Appl Microbiol Biotechnol (2013) 97:53155327
DOI 10.1007/s00253-013-4869-y
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Biosurfactant-enhanced immobilization of hydrocarbon-oxidizing
Rhodococcus ruber on sawdust
Irena B. Ivshina & Maria S. Kuyukina &
Anastasiya V. Krivoruchko & Oleg A. Plekhov &
Oleg B. Naimark & Elena A. Podorozhko &
Vladimir I. Lozinsky
Received: 1 February 2013 / Revised: 17 March 2013 / Accepted: 19 March 2013 / Published online: 13 April 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Immobilization of microorganisms on/in insolu-
ble carriers is widely used to stabilize functional activity of
microbial cells in industrial biotechnology. We immobilized
Rhodococcus ruber, an important hydrocarbon degrader, on
biosurfactant-coated sawdust. A biosurfactant produced by
R. ruber in the presence of liquid hydrocarbons was found
to enhance rhodococcal adhesion to solid surfaces, and thus,
it was used as a hydrophobizing agent to improve bacterial
attachment to a sawdust carrier. Compared to previously
used hydrophobizers (drying oil and n-hexadecane) and
emulsifiers (methyl- and carboxymethyl cellulose, poly(vi-
nyl alcohol), and Tween 80), Rhodococcus biosurfactant
produced more stable and homogenous coatings on wood
surfaces, thus resulting in higher sawdust affinity to hydro-
carbons, uniform monolayer distribution of immobilized R.
ruber cells (immobilization yield 2930 mg dry cells/g), and
twofold increase in hydrocarbon biooxidation rates com-
pared to free rhodococcal cells. Two physical methods,
i.e., high-resolution profilometry and infrared thermogra-
phy, were applied to examine wood surface characteristics
I. B. Ivshina : M. S. Kuyukina : A. V. Krivoruchko
Institute of Ecology and Genetics of Microorganisms,
Russian Academy of Sciences, 13 Golev Street,
614081 Perm, Russia
I. B. Ivshina : M. S. Kuyukina : A. V. Krivoruchko (*)
Microbiology and Immunology Department,
Perm State National Research University, Perm, Russia
e-mail: nast@iegm.ru
O. A. Plekhov : O. B. Naimark
Institute of Continuous Media Mechanics,
Russian Academy of Sciences, Perm, Russia
E. A. Podorozhko : V. I. Lozinsky
A.N. Nesmeyanov Institute of Organoelement Compounds,
Russian Academy of Sciences, Moscow, Russia
and distribution of immobilized R. ruber cells. Sawdust-
immobilized R. ruber can be used as an efficient biocatalyst
for hydrocarbon transformation and degradation.
Keywords Biosurfactant . Immobilization . Rhodococcus
ruber . Sawdust carrier . Hydrophobization . High-resolution
profilometry . Infrared thermography
Introduction
Immobilized microbial cells are widely used in membrane
bioreactors (Cheng et al. 2010), biochips and biosensors
(Borghol et al. 2010), and biofertilizers and biocatalysts
(awniczak et al. 2011) developed for various technological
processes. To increase immobilization efficacy, different engi-
neering approaches are utilized, including cell surface design
(Kondo and Ueda 2004), micro-patterning of carrier surfaces
(Choi and Lee 2011), biospecific attachment (Tk et al. 2005;
Borghol et al. 2010), magnetic and electric fields (Keshavarz
2005; Robatjazi et al. 2012), transition metal activation
(Fernandes 2006), and physicalchemical modification of car-
rier surfaces with polymers (Carnazza 2007; Choi and Lee
2011). Among these approaches, relatively simple and cost-
effective methods of carrier coating with substances which
enhance microbial adhesion, e.g., hydrophobizing agents, are
currently explored. Hydrophobizing coating reduces free sur-
face energy leading to a sharp decrease in the adhesion energy
barrier and therefore facilitates microbial immobilization.
Various chemicals may be used as hydrophobizing agents,
such as light crude oil (Pirog et al. 2005), indium tin oxide
(Bayoudh et al. 2006), and Teflon-like and organosilicon thin
films (Lehock et al. 2009).
It should be noted that optimal hydrophiliclipophilic
balance (HLB) of the hydrophobizer and its homogenous
5316
distribution on a carrier surface are required for successful
hydrophobization. However, excessive hydrophobization
was shown to affect negatively bacterial immobilization
(Podorozhko et al. 2008; Lehock et al. 2009). For instance,
Rhodococcus sp. S3E2 and Rhodococcus sp. S3E3 poorly
adhered to highly (8.620.5 mJ/m2 total surface energy) hy-
drophobic paper coated with Teflon-like or organosilicon films
(Lehock et al. 2009). We previously found that a number of
immobilized Rhodococcus cells on sawdust decreased dramat-
ically after its excessive (in a ratio of carrier/hydrophobizer of
1:2, v/v) treatment with drying oil (Podorozhko et al. 2008).
Homogeneous coating of a carrier surface could be achieved
by applying an organic solvent solution or fine dispersed water
emulsion of the hydrophobizer. However, conventional organ-
ic solvents used for this purpose are toxic, so their application
is hindered by health safety regulations. A preferable way
would be hydrophobizer dispersion in water using emulsifying
agents, e.g., starch (Tesch et al. 2002), methyl celluloses
(Kulicke et al. 1998), or synthetic surfactants (Tadros et al.
2004). In this connection, a search for new solvent-free
hydrophobization techniques and emulsifiers with improved
functional and safety characteristics is of current interest.
