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Title: Identification and characterization of biosurfactantsproduced by the Arctic bacterium Pseudomonas putida BD2
Author: Tomasz Janek Marcin Łukaszewicz Anna Krasowska
PII: S0927-7765(13)00318-4DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2013.05.008Reference: COLSUB 5788
To appear in: Colloids and Surfaces B: Biointerfaces
Received date: 12-12-2012Revised date: 23-4-2013Accepted date: 7-5-2013
Please cite this article as: T. Janek, M. Łukaszewicz, A. Krasowska, Identificationand characterization of biosurfactants produced by the Arctic bacteriumPseudomonas putida BD2, Colloids and Surfaces B: Biointerfaces (2013),http://dx.doi.org/10.1016/j.colsurfb.2013.05.008
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Identification and characterization of biosurfactants produced by the Arctic bacterium
Pseudomonas putida BD2
Tomasz Janeka,*, Marcin Łukaszewicza,b, Anna Krasowskaa
a) Department of Biotransformation, Faculty of Biotechnology, University of Wroclaw,
Przybyszewskiego 63/77, 51-148 Wrocław, Poland
b) Faculty of Chemistry, Wroclaw University of Technology, Gdańska 7/9, 50-344 Wrocław,
Poland
Abstract
One hundred and thirty bacterial strains, isolated from Arctic soil on the Svalbard
Archipelago, were screened for biosurfactant production. Among them, an isolate identified
as Pseudomonas putida BD2 was selected as a potential biosurfactant-producer based on the
surface/interfacial activity of the culture supernatant. The ability of the strain to produce
simultaneously phosphatidylethanolamines and rhamnolipid, using glucose as a sole carbon
source, was demonstrated. The rhamnolipid Rha-Rha-C10-C10 and two homologs of
phosphatidylethanolamine were extracted from cell-free supernatant of P. putida BD2 culture
with ethyl acetate and identified by UPLC-MS analysis. For Rha-Rha-C10-C10 the surface
tension decreased from 72 to 31 mN/m and the critical micelle concentration was 0.130
mg/mL. The Rha-Rha-C10-C10 was able to form stable aggregates (80-121 nm). Pretreatment
of a polystyrene surface with 0.5 mg/mL rhamnolipid inhibited bacterial adhesion by 43-79%
and that of the pathogenic fungal species C. albicans by 89-90%. The same concentration of
phosphatidylethanolamines inhibited bacterial adhesion by 23-72% and that of C. albicans by
96%- 98%. To our knowledge, this is the first report where one type rhamnolipid and two
homologs of phospholipid biosurfactants were produced by Pseudomonas putida isolated
from Arctic soil.
Keywords: Pseudomonas putida BD2; glycolipid; phosphatidylethanolamine; biosurfactant
* Corresponding author. Tel.: +48 713756210; fax: +48 713756234
E-mail address: [email protected] (Tomasz Janek).
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1. Introduction
Biosurfactants are a structurally diverse group of surface-active compounds
synthesized by microorganisms [1, 2]. The structure of these secondary metabolites is strictly
dependent on the medium composition, e.g. carbon and nitrogen sources. Microbial
surfactants encompass a wide spectrum of molecules, such as lipopeptides, glycolipids,
phospholipids, fatty acids, neutral lipids and polymeric biosurfactants [3]. Microorganisms
such as bacteria, filamentous fungi and yeast produce biosurfactants extracellularly or as
compounds associated with cell membrane [4].
These natural compounds have many advantages over synthetic surfactants, such as
non-toxic character or high biodegradability, and have extensive applications in many
industrial fields such as production of food, cosmetics, pesticides, detergents,
pharmaceuticals, oil recovery and bioremediation [5, 6]. Besides their potential application in
industrial and environmental remediation, these surface-active compounds have been reported
to possess several properties of therapeutic and biomedical importance. The features of
biosurfactants allow for, e.g. antimicrobial and anti-adhesive action against several pathogenic
microorganisms on medical implants [7, 8] restoration and maintenance of bacterial
homeostasis in human body [9] or antibiotic activity against bacteria and fungi [10].
