each strain was harvested for use in the
Emulsification test a, b ++ No + biosurfactant activity assay.
Droplet on slides c + No ++
Drop-collapsing test d, e + + ++
2.4. Methods for the determination of biosurfactant activity
Axisymmetric drop shape + + +
analysis by profile f, g
Colorimetric methods h, i, j + No + 2.4.1. Emulsification activity assay
Salt aggregation test k + No + A qualitative biosurfactant activity assay was performed
Bacterial adhesion to hydrocarbon + No + using an emulsification test. This was carried out using 1 mL of
compounds l
supernatant with 0.5 mL n-hexadecane placed in an Eppendorf
Replica plate test m, n + No +
Microplate method o +++ +++ +++ tube and vortexed for 30 s. The supernatants which produced a
stable cloudy appearance in the emulsion layer were chosen to
+++ = highest efficacy.
a
Cooper and Goldenberg (1987).
take part in a larger emulsification test using a final volume of
b
Neu and Poralla (1990). 5 mL supernatant and 5 mL n-hexadecane. This suspension was
c
Persson and Molin (1987) and Persson et al. (1988). then vortexed in a test tube for 2 min and the height of the
d
Jain et al. (1991). emulsion layer was measured after 2 h and again after 48 h. A
e
Bodour Adria and Miller-Maier (1998). higher emulsion layer was taken to indicate greater surface
f
Rotenberg et al. (1983).
g
van der Vegt et al. (1991).
activity.
h
Shulga et al. (1993).
i
Hansoen et al. (1993). 2.4.2. Surface tension measurement
j
Siegmund and Wagner (1991). The surface tension of the supernatant and purified surfactant
k
Lindahl et al. (1981). solutions was determined with a tensiometer (Tracker, Interfa-
l
Dillon et al. (1986).
m
van der Mei et al. (1987). cial Technology Concept, France) at room temperature using the
n
Mozes and Rouxhet (1987). pendant drop shape technique. A 3.5 mL sample of high quality
o
This work, developed from Vaux and Cottingham (2001). water was put into a clean glass cell with the capacity to hold
 C.-Y. Chen et al. / Journal of Microbiological Methods 70 (2007) 503–510 505

8 mL, and placed onto the tensiometer platform. A syringe Table 2

needle of inverted question mark shape was submerged in the Bacterial strains used in this study
water and a small volume of air blown through it to form a Strain number Species
submerged bubble. Surface tension at the bubble surface was BBK001 Stenotrophomonas maltophilia
measured using an automated video imaging system and BBK002 Sporolactobacillus
software, matching bubble shape to that in a solution of BBK003 Stenotrophomonas maltophilia
BBK004 Paenibacillus macerans
known surface tension. The instrument was calibrated with air
BBK005 Paenibacillus macerans
and high quality water to a reading of 72 ± 0.3 mN m− 1. To BBK006 Bacillus subtilis
measure the surface tension as a function of concentration, the BBK006A Bacillus coagulans
test supernatant was added to the 3.5 mL high quality water in BBK007A Pasturella sp.
aliquots of 0.5 mL. The maximum amount of the test liquid that BBK007B Sporolactobacillus sp.
BBK008B Bacillus subtilis
could be added was 4.5 mL due to the size of the glass cell.
BBK009 Paenibacillus macerans
Between each measurement, the syringe needle and the glass BBK011 Unknown Gram positive coccus
cell were rinsed three times with water, three times with acetone BBK012 Unknown Gram positive coccus
and then allowed to dry. BBK013A Bacillus circulans
BBK013B Bacillus sp.
BBK014 Ochrobactrum anthropi
2.4.3. Serial dilution in a 96-well culture plate
BBK015 Sphingobacterium spiritivorum
A mixed culture of 36 strains was inoculated to M9 medium BBK016 Ochrobactrum anthropi
and incubated 48 h at 30 °C, 200 rpm on a rotary shaker. A BBK017A Sphingomonas paucimobillis
bacteria sample of 100 μL was mixed with 900 μL of sterile BBK017B Stenotrophomonas maltophilia
broth, and then successive dilutions were made. The mixture BBK018 Unknown Gram negative rod
Bacillus megaterium/Bacillus circulans a
was serially diluted (8 replicates) from 10− 1 to 10− 12 in a 96-
BBK019
BBK020 Bacillus subtilis
well, 4 mL culture plate (ABgene, Epsom, UK). The plate was BBK023 Bacillus circulans
incubated at 30 °C, 200 rpm on a rotary shaker for 48 h. The BBK024 Bacillus megaterium/Bacillus circulans
supernatant was collected by centrifugation of culture plate at BBK026 Sphingobacterium spiritivorum
8000 rpm for 50 min. 100 μL of each of the diluted bacteria BBK027 Bacillus licheniformis
BBK028 Unknown Gram negative rod
samples was then spread onto an LB agar plate. The number of
BBK029 Stenotrophomonas maltophilia
bacteria colonies that grew on each plate was counted. BBK030 Unknown Gram positive rod
BBK031 Unknown Gram positive coccus
2.4.4. Qualitative microplate analysis BBK032A Brevibacillus brevis
The surfactant activity of individual strains was determined BBK032B Sporolactobacillus
BBK034A Ochrobactrum anthropi
using a multiwell plate assay. A 100 μL sample was taken from BBK034B Sphingobacterium spiritivorum
the supernatant of each strain and was added to a microwell of a BBK038 Bacillus licheniformis
96-microwell plate (Cliniplate, Labsystems). The plate was then
Identification was performed using API 20E.
viewed using a backing sheet of paper with a black and white a
Inconclusive identification.
grid. The optical distortion of the grid provided a qualitative
assay for the presence of surfactant.
identified four isolates as genus Stenotrophomonas species,
2.5. Most-probable-number (MPN) method three as genus Sporolactobacillus, three as genus Paenibacil-
lus, three as genus Sphingobacterium, three as genus Ochro-
A preliminary trial of the MPN method for potential bactrum and the rest of the strains were mostly from the genus
biosurfactant-producing microorganisms was performed using Bacillus as might be expected from a soil sample (Table 2).
a 96-well culture plate described above. After incubation and However, some strains could not be identified by the API
centrifugation, 100 μL supernatant from each well was method because insufficient positive results were available for
transferred to a fresh 96-well microtitre plate and surfactant interpretation. These strains were further characterised by Gram
activity was examined using the same qualitative microplate staining and microscopic morphology, but no conclusive
analysis. Wells with significant optical distortion of the grid identification could be made.
were considered positive. The MPN was calculated using the
8 rows MPN table (Cochran, 1950; Woomer, 1994). 3.2. Evaluation of surface activity