We hypothesized that microbial surfactants could help to
overcome hydrophobization difficulties described above. In
contrast to synthetic analogs, biosurfactants are environmentally
friendly, biodegradable, and less toxic (Mukherjee et al. 2006;
Kuyukina and Ivshina 2010b). Most biosurfactants are nonionic
and bind to a solid carrier through polar or nonpolar groups that
depend on biosurfactant and carrier properties. Nonpolar sur-
factant groups interact with hydrophobic carriers via van der
Waals forces. Simultaneously, polar surfactant groups presented
to a water medium result in surface hydrophilization. With
initially hydrophilic carriers, biosurfactants interact through
polar groups, forming hydrogen bonds with hydroxyl groups
on a carrier surface. Nonpolar biosurfactant groups are
presented to a water medium and increase surface hydropho-
bicity. Effect of hydrophilization/hydrophobization is strongly
related to the HLB value of a biosurfactant (Zhang and
Somasundaran 2006). Biosurfactants with high HLB values
were applied for hydrophilization of medical and industrial
materials, e.g., silicone rubber, polystyrene, stainless steel, or
Teflon, to inhibit adhesion of microorganisms and prevent
microbial contamination (Rodrigues et al. 2004; Shakerifard et
al. 2009; Zeraik and Nitschke 2010). However, in natural
conditions, many microorganisms produce surfactants to pro-
mote colonization of solid substrates, e.g., high-molecular par-
affins, polyaromatics, elemental sulfur, coal particles, and
suspended organic matter via an increase in surface hydropho-
bicity (Neu 1996). Thus, biosurfactants with low HLB values
can be applied as alternative hydrophobizing agents for en-
hancement of microbial immobilization.
Hydrocarbon-oxidizing actinobacteria of the genus
Rhodococcus are industrially important microorganisms
Appl Microbiol Biotechnol (2013) 97:53155327
involved in bioremediation of oil-contaminated ecosystems,
degradation of xenobiotics, production of valuable metabo-
lites, bioindication of oil and gas deposits, microbial enhanced
oil recovery, and biotransformations of hydrophobic organic
compounds (Martnkov et al. 2009; Kuyukina and Ivshina
2010a; Yam et al. 2010). Previously, we immobilized
Rhodococcus ruber into natural (pine sawdust) (Podorozhko
et al. 2008) and synthetic (poly(vinyl alcohol) and poly(acryl-
amide) cryogels) (Kuyukina et al. 2006, 2009) carriers to
stabilize functional activities of rhodococcal cells. In the pres-
ent work, a possibility to enhance R. ruber immobilization
on sawdust using a biosurfactant was studied. A crude
biosurfactant from R. ruber IEGM 231 grown in the presence
of liquid hydrocarbons was used (Kuyukina et al. 2001). The
main active components of this biosurfactant are three treha-
lose lipids with various alkyl chain lengths (Philp et al. 2002),
and other components include 4.3 % of protein and 44.3 % of
nonpolar lipids and a residual hydrocarbon. The HLB value of
the R. ruber biosurfactant was 6.47.5 (Kuyukina et al. 2006).
The advantages of this biosurfactant compared to other micro-
bial surfactants (e.g., rhamnolipids, emulsan, sophorolipids,
surfactin) are as follows: it is produced by nonpathogenic
bacteria, functionally stable under extreme conditions, has
relatively low HLB value, and shows high surface and emul-
sifying activities (Kuyukina and Ivshina 2010b). In the present
paper, applications of Rhodococcus biosurfactant as
hydrophobizing and emulsifying agents for the sawdust sur-
face modification are discussed.
Materials and methods
Bacterial strains, growth conditions, and reagents Five R.
ruber strains IEGM 73, IEGM 77, IEGM 172, IEGM 231,
and IEGM 241 from the Regional Specialized Collection of
Alkanotrophic Microorganisms, Perm, Russia (acronym
IEGM, WFCC # 768, www.iegm.ru/iegmcol/index.html)
were used in this study. The cultures were pre-grown on
nutrient agar at 28 C for 72 h. For experiments, bacterial
cells were grown in 250-mL Erlenmeyer flasks containing
100 mL of Luria-Bertani broth (LB) or Rhodococcus sur-
factant (RS) medium supplemented with 3 % (v/v)
n-hexadecane. The RS medium contained (in grams per
liter): KH2PO4, 2.0; Na2HPO4, 2.0; (NH4)2SO4, 2.0; NaCl,
1.0; KNO3, 1.0; MgSO4 7H2O, 0.2; CaCl2 2H2O, 0.02;
FeCl3 7H2O, 0.01; yeast extract, 0.1; and trace element
solution, 1.0 mL. Inoculated flasks were incubated in a
rotary shaker (160 rpm) at 28 C for 2846 h. Late expo-
nential phase cells were used in all experiments. Absorbance
(A 600 ) measured with the UV/Vis spectrophotometer
Lambda EZ201 (Perkin-Elmer, USA) and the bacterial bio-
mass values calculated (in grams of dry cells per liter) were
used to monitor the bacterial growth.
Appl Microbiol Biotechnol (2013) 97:53155327
All reagents were >97.0 % purity unless otherwise men-
tioned. Hydrocarbons, LB, and iodonitrotetrazolium violet
(INT) were purchased from Sigma-Aldrich. Mineral salts
and crystal violet were purchased from Reachim (Russia).
Organic solvents (acetone, chloroform, ethanol, ethyl ace-
tate, n-hexane, isopropanol) purchased from Sigma-Aldrich
or Acros Organics (Belgium) were GC-MS grade.
Biosurfactant production R. ruber strains were grown in RS
medium supplemented with n-hexadecane for 5 days, as
described above for growth conditions. Rhodococcus
biosurfactant was extracted from the bacterial culture with
methyl tert-butyl ether (Kuyukina et al. 2001), and the
amount of the crude biosurfactant recovered was determined
gravimetrically.