Glycolipids are one of the most promising biosurfactants. They consist of
carbohydrates and long-chain aliphatic or hydroxyaliphatic acids. Rhamnolipids (RLs)
produced by Pseudomonas strains have a glycosyl head group (a rhamnose moiety) and a 3-
(hydroxyalkanoyloxy)alkanoic acid (HAA) [11]. The two most abundant RLs congeners are
mono-rhamnolipids and di-rhamnolipids often referred to as Rha-C10-C10 and Rha-Rha-C10-
C10, respectively [12]. Factors influencing RLs production in the genus Pseudomonas were
reviewed by Muller et al. [13]. Pseudomonas was shown to be capable of using both water
soluble carbon substrates, such as glycerol, mannitol and fructose [14] as well as water
immiscible n-alkanes to produce rhamnolipid-type biosurfactants [15]. Not only the type of
carbon and nitrogen source but also the respective C/N ratio strongly influence total RL
production [16, 17].
Desai and Banat [1] have reviewed a wide range of techniques to determine the
presence of biosurfactants in culture media. The chemical and physico-chemical methods
used for this purpose include determination of surface tension (ST), emulsification index (E24)
determined over a 24-h period [18], drop-collapsing test [19], thin layer chromatography
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(TLC), high performance liquid chromatography (HPLC), Fourier transform infrared
spectroscopy (FTIR) and mass spectrometry (MS).
The aims of this work included identification of a new Arctic strain, purification of
biosurfactants secreted to minimal medium with glucose as a sole carbon source,
characterization of chemical structures of isolated biosurfactants and their physico-chemical
properties (minimum surface tension, critical micelle concentration, emulsification activity)
and the anti-adhesive activities against several microorganisms. Furthermore, the size of
micelles of Rha-Rha-C10-C10 was also studied.
2. Experimental
2.1. Microorganism and growth culture
Pseudomonas putida BD2 was isolated from a soil sample from the Arctic
Archipelago of Svalbard (latitude 77o 04' N, longitude 15o 14' E). After being grown on LB
medium containing 5 g/L yeast extract, 10 g/L bacto-tryptone and 10 g/L NaCl, the bacterial
strain was maintained at −70 °C in 15% glycerol. Experiments were carried out in 1000 mL
baffled Erlenmeyer flasks containing 500 mL of medium with the following composition: 7
g/L K2HPO4, 2 g/L KH2PO4, 1 g/L (NH4)2SO4, 0.5 g/L sodium citrate x 2H2O, and 0.1 g/L
MgSO4 x 7H2O (pH 7.0) [20]. Finally, 20 g/L of glucose was used as the carbon source.
Medium components were sterilized separately at 120 °C, 1 atm for 20 min. The flasks were
inoculated with 5 ml overnight pre-culture of the strain and incubated at 28 °C on a reciprocal
rotary shaker at 170 rpm.
2.2. Identification of bacterial strain
Strain BD2 was identified by the API 20E test for Enterobacteriacae (BioMerieux,
Marcy l’Etoile, France) and genomic DNA was obtained by the method of Janek et al. [20].
Genomic DNA was isolated from a one-day liquid culture using a GeneMATRIX Bacterial
and Yeast Genomic DNA Purification kit 50 (EURx, Gdansk, Poland) following the
manufacturer’s standard protocol, and used as a target for polymerase chain reaction (PCR)
amplification using primers 27F (5’-AGR GTT YGA TYM TGG CTC AG-3’) and 1492R
(5’-GGT TAC CTT GTT ACG ACT T-3’). The PCR products were subjected to agarose gel
electrophoresis, purified using a GeneMATRIX PCR/DNA Clean-up Purification kits (EURx,
Gdansk, Poland), and their nucleotide sequence was determined at the Institute of
Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland) using the
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standard shotgun sequencing reagents and a 454 GS FLX Titanium Sequencing System
(Roche, Basel, Switzerland), according to the manufacturer's instructions. The rDNA gene
sequence obtained from the BD2 isolate was compared with other bacterial sequences by
using GenBank (http://www.ncbi.nlm.nih.gov/BLAST/).