3. Results and discussion 3.2.1. Emulsification activity assays

3.1. Identification of bacteria
The API 20E method of numerical taxonomy was applied for
a rapid identification of all the 36 strains. The results are listed
in Table 2. From the 36 strains studied, the API protocol
The most widely used methods for the measurement of
surface activity are based on emulsification tests. Of the 36
strains tested, the majority produced extracellular emulsificants,
but only 10 organisms produced a strong surfactant capable of
generating a stable emulsion over several hours. These
organisms (BBK006, -008B, -013A, -016, -017A, -019, -020,
506 C.-Y. Chen et al. / Journal of Microbiological Methods 70 (2007) 503–510

-023, -024 and -027) were harvested for larger scale
emulsification analysis with the purpose of precise confirmation
(shown in Fig. 1A and B).
Both Cooper and Goldenberg (1987) and Neu and Poralla
(1990) used an emulsification test to study bacteria capable of
biosurfactant production in a medium containing tryptic soy broth
(TSB) and yeast extract (YE). However, there are compounds in
TSB which have some surfactant activity, and can in some
circumstances give a spurious positive result in an emulsification
test. In this study, M9 minimal medium supplemented with
glucose was employed as a selective medium to culture bacteria as
M9 minimal medium was found to have no intrinsic surfactant
activity (assayed via tensiometry). Furthermore, biosurfactants
might stabilise (emulsifiers) or destabilise (de-emulsifiers) the
emulsion, so that a screening test based on the emulsification test
alone will fail to identify compounds with surfactant activity
which destabilise emulsions.
3.2.2. Surface tension measurements
The ten potential producers of biosurfactant were identified
from those that gave the most promising results in the
emulsification activity assay. Culture supernatants were col-
lected and for each the relationship between surface tension and
supernatant concentration was determined (Fig. 2). As
expected, the surface tension decreased with an increase in
surfactant concentration until a limiting value of the surface
tension was reached at some critical concentration. This is the
critical micellar concentration (CMC). It is clear that BBK006
exhibited the highest surface activity with the lowest surface
tension of 28.75 mN m− 1. Supernatant 19 had the second

Fig. 1. A. Height of emulsion layer of 5 mL supernatants when vortexed with the same volume of n-hexadecane. B. Emulsification test after settling time of 48 h (note
8A is BBK006).
 C.-Y. Chen et al. / Journal of Microbiological Methods 70 (2007) 503–510 507

Fig. 2. Surface tension curve after the addition of supernatant.

strongest effect, with 8B and 20 also showing a similar strong
ability to reduce surface tension. In contrast, the emulsion layer
of 16 was dilute and sparse, which showed that the surfactant
from 16 is not as good an emulsifying agent. However, the
surface tension result indicated that the biosurfactant from
supernatant 16 was strong but farther from CMC than the
others. No biosurfactant from species similar to strain 16
(Ochrobactrum anthropi) has previously been described. We
conclude that this could be an interesting candidate for further
investigation, although it would not have been easily detected
from the emulsification test. Therefore a new method was
required.
3.2.3. Quantitative microplate analysis
We have used a simple microplate assay based on the
proposal of Vaux and Cottingham (2001), the principle of which
is as follows. Pure water in a well has a flat surface which meets
the sides of the well at 90°. The presence of surfactant in the