Bacterial adhesion measurements Adhesive activities of R.
ruber strains were determined according to Huber et al.
(2001) in 96-well polystyrene microplates (Medpolymer,
Russia) coated with Rhodococcus biosurfactant. The
biosurfactant was dissolved in isopropanol at concentrations
of 0.01, 0.1, 1.0, 10, or 100 mg/L, and 200 L of these
solutions were added into microplate wells. The plates were
then dried overnight under sterile air to evaporate
isopropanol. Biosurfactant-coated and control (without
biosurfactant) plates were filled up with 200 L of RS
medium supplemented with n-hexadecane and inoculated
with 5 L of rhodococcal cell suspension (109 cells/mL).
Inoculated plates were incubated in a Titramax 1000 incu-
bator (Heidolph Instruments, Germany) at 150 rpm, 28 C
for 48 h. After incubation, the culture medium with non-
adhered bacteria was carefully removed and the plates were
washed twice with phosphate buffer (3.53 g/L of Na2HPO4,
3.39 g/L of KH2PO4, pH 7.0) using a Stat Fax 2600
washer (Awareness Technology Inc., USA). After washing,
1 % (w/v) aqueous crystal violet solution was added into the
plate wells. Following 20-min staining at room temperature,
the dye was removed and the plates were washed twice with
phosphate buffer. Crystal violet incorporated into adhered
cells was extracted with the acetone/ethanol mixture (1:4, v/v)
and A630 was measured with a Multiscan Ascent photometer
(Thermo Electron Corporation, Finland). Calibration curves
for A630 and numbers of colony-forming units (CFU) were
used for quantification of bacterial adhesion (data not shown).
Sawdust hydrophobization procedure Pine sawdust was
sieved to obtain 12 mm particles. Rhodococcus biosurfactant,
5.0 % (w/v) as well as sunflower seed-based drying oil (Oksol,
Russia) and n-hexadecane both at concentrations of 10.0 %
(w/v) were used as hydrophobizing agents (Podorozhko et al.
2008). Hydrophobization procedures are summarized in
Table 1. A water emulsion of the biosurfactant was prepared
using a Soniprep 150 sonicator (Sanyo, Japan) at 23 kHz for
5317
1 min. Water emulsions of drying oil were prepared by thor-
ough mixing with Rhodococcus biosurfactant, methyl cellu-
lose (Reachim, Russia), carboxymethyl cellulose (Reachim,
Russia), poly(vinyl alcohol) of 16/1 trade mark (viscosity
average molecular weight 69,000, 99 % hydrolyzed) (NPO
Azot, Ukraine), or Tween 80 (Sigma-Aldrich) added into
water at concentrations of 1.03.0 % (w/v). Alternatively,
drying oil was dissolved in a Stoddard solvent (Polychim-
Invest, Russia). Sawdust and a hydrophobizer in a ratio of
1:1 (w/w) were mixed thoroughly for 10 min, dried under
sterile air at 25 C for 24 h, and then dried at 110 C for 1 h.
In some cases, sawdust hydrophobization was performed by
exposing sawdust to n-hexadecane vapors (Podorozhko et al.
2008). Hydrophobized sawdust samples were sterilized by
autoclaving at 105 C for 15 min. Since most biosurfactants
are thermostable (Rapp and Gabriel-Jrgens 2003; Khopade et
al. 2012), this autoclaving regime was expected to not influ-
ence biosurfactant-hydrophobized sawdust properties.
Relative hydrophobicity of native and hydrophobized
sawdust was estimated by measuring contact wetting angles
of water droplets using Axiostar plus microscope (Carl
Zeiss, Germany) equipped with VideoTesT-Size software
(VideoTesT, Russia) at 10 magnification. Examination
of the location of hydrophobized sawdust samples in the
n-hexadecane/water (1:1, v/v) two-phase system was
performed after 5-day incubation in a rotary shaker at
130 rpm, 25 C. Hydrocarbon adsorption to sawdust was
determined after the liquid phase removal followed by fil-
tration through the filter paper (Whatman no. 1). Adsorbed
n-hexadecane was extracted with n-hexane and measured
gravimetrically. Amounts of water vapors adsorbed by
hydrophobized sawdust were determined as described pre-
viously (Podorozhko et al. 2008).
Bacterial immobilization on hydrophobized sawdust R.
ruber cells grown in LB were washed twice with phosphate
buffer and resuspended in the same buffer to the concentration
corresponding to A600 1. Immobilization was performed in
250-mL Erlenmeyer flasks containing 1 g of hydrophobized
sawdust and 50 mL of the bacterial suspension in a rotary
shaker (130 rpm) at room temperature for 6 days. The A600 of
R. ruber suspension was measured daily to monitor cell im-
mobilization. Control flasks containing hydrophobized saw-
dust and the buffer without bacterial cells were used to
estimate the resistance of coatings to mechanical stress (shak-
ing and long contact with water) during incubation. Numbers
of immobilized R. ruber cells on sawdust were determined
using the modified INT staining method (Wrenn and Venosa
1996). For this, sawdust samples with immobilized cells were
washed three times with water and incubated with 0.01 %
(w/v) INT solution at room temperature for 24 h, thus allowing
the reduction of INT into insoluble redviolet INT-formazan.
After the liquid phase removal, INT-formazan was extracted
5318
Table 1 Hydrophobization var-
iants for sawdust carriers Hydrophobizing agent Concentration of a
hydrophobizing agent (%)
Rhodococcus biosurfactant 5.0
n-Hexadecane 10.0
Drying oil 10.0
Drying oil 10.0
Drying oil 10.0
Drying oil 10.0
Drying oil 10.0
Drying oil 10.0
with ethyl acetate, and A490 was measured. Calibration curves
for A490 and CFU were used to calculate numbers of viable
immobilized cells (data not shown). Numbers of cells tightly
bound to hydrophobized sawdust were determined after the
washing thrice with 1 M NaCl.