2.3. Biosurfactant production and purification
Microbial growth was evaluated by measuring the absorbance of the culture at 600
nm. Glucose released was measured enzymatically with the Glucose kit from Biosystems
(Barcelona, Spain). Emulsification assays of the biosurfactants were performed using a
previously described method [18]. The emulsification activity of the supernatant was
measured by adding 6 mL petroleum ether to 4 ml of aqueous sample in a test tube, vortexing
for 2 min, and then leaving it to settle for 24 h. The emulsification index (E24) was estimated
as the height of the emulsion layer, divided by the total height, multiplied by 100. The surface
tension of the free culture supernatant was measured with a tensiometer using the du Nouy
procedure with a platinum ring at 25 °C.
Cell-free supernatant was obtained from the culture by centrifugation (8000g) at 4 °C
for 30 min. It was extracted three times with ethyl acetate and evaporated under vacuum. The
crude biosurfactants were dissolved in 5 mL of methanol, filtered through a 0.2 µm syringe
filter and 40 mg crude biosurfactants were separated by preparative layer chromatography
(PLC) (silica gel 60 F254 20 cm x 20 cm with concentrating zone, 1 mm x 20 cm x 4 cm,
Merck; chloroform/methanol/water 65:15:2). Visualization was carried out by UV
transilluminator. In preparative mode, visualized spots were scraped off and the biosurfactants
were extracted with 15 mL of chloroform/methanol (2:1). The surface-active fractions were
evaporated under vacuum and stored at -20 °C for further studies.
2.4. Thin-layer chromatography
The purified biosurfactant fractions obtained after preparative TLC purification were
separated by thin layer chromatography (Silica gel 60; Merck, Darmstadt, Germany). The
compounds were separated using a mobile phase of chloroform/methanol/water in a 65:15:2
ratio (vol/vol/vol). The resulting spots on the TLC were visualized by spraying with a solution
of 0.25% ninhydrin in acetone [20] for amine groups and orcinol solutions [8] for sugar
detection. For the detection of lipids, the plates were treated with 0.1% bromothymol blue in
10% aqueous ethanol [21] and iodine.
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2.5. Liquid chromatography
Aliquots of the biosurfactant extracts or of the chromatographed samples were
dissolved in methanol to obtain 1 mg/mL solutions.
Chromatographic equipment consisted of an Acquity ultra-performance liquid
chromatography (UPLC) system (Waters, Milford, MA, USA). Separation was performed on
a BEH C18 column (1.7 µm, 2.1 x 100 mm, Waters) held at 40 oC. A multistep linear gradient
composed of eluent A (water + 0.1% trifluoroacetic acid) and eluent B (acetonitrile + 0.1%
trifluoroacetic acid) was applied. The autosampler temperature was maintained at 10 oC and
10 µL of sample solution was injected. From 0.00 min to 13.00 min a linear gradient was
applied from the mixture A:B (70:30, vol/vol) to A:B (0:100, vol/vol). A plateau of 100%
eluent B from 13.00 min to 15.00 min was set before going back to 70% eluent A from 15.00
min to 16.00 min. Flow rate was 0.3 mL/min.
2.5. Mass spectrometry analysis
The LC system was coupled to a Waters Xevo QTOF MS mass spectrometer with an
atmospheric pressure electrospray interface. The ESI source was set in positive and negative
ionization mode. The parameters used for the mass spectrometer under the ESI mode were as
follows: ESI+ (capillary voltage 4.50 kV, source temperature 80 °C, desolvation temperature
250 °C, desolvation gas flow 650 L/h, cone gas flow 18 L/h) and ESI- (capillary voltage 3.50
kV, source temperature 120 °C, desolvation temperature 250 °C, desolvation gas flow 700 L/h,
cone gas flow 20 L/h). Different cone voltages were tested to study the fragmentation of
biosurfactants. LC/MS full scan positive and negative modes were performed from m/z 200 to
800; alternatively, LC/ESI-MS/MS modalities were applied to the selected precursor ions,
following the conditions set during the infusion analysis. Waters MassLynx version NT 4.1
was used for LC/MS system control and data analysis.