Fig. 3. Qualitative microplate assay of a high quality water control (A) and biosurfactant from strain BBK016 (B).
508 C.-Y. Chen et al. / Journal of Microbiological Methods 70 (2007) 503–510

water causes some wetting at the edge of the well, and the fluid
surface becomes concave. The fluid takes the shape of a single
diverging lens, which distorts the image of the grid below the
well, when viewed from above (Fig. 3).
The supernatant of strain 16 was tested for surfactant activity
using the qualitative microplate assay and surfactant activity
could be easily detected by optical distortion of grids compared
with a control as shown in Fig. 3. Compared to the emulsification
test, the microplate assay proved to be sensitive, rapid and easy
to perform and could be used without specialised equipment or
chemicals. This assay offers the potential for conversion to high
throughput screening for biosurfactant-producing microbes
before undertaking extensive experiments in biosurfactant
production.
3.3. Adaptation to high throughput screening
A study was then designed to investigate the potential of the
microplate method for high throughput screening. A mixture of
bacterial species was grown in a culture microtitre plate (4 mL
maximum capacity, a type of different plate from the assay
microtitre plate) with 0.2% (w/w) glucose as the only source of
energy and carbon. A serial dilution was carried out to estimate
the most-probable-number of biosurfactant-producing organ-
isms and to isolate a pure strain from the mixed culture. Eight
series of wells were inoculated with each of the twelve dilutions
and after incubation, counts of the numbers of wells that showed
growth and surfactant activity at each dilution were made (see
Fig. 4A).
After centrifugation, the supernatants were transferred to a
fresh 96-well microtitre plate (see Fig. 4B) for the purpose of
assay. Positive and negative wells at each dilution step were
used to quantify the number of microorganisms for potential
biosurfactant production in the original suspension using a
most-probable-number (MPN) statistical analysis (based on
Cochran, 1950; Woomer, 1994). In order to know the number of
viable bacteria in each dilution step, dilution plating was carried
out using the same dilution factor.

Fig. 4. The mixture was serially diluted from 10− 1 to 10− 12 in a 96-well (A) 4 mL culture plate. After incubation and centrifugation, the supernatant was transferred to a
fresh 96-well microtitre plate (B). The optical distortion of the grid in qualitative microplate assay provided a qualitative assay to screen for surfactant-producing
microorganisms.
 C.-Y. Chen et al. / Journal of Microbiological Methods 70 (2007) 503–510 509

Table 3
Summary of MPN method and dilution plating
Set number (8 wells in each set) 1 2 3 4 5 6 7 8 9 10 11 12 Combination of MPN/
positives mL
Amount inoculated into agar plates and 100 100 100 100 100 100 100 100 100 100 100 100
each well of plate (μL)
Dilution factor 10− 1 10− 2 10− 3 10− 4 10− 5 10− 6 10− 7 10− 8 10− 9 10− 10 10− 11 10− 12
Colony count on agar plate TNTC a TNTC TNTC TNTC TNTC 263 81 15 1 0 0 0
No. of wells showing growth 8 8 8 8 7 7 4 4 2 1 0 1
No. of wells showing surfactant production 8 8 8 8 7 7 4 4 2 1 0 1
No. of wells showing surfactant production 8 8 8 8 7 7 4 4 2 0 0 0 4-4-2 15
after correction b
a
TNTC indicated as “too numerous to count”.
b
Woodward (1957) recommended that MPN tables should be corrected by omission of those combinations of positive wells that are so improbable that they raise
concerns about laboratory error or contamination.

The result of a typical microplate assay shown in Fig. 4 References

showed that the optical distortion of the grid provided a
qualitative detection of surfactant-producing microorganisms.
The serial dilution method and MPN analysis allowed us to
estimate the concentration of microbes in a broth (summarized
in Table 3).
The published MPN method of Cochran (1950) and Woomer
(1994) for 8 tubes, (4-4-2) indicated that an average of 15
organisms were inoculated into each of the wells in the middle
set (i.e. set number 8). As shown in Table 3, set number 8 was
inoculated with 100 μL at 108 dilution factor. Therefore the
most-probable-number of biosurfactant-producing organisms
per mL of the original, undiluted sample was 1.5 × 1010, a result
substantiated by parallel plate counts.
4. Conclusions
Interest in biosurfactants from microorganisms has led to the
development of several screening methods. However, the
existing methods all have some disadvantages such as being
time-consuming, requiring specific equipment and chemicals,
or complicated preparation. The screening method we describe
here requires no specialised equipment or chemicals, and no
prior structural information about the biosurfactants (as would
be needed for colorimetry). The presence of biosurfactants
produced by microbes is easy and fast to investigate by visual
inspection. The results show that the microplate assay has
several advantages over the other screening assays: (1)
instantaneous detection; (2) small volume of sample (just
100 ìL needed); (3) easy and quick to perform; and (4) more
reliable and cheaper than the others. It is thus suitable for
incorporating in an automatic procedure for high throughput
screening. The B. subtilis strain of BBK006 identified in this
paper was chosen as a research target for biosurfactant
production, to be described in a further work.
Acknowledgement
The authors are very grateful to the National Science Council
of Taiwan for sponsoring this research (NSC 95-2119-M-194-
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