High-resolution profilometry and infrared thermography
Physical methods of high-resolution profilometry with a
New View 5000 interference microscope (Zygo, USA) and
infrared (IR) thermography with a CEDIP Silver 450 M
camera (CEDIP Infrared Systems, France) were used to
analyze wood surfaces with immobilized rhodococcal cells.
It is essential for these methods to use flat (smooth) surfaces
with low values of roughness allowing the obtaining of
readable images. For this, 222 cm pine wood blocks
were polished with fine sand paper (58.5 m, average grit
size) to remove surface imperfections. As a result, the
polished wood blocks with more than 80 % readable areas
were obtained. The blocks were hydrophobized using the
same procedure as described for sawdust hydrophobization.
Rhodococci were immobilized onto hydrophobized blocks
and glass slides used as reference smooth surfaces under the
conditions similar those described above for bacterial im-
mobilization on sawdust. After immobilization, blocks were
washed twice with phosphate buffer, dried at room temper-
ature for 1 h, and scanned using the interference microscope
under a 50-W halogen daylight lamp by moving the micro-
scope objective in a vertical position with a scanning pitch
of 75 nm. Interference images captured during scanning
with a video analog camera were analyzed with the
MetroPro software (Zygo, USA) generating topographic
maps and surface profiles.
The IR camera (with the following characteristics: sensi-
tivity to the passive emission of 35 m wavelength IR
photons, 14 bits, 0.025 C thermal resolution, 320256 pixels
per image, and 380 Hz full frame) was used for thermal
imaging of the wood blocks. Imaging was performed at day-
light illumination, temperature of 221 C, camera exposure
of 2,100 s, and the distance between the camera and the
sample of 15 cm. Hydrophobized wood blocks without
Appl Microbiol Biotechnol (2013) 97:53155327
Emulsifier Concentration of
an emulsifier (%)
No No
No No
No No
Rhodococcus biosurfactant 2.5
Methyl cellulose 1.0
Carboxymethyl cellulose 1.0
Polyvinyl alcohol 3.0
Tween 80 3.0
immobilized bacterial cells served as abiotic controls.
Thermal images were processed using Altair (FLIR Systems,
Netherlands) and MathCad 15.0 (Parametric Technology
Corporation, USA) software. Temperature differences (t,
in degrees Celsius) between inoculated and non-inoculated
block surfaces were determined for all experimental variants.
Since bacterial cells represent weak heat sources, statistically
significant positive t values were used as indicators for
viable immobilized bacteria. Additionally, wood blocks
hydrophobized with drying oil in excess (at ratio of 1:2
(w/w)) and adsorbed a priori low numbers of R. ruber cells
(Podorozhko et al. 2008) were prepared and analyzed in a
similar way. The numbers of immobilized R. ruber cells on
wood blocks were also controlled by the INT staining.
Hydrocarbon oxidation measurements The hydrocarbon-
oxidizing activity of immobilized R. ruber cells was deter-
mined in 250-mL Erlenmeyer flasks containing 100 mL RS
medium supplemented with n-hexadecane after incubation
in a rotary shaker (160 rpm) at 28 C for 10 days. Prior to
incubation, the flasks were inoculated with free or
immobilized cells taken at equal concentrations. Flasks were
closed with cotton wool stoppers to keep sterility and pro-
vide optimal aeration conditions. Non-inoculated flasks with
sawdust were used as controls. Residual n-hexadecane was
extracted with n-hexane and analyzed by GC-MS using the
Agilent 6890N chromatograph equipped with a quadrupole
mass spectrometric detector Agilent MSD 5973N (Agilent
Technologies, USA) and a quartz column HP-5MS having a
length of 30 m, diameter of 0.25 mm, and a film thickness of
0.25 km. GC-MS conditions were as follows: 80 C for
2 min, 10 C/min, 300 C for 5 min, temperature of injector
280 C, injection mode is splitless, the carrier gas is He, rate
1 mL/min, and m/z range of 40600.
Results
Biosurfactant effect on R. ruber adhesion Rhodococcus
biosurfactant was found to affect the R. ruber cell adhesion.
Appl Microbiol Biotechnol (2013) 97:53155327
Strong positive correlations (R = 0.92, p =0.03) between
biosurfactant production and adhesive activities towards
the polystyrene surface were revealed for all five R. ruber
strains studied (Fig. 1). Namely, R. ruber IEGM 73, IEGM
77, and IEGM 231 with high (5972 %) adhesive activities
demonstrated higher (8.213.7 g/L) biosurfactant produc-
tion, whereas R. ruber IEGM 172 and IEGM 241 with
relatively low (3031 %) adhesive activities produced lower
(4.55.8 g/L) amounts of the biosurfactant. Furthermore, in
the experiments with native and Rhodococcus biosurfactant-
coated polystyrene microplates, a 1.83.4-fold increase in R.
ruber adhesion was registered after polystyrene treatment
with the biosurfactant (Fig. 2). Adhesion of rhodococcal
cells increased exponentially at biosurfactant concentrations
ranging between 0.01 and 1.0 mg/L, then amounted to the
maximum value at 1.0 mg/L and did not change further at
higher (10100 mg/L) biosurfactant concentrations.