2.6. Physico-chemical characterization
The surface tension was measured at 25 °C with a Kruss K100 (Kruss GmbH,
Hamburg, Germany), tensiometer by the du Nouy’s ring method. The instrument was
calibrated against Mili-Q ultrapure distilled water. Aqueous solutions of purified rhamnolipids
in the concentration range of 180–5 mg/L were obtained by successive dilutions of a
concentrated sample prepared by weight in ultrapure water.
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The mean particle size and polydispersity index (PDI) of rhamnolipids diluted in
Milli-Q water were determined using a Zetasizer Nano-ZS (Malvern Instruments Ltd.,
Malvern, UK) and PCS software. The analysis of particle size and PDI, determined by photon
correlation spectroscopy, was done using the volume distribution algorithm. The
polydispersity index qualifies the particle size distribution, which here ranged from 0 for
monodisperse to 1.0 for entirely heterodisperse emulsions. In order to obtain the optimum
light scattering intensity and for the size analysis, approximately 100 μL of the rhamnolipid
was added to 900 µL of Milli-Q water. All the measurements were carried out at 25 ºC.
Emulsification activity of phospholipids and rhamnolipids was tested on different
organic compounds: petroleum ether, benzene, n-hexane, cyclohexane, n-hexadecane, xylene,
toluene, sunflower, olive and rapeseed oil. The purified biosurfactants dissolved in 5.0 mL
distilled water (0.2% w/v) were mixed with 5 mL of each hydrophobic compound, and then
vortexed at high speed for 2 min, after which the mixture was kept at 25 °C for 24 h.
2.7. Biological assays
The anti-adhesive properties of phospholipids and rhamnolipids were tested on several
pathogenic strains that colonize medical devices or human body. A wide range of Gram-
positive and Gram-negative bacteria were tested: Escherichia coli ATCC 25922, Escherichia
coli ATCC 10536, Escherichia coli 17-2 (clinical isolate, Wroclaw Medical University),
Enterococcus faecalis ATCC 29212, Enterococcus faecalis JA/3 (clinical isolate, Wroclaw
Medical University), Eenterococcus hirae ATCC 10541, Staphylococcus epidermidis KCTC
1917 and Proteus mirabilis ATCC 21100. Yeast strains: Candida albicans ATCC 20231,
Candida albicans SC5314.
Inhibition of microbial adhesion by the biosurfactants was tested in 96-well plates
(Sarstedt, Nümbrecht, Germany) by the method of Janek et al. [7]. The wells of a sterile 96-
well flat-bottom plate were filled with biosurfactants dissolved in PBS. Next, the plates were
incubated for 2 h at 37 °C on a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at
300 rpm and subsequently washed twice with PBS. The overnight cultures of microbial
strains were centrifuged, washed twice with PBS (pH 7.4) and re-suspended in PBS. The
microbial suspension was added to each well of the microtiter plate. After a 2-h incubation at
37 °C in a rotary shaker at 300 rpm, nonadherent cells were removed by three washes with
PBS. Then the plates were stained with 0.1% crystal violet for 5 min and again washed three
times with PBS. The adherent microorganisms were permeabilized and the dye was
resolubilized with 150 μL of isopropanol-0.04 N HCl and 50 μL of 0.25% SDS per well.
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Crystal violet optical density readings of each well were taken at 590 nm on the Asys UVM
340 (Biogenet) microplate. All the experiments were carried out in triplicate with suitable
controls (uninoculated microtiter plates).
3. Results and discussion
3.1. Characterization of Pseudomonas putida BD2 and purification of secreted biosurfactants
The strain BD2 isolated from Arctic soil was Gram-negative, aerobic, oxidase-
positive and these results classified it as belonging to the Pseudomonas genus. The optimal
temperature for growth of strain BD2 in MSM with 2% glucose was 28 oC. Comparison of
16S rRNA nucleotide sequence from strain BD2 with sequences in the GenBank database
revealed 100% identity with the corresponding sequences from Pseudomonas putida [22].
