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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.
-023, -024 and -027) were harvested for larger scale alone will fail to identify compounds with surfactant activity
emulsification analysis with the purpose of precise confirmation which destabilise emulsions.
(shown in Fig. 1A and B).
Both Cooper and Goldenberg (1987) and Neu and Poralla 3.2.2. Surface tension measurements
(1990) used an emulsification test to study bacteria capable of The ten potential producers of biosurfactant were identified
biosurfactant production in a medium containing tryptic soy broth from those that gave the most promising results in the
(TSB) and yeast extract (YE). However, there are compounds in emulsification activity assay. Culture supernatants were col-
TSB which have some surfactant activity, and can in some lected and for each the relationship between surface tension and
circumstances give a spurious positive result in an emulsification supernatant concentration was determined (Fig. 2). As
test. In this study, M9 minimal medium supplemented with expected, the surface tension decreased with an increase in
glucose was employed as a selective medium to culture bacteria as surfactant concentration until a limiting value of the surface
M9 minimal medium was found to have no intrinsic surfactant tension was reached at some critical concentration. This is the
activity (assayed via tensiometry). Furthermore, biosurfactants critical micellar concentration (CMC). It is clear that BBK006
might stabilise (emulsifiers) or destabilise (de-emulsifiers) the exhibited the highest surface activity with the lowest surface
emulsion, so that a screening test based on the emulsification test 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
strongest effect, with 8B and 20 also showing a similar strong investigation, although it would not have been easily detected
ability to reduce surface tension. In contrast, the emulsion layer from the emulsification test. Therefore a new method was
of 16 was dilute and sparse, which showed that the surfactant required.
from 16 is not as good an emulsifying agent. However, the
surface tension result indicated that the biosurfactant from 3.2.3. Quantitative microplate analysis
supernatant 16 was strong but farther from CMC than the We have used a simple microplate assay based on the
others. No biosurfactant from species similar to strain 16 proposal of Vaux and Cottingham (2001), the principle of which
(Ochrobactrum anthropi) has previously been described. We is as follows. Pure water in a well has a flat surface which meets
conclude that this could be an interesting candidate for further 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 bacterial species was grown in a culture microtitre plate (4 mL
surface becomes concave. The fluid takes the shape of a single maximum capacity, a type of different plate from the assay
diverging lens, which distorts the image of the grid below the microtitre plate) with 0.2% (w/w) glucose as the only source of
well, when viewed from above (Fig. 3). energy and carbon. A serial dilution was carried out to estimate
The supernatant of strain 16 was tested for surfactant activity the most-probable-number of biosurfactant-producing organ-
using the qualitative microplate assay and surfactant activity isms and to isolate a pure strain from the mixed culture. Eight
could be easily detected by optical distortion of grids compared series of wells were inoculated with each of the twelve dilutions
with a control as shown in Fig. 3. Compared to the emulsification and after incubation, counts of the numbers of wells that showed
test, the microplate assay proved to be sensitive, rapid and easy growth and surfactant activity at each dilution were made (see
to perform and could be used without specialised equipment or Fig. 4A).
chemicals. This assay offers the potential for conversion to high After centrifugation, the supernatants were transferred to a
throughput screening for biosurfactant-producing microbes fresh 96-well microtitre plate (see Fig. 4B) for the purpose of
before undertaking extensive experiments in biosurfactant assay. Positive and negative wells at each dilution step were
production. used to quantify the number of microorganisms for potential
biosurfactant production in the original suspension using a
3.3. Adaptation to high throughput screening most-probable-number (MPN) statistical analysis (based on
Cochran, 1950; Woomer, 1994). In order to know the number of
A study was then designed to investigate the potential of the viable bacteria in each dilution step, dilution plating was carried
microplate method for high throughput screening. A mixture of 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.
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