Physicochemical properties of hydrophobized sawdust As
seen in Table 2, Rhodococcus biosurfactant increased the
hydrophobicity of sawdust. Sawdust samples hydrophobized
with the biosurfactant located at the n-hexadecane/water in-
terface had a water contact angle 86, adsorbed 0.34 g/g water
vapors, and 32 % n-hexadecane. For comparison, native saw-
dust located in water had a contact angle of 67 and adsorbed
5 % n-hexadecane. Interestingly, the relative hydrophobicity,
hydrocarbon/water location, and water vapor-adsorbing ca-
pacity of biosurfactant-coated sawdust were similar to those
of sawdust samples hydrophobized with drying oil or n-
hexadecane which is also located at the interface, adsorbed
0.310.33 g/g H2O, and had a water contact angle of 9496.
However, hydrocarbon adsorption to biosurfactant-coated
sawdust was three to five times higher compared to drying
oil and n-hexadecane-treated carriers.
100
90
80 R. ruber
Adhesive activity (%)
R. ruber IEGM 73
70 IEGM 77
R. ruber
60 IEGM 231
R. ruber
50 IEGM 241
40
R. ruber
30 IEGM 172
y = 4.8709x + 10.083
20 R = 0.92, p = 0.03
10
0 lization curve with poorly distinguished phases (Fig. 3).
3 6 9 12
Produ ction of Rhodococcus biosu rfactan t (g/L)
Fig. 1 Correlation between R. ruber biosurfactant production and
adhesion to polystyrene. Meansstandard deviations of triplicates are
shown; R is a Pearson correlation coefficient
5319
100
90 *
* *
80
Adhesive activity (%)
70 *
60
50 *
40
30
20
10
0
0 0.01 0.1 1 10 100
Rhodococcus biosurfactant concentrations (mg/L)
Fig. 2 Adhesion of R. ruber IEGM 231 to polystyrene coated with
Rhodococcus biosurfactant at different concentrations. Meansstandard
deviations of triplicates are shown. Asterisk: statistically significant
(p<0.05) differences between untreated (control) and biosurfactant-coat-
ed polystyrene surfaces
As an emulsifying agent, Rhodococcus biosurfactant
acted similarly to methyl cellulose, carboxymethyl cellu-
lose, and poly(vinyl alcohol) and did not influence signifi-
cantly physicochemical properties of sawdust carriers
(Table 2). However, hydrocarbon adsorption to sawdust
treated with drying oil emulsified by the biosurfactant was
two to five times higher than that of sawdust hydrophobized
with drying oil in the presence of other emulsifiers. It should
be noted that the application of synthetic surfactant Tween
80 resulted in the lowest (only 5 %) adsorption of n-
hexadecane, carrier location in the water phase, and decreas-
ing a contact wetting angle to 67, similar to unmodified
sawdust (Table 2).
R. ruber immobilization results As seen in Fig. 3, the aver-
age numbers of immobilized R. ruber cells on sawdust
treated with different hydrophobizing formulations were
similar (3.5 10 9 , 2.8 10 9 , and 4.0 10 9 CFU/g for
biosurfactant, drying oil, and n-hexadecane, respectively).
However, differences in bacterial immobilization dynamics
were revealed. Namely, 5274 % of rhodococcal cells adsorbed
on sawdust samples hydrophobized with Rhodococcus
biosurfactant or n-hexadecane after 12 days, then immobiliza-
tion process slowed down and the rest of bacterial cells were
adsorbed within the next 45 days, thus demonstrating two-
phase immobilization. Using the sawdust hydrophobized with
drying oil, we observed rather a monotonous bacterial immobi-
15 Averagely, 9299 % of R. ruber cells remained tightly bound
to hydrophobized sawdust after washing it with 1 M NaCl (data
not shown).
Applied as an emulsifying agent for drying oil,
Rhodococcus biosurfactant provided significant time reduction
5320 Appl Microbiol Biotechnol (2013) 97:53155327

Table 2 Physicochemical properties of hydrophobized sawdust carriers

Hydrophobizer Contact wetting Location in the Water vapors Adsorption of

angle, deg n-hexadecane/water system adsorbed (g H2O/g) n-hexadecane (%)

Rhodococcus biosurfactant 866 Interface 0.340.03 323

Drying oil 949 Interface 0.330.02 101
n-Hexadecane 9610 Interface 0.310.02 71
Drying oil + Rhodococcus biosurfactant 916 Interface 0.380.03 232
Drying oil + methyl cellulose 965 Interface 0.350.01 111
Drying oil + carboxymethyl cellulose 958 Interface 0.310.01 81
Drying oil + poly(vinyl alcohol) 898 Interface 0.280.01 101
Drying oil + Tween 80 627 Water 0.280.01 52
Without hydrophobizer 675 Water 0.340.02 52

(up to 2 days) for bacterial immobilization on hydrophobized
sawdust compared to other emulsifiers studied (Fig. 4a).
Moreover, R. ruber immobilization efficiency on sawdust
treated with biosurfactant-emulsified drying oil was 2.75.7
times higher than corresponding values for Tween 80, methyl
cellulose, or carboxymethyl cellulose used as emulsifiers. As
seen in Fig. 4a, sawdust treated with methyl cellulose-
emulsified drying oil did not adsorb R. ruber cells and even a
slight increase in the cell suspension A600 value by 0.180
optical units was observed. Furthermore, most stable drying
oil coating on sawdust was formed in the presence of the
biosurfactant as evident from the low release of coating resi-
dues into the liquid medium (A600 of 0.166) in uninoculated
flasks (Fig. 4b). In comparison, conventional emulsifiers, such
as poly(vinyl alcohol), Tween 80, methyl cellulose, and
carboxymethyl cellulose led to the formation of less stable
4.0
3.5
Adsorption (x10 9 CFU/g)
coatings resulting in liquid medium A600 increases to 0.321,
0.442, 0.537, and 0.929 optical units, respectively (Fig. 4b).