When P. putida BD2 was grown at 28 oC in submerged culture in a sterile mineral salts
medium with 2% (w/v) glucose, 0.15 g/L of the crude biosurfactant was produced after 7 days
of culture (Fig. 1). The maximum decrease in the surface tension of the culture medium (45.8
mN/m) occurred after 48 h. The emulsification index (E24) of the culture filtrate with
petroleum ether increased from 2% to 62.9% and stabilized after 144 h. The extracted
biosurfactants were then subjected to PLC. A series of bands were observed after PLC (Fig.
2). These fractions were divided into 4 groups and scraped off from the preparative plates, and
then eluted by a chloroform–methanol mixture (2:1) and evaporated. These 4 samples,
designated A to D, were re-dissolved in 200 μL of methanol and stored for future studies.
Further assays were carried out with these 4 samples in order to determine their surface
activity by the drop-collapsing assay. Due to their strong surface tension reducing activity,
samples A (5 mg) and B (20.5 mg) from 40 mg crude extract were selected and subjected to
further analytical UPLC. Four more cycles of the biosurfactant separation and extraction
experiments had to be repeated in order to obtain sufficient quantity of samples for the
analytical UPLC, physico-chemical and biological assays.
Until recently, Pseudomonas aeruginosa was considered as potentially the main
producer of rhamnolipids [23, 24]. However, P. aeruginosa species is pathogenic. Thus, our
research was focused on characterizing other RLs producer species. This paper describes a
new strain of P. putida BD2 which is generally considered non-pathogenic [25]. Besides
production of the rhamnolipid, another important tenet is the finding that P. putida BD2 also
produces phospholipids.
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Strain BD2 grew in culture media containing different types of carbon source (data not
shown) but maximum cell growth and biosurfactant production occurred with glucose used as
a sole carbon source. While this finding was in agreement with that of Rendell et al. [26],
some other strains have shown the highest biosurfactant production on hydrophobic substrates
such as soybean oil [27, 28]. To our knowledge, non-pathogenic P. putida strains produce less
rhamnolipids than the pathogenic P. aeruginosa. The production of rhamnolipids was
observed when a strain of P. putida was grown on soluble substrates such as glucose,
molasses or on poorly soluble substrates such as hexadecane, reaching values from 0.52 to 1.2
g/L; however the exact structures of the produced rhamnolipids were not determined [29, 30].
3.2. UPLC-MS analysis
The molecular mass of the purified active compounds, i.e. fractions A and B, was
measured using UPLC/ESI-MS/MS. Previous studies revealed that the active ingredient of
biosurfactants produced by Pseudomonas was rhamnolipids [31, 32]. UPLC/MS analysis
revealed the presence of one major component with retention time 5.23 min. The mass
spectrum and chemical structure of the component is illustrated in Fig. 3A. One [M-H]-
pseudomolecular ion with m/z 649 was observed. ESI-MS showed a molecular ion of Rha-
Rha-C10-C10, which gave rise to fragments with m/z 479 and 310. The possible di-
rhamnolipid structure and fragmentation is also shown in Fig. 3A.
In the generally accepted pathway for the rhamnolipid synthesis, Rha-C10-C10 is the
precursor for Rha-Rha-C10-C10 [33]. This would suggest that for each di-rhamnolipid
detected, the mono-rhamnolipid congener should also be detected, although this is not always
the case [34]. In our work the di-rhamnolipid did not have a mono-rhamnolipid congener.
Rhamnolipids containing only one hydroxyaliphatic acid were also found by other authors
[35, 36]. In our study we did not find glycolipids with one -hydroxy fatty acid connected,
which might be due to differences either in the culture conditions or in the strain used.
ESI-MS spectra of P. putida BD2 phospholipids showed that the dominant species are
protonated phosphatidylethanolamines (PE(32:1)) (PE(33:1)) at m/z 690.5 and 704.5,
respectively. Dimers of phospholipids were observed in the range of m/z 1300–1500 and
verified by tandem mass spectrometry (MS/MS). Tandem mass spectra display the
fragmentation pattern of [PE(16:1/16:0) + H]+ and [PE(16:1/17:0) + H]+, which mainly lose
neutral phosphoethanolamine (141 mass units) to yield the fragment ion at m/z 549.5 and
563.5 respectively. The peak of m/z 549.5 was dissociated to form two major fragment ions to
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form [C16H31O]+ of m/z 239.2, and [C16H29O]+ of m/z 237.2 (Fig. 3B). The m/z 563.5 was
used as precursor ions for further ESI-MS/MS analyses (Fig. 3C).
3.3. TLC analysis
Thin-layer chromatography showed that the crude extract was a mixture of compounds
that could be classified as glycolipid and phospholipid. Visualization was carried out with
orcinol and ninhydrin followed by heating. The lower spot (Rf = 0.26) is from the di-
rhamnolipid, while the higher spot (Rf = 0.47) comes from phosphatidylethanolamines (data
not shown). Our results are similar to previously reported results of TLC of rhamnolipids
from different strains of Pseudomonas [23]. The Rf values obtained by these authors are 0.27
and 0.57 for di- and mono-rhamnolipid, respectively.
3.4. Physico-chemical properties of biosurfactants
Biosurfactants tend to form aggregates with their hydrophilic head groups in contact
with water and their hydrophobic tails directed toward the interior of the micelles and away
from water. These aggregates are generally called micelles. Depending on their shape and
structure, micelles can be generally divided into four classes: spheres, rods, lamellar
structures, and vesicles. The aggregation behavior of Rha-Rha-C10-C10 was characterized by
measuring the change in the size of aggregates with changing compound concentration. Fig. 4
shows the aggregation behavior of Rha-Rha-C10-C10. The size of the aggregates increased
with concentration in the range of 0.15–0.5 mg/mL and reached 80-121 nm. In previous
studies, we have demonstrated that aggregation above the CMC was also observed for
pseudofactin II, a surface active lipopeptide produced by Pseudomonas fluorescens [37]. The
mean hydrodynamic diameter of pseudofactin-biosurfactant micelles in water ranged from
40.2 nm to 60.3 nm. Xu et al. [38] demonstrated that the type and concentration of surfactant
significantly influence the properties of aggregates. The aggregation behavior of the surface
active compounds affects the size distribution and stability of aggregates. There is still little
information available about aggregation and polydispersity index (PDI) of various
rhamnolipids. In this work, we show the size of aggregates for one type of rhamnolipid (Rha-
Rha-C10-C10), while other reports focused on mixtures of rhamnolipid homologs [39, 40].
The new data about each described biosurfactant will contribute to understanding the
aggregation process and compounds the final activity of the product.
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The emulsifying properties of 0.2% aqueous solution of biosurfactants produced by
BD2 strain were examined with different vegetable oils and hydrocarbons. The purified
phospholipids and the rhamnolipid emulsified vegetable oils more efficiently than
hydrocarbons (Fig. 5). Biosurfactants could emulsify 70% of olive oil, while emulsification of
n-hexane, xylene, hexadecane, and petroleum ether ranged from 51 to 65%.
The rhamnolipid (Rha-Rha C10 C10) reduced surface tension of the water from 72 to
31 mN/m. Critical micelle concentration (CMC) determined with the aid of a series of
concentrations was around 0.130 mg/mL (Fig. 6). The surface tension properties of
rhamnolipids are dependent on bacterial strain, medium composition, and culture conditions
that determine the composition and distribution of homolog molecules present in the final
product [41]. CMC values in a wide range from 5 to 386 mg/L and surface tension from 25 to
31 mN/m have been reported for different rhamnolipid mixtures [42]. Abalos et al. [43]
reported CMC values of 106, 150 and 234 mg/L for different mixtures of mono- and di-
rhamnolipids. More hydrophilic rhamnolipids like Rha-C10 or Rha-Rha-C10 yielded CMC as
high as 200 mg/L whereas lower values of 5-60 mg/L have been reported for mixtures
containing mainly mono-rhamnolipid Rha-C10-C10 [44].