Results of high-resolution profilometry and IR thermography
Using high-resolution topographic maps obtained by the in-
terference microscopy, the concentration and distribution of
rhodococcal cells on the surface of wood blocks treated with
different hydrophobizers were determined (Fig. 5). An aver-
age number of R. ruber cells on biosurfactant-treated blocks
was (1.30.1)107 cells/cm2. As seen in Fig. 5a, rhodococcal
cells were uniformly distributed across the surface forming cell
monolayers with distances between the cells averaging 0.5
2.0 m. Topographical maps of R. ruber cells immobilized on
biosurfactant-treated blocks were similar to those on wood
blocks hydrophobized with drying oil (Fig. 5a, b). However,
in the case of blocks hydrophobized with n-hexadecane, R.
ruber formed clusters consisting of 6080 cells with intercluster
distances from 0.5 to 5.0 m and intercellular distances from
0.2 to 1.0 m (Fig. 5c). Smaller cell clusters consisting of three
to ten cells were observed on glass slides used as the reference
smooth surfaces (Fig. 5d). It could be resumed that although the

3.0 concentration of R. ruber cells immobilized on differently

2.5 treated pine blocks and glass slides did not vary significantly
(average value was 107 cells/cm2), their distribution across
2.0 surfaces depended on the material to be hydrophobized and
1.5 the hydrophobization procedure applied.
Analyzing surface profiles of pine blocks, it was found
1.0 that the initial (non-hydrophobized) wood surface was quite
0.5 rough, which roughness was 39 times higher than that of a
control (glass slide) surface (Fig. 6). Particularly, small
0.0 irregular pores (0.10.5 m wide and 0.62.3 m deep)
0 1 2 3 4 5 6
Time (days) were observed on the surface of non-hydrophobized pine
blocks (Fig. 6a). Rhodococcus biosurfactant was shown to
Fig. 3 Dynamics of R. ruber IEGM 231 immobilization on sawdust fill up these pores and smooth out the pine block surface to
hydrophobized with the biosurfactant and conventional hydrophobizers.
Hydrophobizers: black up-pointing triangleRhodococcus biosurfactant,
the level of glass slides (Fig. 6b, e), thus suggesting a
white squaren-hexadecane, white circledrying oil. Meansstandard formation of homogenous hydrophobic coating. However,
deviations of triplicates are shown drying oil and n-hexadecane were revealed to produce
Appl Microbiol Biotechnol (2013) 97:53155327
1.6 a 1.6
1.4
1.2
1.0
A600 0.8
0.6
0.4
0.2
0.0
0 1 2 3 4 5
Time (days)
Fig. 4 Dynamics of R. ruber IEGM 231 immobilization on sawdust
hydrophobized with drying oil in the presence of different emulsifiers.
Emulsifiers: black circleRhodococcus biosurfactant, white circle
poly(vinyl alcohol), lozengeTween 80, multiplication signmethyl
disruptive coatings on pine blocks, which appeared on sur-
face profiles as broken lines of 0.4 to 1.4 m amplitude
(Fig. 6c, d). It should be noted that drying oil coating
produced more regular peaks on the surface profile com-
pared to n-hexadecane coating which was characterized by
irregular peaks and valleys on a profilometry curve.
As seen in the thermal images obtained (Fig. 7), R. ruber
cells immobilized on biosurfactant-hydrophobized wood
blocks generated heat as evident from the temperature shift
t=2.97 C similar to that produced by bacteria immobilized
on drying oil-hydrophobized blocks (t=3.14 C). However,
on blocks hydrophobized with n-hexadecane, R. ruber cells
produced a lower temperature shift (t=2.29 C). The lowest
heat release (t=1.38 C) was registered for rhodococcal cells
immobilized on blocks hydrophobized with drying oil in
excess that correlated with the low number of viable R. ruber
cells (4.0106 CFU/cm2) on this carrier as determined by the
INT staining (data not shown). In comparison, numbers of
viable bacteria on wood blocks hydrophobized with a
biosurfactant, drying oil, and n-hexadecane ranged from
1.1107 to 1.9107 CFU/cm2 (data not shown).
n-Hexadecane oxidation results Data presented in Fig. 8
suggest that the hydrocarbon-oxidizing activity of R. ruber
immobilized on hydrophobized sawdust was 1.62.5 times
higher than that of freely suspended bacteria. This finding
was in agreement with our previous results (Podorozhko et
al. 2008). Treatment of a sawdust carrier with Rhodococcus
biosurfactant was shown to enhance n-hexadecane oxidation
by immobilized R. ruber up to 35 mg/(Lh). This was
compared (3940 mg/(Lh) to n-hexadecane oxidation rates
of R. ruber immobilized on sawdust modified using drying
oil. For comparison, the hydrocarbon-oxidizing activity of
R. ruber immobilized on sawdust hydrophobized with n-
hexadecane was 1.5 times lower than that of rhodococci
immobilized on biosurfactant-hydrophobized sawdust.
5321
b
1.4
1.2
1.0
A600
0.8
0.6
0.4
0.2
0.0
6 0 1 2 3 4 5 6
Time (days)
cellulose, Greek capital deltacarboxymethyl cellulose. Experimental
variants: a cell suspension, b control. Meansstandard deviations of
triplicates are shown
Discussion
One of the physiological roles of biosurfactants, apart from
hydrocarbon solubilization, is to enhance bacterial adhesion
and subsequent surface colonization (Neu 1996; Rodrigues
et al. 2006). Biosurfactant molecules are adsorbed onto solid
substrates and coat them with a conditioning film having
high affinity to microbial cells. Moreover, biosurfactant
molecules can saturate a bacterial cell wall, increase cell
surface hydrophobicity, and thereby enhance adhesion
(kvarla et al. 1998). Biosurfactant-producing bacteria in-
volve these mechanisms to regulate adhesion, biofilm for-
mation, and substrate colonization processes (Kinsinger et
al. 2003; Mehdi and Giti 2008). Effects of Rhodococcus
biosurfactants on the adhesion of producing strains were
not studied previously. In the present work, a strong (R=
0.92, p=0.03) correlation between adhesive activities and
biosurfactant-producing abilities and an increase in cell
adhesion to polystyrene coated with Rhodococcus
biosurfactant were demonstrated for R. ruber strains.
These findings suggest that the biosurfactant is implicated
in the rhodococcal adhesion, and therefore, it could be used
to enhance R. ruber immobilization on solid carriers.
Furthermore, the adhesion-promoting effect of the
biosurfactant was found to be concentration dependent.
The highest number of R. ruber cells adhered on polysty-
rene was detected at 1.0 mg/L biosurfactant, whereas sub-
sequent increase in biosurfactant concentration did not
affect bacterial adhesion (see Fig. 2). Presumably, at low
biosurfactant concentrations (<1.0 mg/L), heterogeneous
adsorption of biosurfactant molecules on polystyrene sur-
face led to the formation of biosurfactant-based sites with
high affinity to R. ruber cells. Similar ununiform coatings of
SiO2 on ferric (III) oxide particles were described at low
(0.04 M) SiO2 concentrations, whereas at high (0.68 M)
SiO2 concentrations, Fe2O3 particles were smooth and fully
5322 Appl Microbiol Biotechnol (2013) 97:53155327

Fig. 5 Topographic maps of

solid surfaces with immobilized
R. ruber IEGM 231 cells (on
the left) and free of cells (on the
right). Surfaces: a pine blocks
hydrophobized with
Rhodococcus biosurfactant, b
pine blocks hydrophobized with
drying oil, c pine blocks
hydrophobized with n-
hexadecane, d glass slides.
Color scales show the height
gradient from minimal to
maximal. Black color indicates
unreadable areas. Arrows point
to immobilized cells

coated (Han et al. 2000). At threshold biosurfactant concen-
tration (1.0 mg/L), polystyrene was fully coated with a
homogenous biosurfactant film, and the whole surface be-
came available for bacterial adhesion. Similarly, homoge-
nous distribution of the biosurfactant on solid surfaces (pine
blocks) was confirmed by high-resolution profilometry
(Figs. 5 and 6). Apparently, further increase in the
biosurfactant concentration did not increase the surface area
available for bacterial adhesion.
Studying physicochemical properties of sawdust carriers,
a significant increase in hydrophobicity of sawdust after
Rhodococcus biosurfactant treatment was revealed.
Biosurfactant-treated sawdust was moderately hydrophobic
with high affinity to hydrocarbons hence suggesting its
optimal hydrophiliclipophilic properties for bacterial im-
mobilization and hydrocarbon oxidation. Acting as solubi-
lizing agents for hydrocarbons, biosurfactants form micelles
with hydrocarbon molecules located in a core volume of the
micelles (Neu 1996; Page et al. 1999). Biosurfactant satura-
tion with hydrocarbons depends on a structure of nonpolar
groups of the biosurfactant, mainly hydrophobic tail length
(Gambi et al. 2005). R. ruber trehalose lipids contain long
chain (2941 carbon atoms) branched fatty acids (Philp et
al. 2002), which provide a large micelle core volume for
hydrocarbons (Page et al. 1999). For comparison, n-
hexadecane chain length is 16 carbon atoms, and drying
Appl Microbiol Biotechnol (2013) 97:53155327 5323

Fig. 6 Surface profiles of

uninoculated solid carriers.
Carriers: a pine blocks without
hydrophobization, b pine
blocks hydrophobized with
Rhodococcus biosurfactant, c
pine blocks hydrophobized with
drying oil, d pine blocks
hydrophobized with n-
hexadecane, e glass slides
5324
Fig. 7 Thermal images of hydrophobized pine blocks without cells (a)
and with immobilized R. ruber IEGM 231 cells (b). The hydrophobizer
used and mean t are shown below the corresponding image. Color
oil unsaturated fatty acids consist of 1618 carbon atoms.
Similarly, a glycolipid biosurfactant from Rhodococcus
strain H13-A formed micelles with four time larger diameter
than Tween 80 micelles, which resulted in higher saturation
of polyaromatic hydrocarbons (Page et al. 1999). In our
study, the hydrocarbon adsorbed by biosurfactant-treated
sawdust remains available for oxidation by immobilized R.
ruber that resulted in its efficient biodegradation, similarly
to previously described for crude oil, diesel, oil slops, and
phenanthrene degradation (Quek et al. 2006; Xia et al.
2010). Previously Rhodococcus sp. F92 immobilized on
polyurethane foam with high oil-adsorbing capability was
shown to reduce hydrocarbon concentrations in the medium
and their toxic effects on suspended microorganisms (Quek
et al. 2006).