3.5. Biological activity of biosurfactants
The microtiter-plate based anti-adhesion assay estimated the rhamnolipid and the
phospholipids concentrations that were effective in inhibiting adhesion of the pathogenic
microorganisms. The highest percentage of microbial adhesion inhibition was obtained
against C. albicans SC5314, 90% for the rhamnolipid (Table 1) and 98% for the
phospholipids (Table 2) at 0.5 mg/mL concentration of the biosurfactants. The order of anti-
adhesive action against microorganisms studied was C. albicans>E. faecalis>P. mirabilis>E.
coli>S. epidermidis>E. hirae.
Attempts to reduce or inhibit microbial adherence is a viable means to control
infection, since such adherence is one of the initial stages of the infectious process. Thus, pre-
coating of solid surfaces by biosurfactants might constitute a new and effective means of
combating colonization by pathogens and an effective strategy to reduce microbial adhesion
[10]. Thanks to their amphiphilic structure, surface active compounds reduce the surface
tension at interfaces, and thus affect the adhesion and detachment of microorganisms [10].
Gudina et al. [45] characterized the anti-adhesive activity of biosurfactants against several
microorganisms including Gram-positive and Gram-negative bacteria. Several investigations
have pointed that biosurfactants produced by probiotic microorganisms inhibited adhesion of
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bacteria or fungi to artificial surfaces [46-48]. For example, biosurfactants from Streptococcus
thermophilus and Lactobacillus acidophilus suppressed C. albicans adhesion to silicone
rubber [8, 49].
In conclusion, we suggested that the phospholipids and rhamnolipid produced by
nonpathogenic P. putida BD2 could be used as alternative anti-adhesive agents against
pathogenic microorganisms responsible for diseases and infections in the urinary, vaginal and
gastrointestinal tracts. This points to their potential utility as coating agents for medical
insertional materials.
4. Conclusions
Our data allow several conclusions:
- The new bacterial isolate BD2 was identified as Pseudomonas putida.
- Two distinct fractions of the crude biosurfactants correspond to a mixture of
phosphatidylethanolamines PE(32:1), PE(33:1) and di-rhamnolipid (Rha-Rha–C10–C10).
- P. putida species has not been previously described as rhamnolipid and phospholipid
producer.
- The physico-chemical properties such as CMC (0.130 mg/mL), surface tension (31 mN/m),
emulsification (51- 70%), and size of aggregates above CMC 80-121 nm were evaluate.
- Described biosurfactants have strong anti-adhesive activities against bacterial and yeast
strains on a polystyrene surface.
Acknowledgments
This work was supported by grant from the University of Wroclaw 1498/M/WB/11 and
by the Polish National Centre for Science No N N302 640940.
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Tables
Table 1. Inhibition of microbial adhesion in the microtiter plate by purified di-rhamnolipid.
PBS was used as control and set at 0% as no inhibition occurs. Values ± confidence interval,
n=9, =0.05.
Microorganism Inhibition of microbial adhesion (%)
Rhamnolipid concentration (mg/ml) Control (PBS)
0.500 0.250 0.150 0.075 0.035 0
Escherichia coliATCC 25922 65±0.2 30±0.2 21±0.1 13±0.2 9±0.1 0
Escherichia coliATCC 10536 52±0.2 44±0.1 42±0.1 42±0.1 39±0.2 0
Escherichia coli17-2 62±0.1 61±0.2 58±0.5 41±0.1 34±0.3 0
Enterococcus faecalis ATCC 29212 79±0.3 67±0.1 55±0.1 10±0.2 7±0.2 0
Enterococcus faecalis JA/3 55±0.1 26±0.1 18±0.2 16±0.3 8±0.2 0
Enterococcus hiraeATCC 10541 43±0.1 21±0.1 19±0.3 14±0.3 9±0.3 0
Staphylococcus epidermidis KCTC 1917 45±0.1 41±0.1 22±0.1 11±0.1 10±0.2 0
Proteus mirabilisATCC 21100 75±0.5 72±0.1 71±0.1 65±0.1 54±0.1 0
Candida albicans ATCC 20231 89±0.2 84±0.2 72±0.1 36±0.1 25±0.1 0
Candida albicansSC5314 90±0.2 72±0.2 69±0.1 40±0.1 37±0.2 0
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Table 2. Inhibition of microbial adhesion in the microtiter plate by purified
phosphatidylethanolamines. PBS was used as control and set at 0% as no inhibition occurs.