In experiments with different emulsifiers for drying oil,
Rhodococcus biosurfactant was shown to produce a stable
hydrophobic coating on a sawdust surface compared to
poly(vinyl alcohol, methyl cellulose, carboxymethyl cellu-
lose, and Tween 80). Moreover, a stable homogenous coat-
ing in the presence of the biosurfactant resulted in three- to
n-Hexadecane oxidation, mg/(Lh)
45
40
35
30
25
20
15
10
5 with the biosurfactant (see Fig. 6). Topographic maps
0 recorded for blocks with immobilized bacteria confirmed
a b c d
Experimental variant
Fig. 8 Rates of n-hexadecane oxidation by R. ruber cells. Experimen-
tal variants: a freely suspended cells, be cells immobilized on sawdust
hydrophobized with n-hexadecane (b), Rhodococcus biosurfactant (c),
drying oil in the presence of Rhodococcus biosurfactant used as an
emulsifier (d), and drying oil (e). Meansstandard deviations of trip-
licates are shown
Appl Microbiol Biotechnol (2013) 97:53155327
scales show the temperature gradient from minimal to maximal. Means
standard deviations of triplicates are shown. Asterisk: statistically
significant (p<0.05) differences
sixfold increase in bacterial immobilization efficacy that
was supposed to be due to sawdust surface hydrophobization
(Fig. 4). Based on the low toxicity and high emulsifying
activity of Rhodococcus biosurfactant (Ivshina et al. 1998),
it can be recommended as an emulsifying agent for carrier
modifications rather than chemical solvents, polymers, or
surfactants.
It is known that attachment of microbial cells to solid
carriers involves two phases. The first reversible phase is the
initial contact of cells with a carrier, and the second irreversible
phase is slow multipoint binding of cells to the carrier surface.
The first phase depends on the number of cell binding sites,
while the second phase is determined by affinities of these sites
to bacteria (Bos et al. 1999; Adamczyk et al. 2005; Hori and
Matsumoto 2010). The differences revealed in R. ruber immo-
bilization dynamics between sawdust samples (see Fig. 3)
could be explained by different binding site numbers and
affinities of hydrophobic coatings to rhodococcal cells.
For visualization of hydrophobized carrier surfaces with
immobilized R. ruber cells, relatively new in biological
practice methods of high-resolution profilometry and IR
thermography were used. These methods are non-
contacting and non-destructive, highly sensitive, and soft-
ware equipped; also, no special sample preparation is re-
quired. High-resolution profilometry with a white light
interferometer was previously used for topographical anal-
ysis of corrosive bacterial biofilms on polished steel, calcite,
and hydroxyapatite surfaces (Waters et al. 2009). In this
work, the initially high roughness of polished wood blocks
was decreased to the level of glass slides by the treatment
e
that rhodococci were uniformly distributed and formed cell
monolayers on the biosurfactant-hydrophobized carrier
(Fig. 5). Such monolayer distribution of bacteria on a carrier
surface was suggested to be most favorable for hydrocarbon
assimilation (Hori et al. 2008). Opposite cell arrangement
was observed on wood blocks hydrophobized with
n-hexadecane where R. ruber formed dense cell clusters
Appl Microbiol Biotechnol (2013) 97:53155327
(Fig. 5). A possible reason for cell clustering lays in the
formation of heterogeneous n-hexadecane coating on the
wood surface recorded by profilometry which resulted in
bacterial attachment mostly to coated sites served as adsorp-
tion centers. In our study, we used n-hexadecane vapors
condensed on the sawdust surface in separate drops, serving
as sites for bacterial adhesion (Rose 2002). Apparently,
bacterial co-aggregation led to a decrease in n-hexadecane
oxidation rates due to hydrocarbon bioavailability limita-
tions. In our experiments, R. ruber immobilized on n-
hexadecane-hydrophobized sawdust was found to oxidize
the hydrocarbon with 30 % less efficiency compared to the
cells immobilized on biosurfactant- or drying oil-
hydrophobized sawdust (Fig. 8).
By IR thermography, heat production by immobilized R.
ruber cells was determined. Higher t values were recorded
for bacteria immobilized on wood hydrophobized with the
biosurfactant or drying oil compared to n-hexadecane-treat-
ed wood surfaces (Fig. 7). We presumed that such heat
production differences resulted from different numbers of
bacterial cells immobilized. However, INT staining data
showed similar viable cell numbers on wood blocks treated
with all hydrophobizers tested, suggesting that the heat
produced also depends on metabolic activities of individual
bacterial cells. IR thermography results correlated with
n-hexadecane oxidation data and confirmed higher met-
abolic activities of R. ruber immobilized on sawdust
hydrophobized with the biosurfactant or drying oil com-
pared to n-hexadecane-treated carriers. Further detailed
studies are required to reveal relationships between the
heat production by immobilized bacterial cells and their
metabolic activities.
Interestingly, immobilized R. ruber cells had higher
hydrocarbon-oxidizing activities than suspended ones.
We supposed that it is due to the contact area between
n-hexadecane and R. ruber cells at the interface of
hydrocarbon/water. Since hydrophobized sawdust with
immobilized rhodococci located at the interface, the hy-
drocarbon substrate was more available for immobilized
bacteria than for planktonic cells located in the bulk
water phase. Van Loosdrecht et al. (1990) mentioned
strong indirect effects of the microenvironment around
bacteria adhered to solid surfaces which could enhance a
catalytic activity of immobilized cells.
In summary, Rhodococcus biosurfactant was applied as a
hydrophobizing agent for sawdust resulting in the formation of
a stable homogeneous coating with increased affinity to hy-
drocarbons and hydrocarbon-oxidizing bacteria. Despite some
similar characteristics with conventional hydrophobizers (dry-
ing oil and n-hexadecane), the biosurfactant has certain advan-
tages such as low toxicity and incombustibility, the possibility
to be used at lower concentration, and without additional
solvents or surfactants. R. ruber cells were distributed
5325
uniformly on the surface of biosurfactant-treated sawdust and
demonstrated high hydrocarbon-oxidizing activity.
Acknowledgments The research was partially supported by the grants
of the Russian Academy of Sciences Presidium Program Leaving Na-
ture: Current State and Development Problems (no. 01201256869) and
Molecular and Cell Biology (no. 01201256863) and the Russian Foun-
dation for Basic Research grant (11-04-96045-r_ural_).
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