Values ± confidence interval, n=9, =0.05.
Microorganism Inhibition of microbial adhesion (%)
Phosphatidylethanolamines concentration (mg/ml) Control (PBS)
0.500 0.250 0.150 0.075 0.035 0
Escherichia coliATCC 25922 35±0.2 20±0.1 11±0.1 9±0.2 4±0.2 0
Escherichia coliATCC 10536 47±0.2 42±0.2 38±0.2 24±0.1 19±0.2 0
Escherichia coli17-2 42±0.2 36±0.2 28±0.1 25±0.2 19±0.1 0
Enterococcus faecalis ATCC 29212 72±0.1 70±0.1 54±0.1 43±0.1 38±0.3 0
Enterococcus faecalis JA/3 40±0.2 31±0.1 25±0.3 9±0.2 2±0.3 0
Enterococcus hiraeATCC 10541 23±0.1 19±0.2 15±0.1 11±0.3 8±0.1 0
Staphylococcus epidermidis KCTC 1917 41±0.2 38±0.1 33±0.2 14±0.1 6±0.2 0
Proteus mirabilisATCC 21100 63±0.2 58±0.1 52±0.1 45±0.2 35±0.2 0
Candida albicans ATCC 20231 96±0.1 88±0.2 82±0.2 76±0.1 75±0.1 0
Candida albicansSC5314 98±0.1 93±0.1 90±0.1 84±0.1 79±0.1 0
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Figures
Fig. 1. Time course of biosurfactant production, cell growth, surface tension and
emulsification activity of P. fluorescens BD5 grown on mineral salt medium with 2% glucose
as carbon source at 28 oC.
Fig. 2. Preparative layer chromatography (PLC) of compounds obtained from P. putida BD2
by using a mobile phase of chloroform/methanol/water in the 65:15:2 ratio (vol/vol/vol).
Fractions were visualized by UV transilluminator.
Fig. 3. UPLC-ESI–MS spectrum, negative and positive ion mode of rhamnolipid and
phospholipids respectively. (a) Negative mass spectrometry showing the predominance
(retention time of 5.23 min) of m/z 649 (Rha-Rha-C10-C10). Product ion spectra of the
protonated molecules [M + H]+ of (b) PE(32:1) at retention time 10.13 min and (c) PE(33:1)
at retention time 10.64 min.
Fig. 4. The effect of di-rhamnolipid concentration on aggregate size.
Fig. 5. Emulsifying index (E24) of biosurfactants with different hydrocarbons, mean ± SD
(n=3).
Fig. 6. Effect of Rha-Rha-C10-C10 concentration on surface tension. The CMC was
determined from the intersection of regression lines that describe the two parts of the curve,
below and above CMC. Results represent the average of three independent measurements.
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Graphical abstract
Highlights
1. Pseudomonas putida BD2 was isolated and identified from an Arctic soil.
2. The strain was found to release to growth medium biosurfactants.
3. Biosurfactants were purified and indentified using various physico-chemical methods.
4. Structures were identified using UPLC-MS method.
5. One glycolipid and two phospholipids were identified.
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*Graphical Abstract (for review)
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Fig. 1.
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Fig. 2.
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Fig. 3.
Fig. 4.
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Fig. 5.
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Fig. 6.
