Process Biochemistry 49 (2014) 1606–1611

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Short communication

Growth of the fungus Paecilomyces lilacinus with n-hexadecane

in submerged and solid-state cultures and recovery of
hydrophobin proteins
Gabriel Vigueras a,∗ , Keiko Shirai b , Maribel Hernández-Guerrero a ,
Marcia Morales a , Sergio Revah a,∗
a
Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Avenida Vasco de Quiroga 4871, 05348 México D.F., México
b
Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, 09340 México D.F.,México

a r t i c l e i n f o a b s t r a c t

Article history: The filamentous fungus Paecilomyces lilacinus was grown on n-hexadecane in submerged (SmC) and solid-
Received 20 November 2013 state (SSC) cultures. The maximum CO2 production rate in SmC (Vmax = 11.7 mg CO2 Lg −1 day−1 ) was three
Received in revised form 5 June 2014 times lower than in SSC (Vmax = 40.4 mg CO2 Lg −1 day−1 ). The P. lilacinus hydrophobin (PLHYD) yield from
Accepted 7 June 2014
the SSC was 1.3 mg PLHYD g protein−1 , but in SmC, this protein was not detected. The PLHYD showed a
Available online 23 June 2014
critical micelle concentration of 0.45 mg mL−1 . In addition, the PLHYD modified the hydrophobicity of
Teflon from 130.1 ± 2◦ to 47 ± 2◦ , forming porous structures with some filaments <1 ␮m and globular
Keywords:
aggregates <0.25 ␮m diameter. The interfacial studies of this PLHYD could be the basis for the use of the
Paecilomyces lilacinus
n-Hexadecane
protein to modify surfaces and to stabilize compounds in emulsions.
Submerged and solid-state cultures © 2014 Elsevier Ltd. All rights reserved.
Hydrophobin
Surface activity

1. Introduction
Filamentous fungi produce amphipathic proteins, called
hydrophobins, having both low molecular weight (∼10 kDa) and
surface activity. The interfacial activity of these proteins is of
great interest for biotechnological and medical applications such
as the immobilization of biomolecules in solid surfaces, as sur-
factants in biphasic solid–liquid systems, in biosensors, etc. [1–3].
Hydrophobins are moderately to strongly hydrophobic and have
eight conserved cysteine (Cys) residues, providing high stability.
Class I hydrophobins form highly stable monolayers, tolerating 2%
sodium dodecyl sulfate (SDS) at 100 ◦ C, and are dissolved only with
formic (FA) or trifluoroacetic (TFA) acids. On the other hand, pro-
tein aggregates formed by class II hydrophobins are less stable and
can be dissolved with 60% ethanol or 2% SDS [2]. These differences
in stability have been explained by their hydropathy patterns and
structure The loops formed in the class I hydrophobins between
Cys residues in the beta barrel structure are much larger, which
form rodlet structures similar to those of the amyloid fibrils [4].
∗ Corresponding authors. Tel.: +52 55.5814 - 6536
E-mail addresses: jvigueras@correo.cua.uam.mx (G. Vigueras),
srevah@xanum.uam.mx, srevah@correo.cua.uam.mx (S. Revah).
X-ray crystal structures show that both hydrophobins classes exist
as monomers, dimers and tetramers in solution [5,6]. The ability of
hydrophobins to self-assemble at the air–water interface reduces
the water surface tension, allowing the emergence of the hyphae
from the liquid media to air [2,7]. Furthermore, these proteins form
a hydrophobic coating on the hyphae, protecting them against both
excessive cytoplasmic water evaporation and wetting.
An important biological role of hydrophobins is to cover coni-
dia allowing their adhesion to hydrophobic surfaces such as the
cuticle from insect or nematode hosts [1,8]. Consequently, these
proteins enable entomopathogeneous and nematophagous fungi
to grow on the hydrophobic surfaces of the host facilitating the
enzymatic degradation of the cuticular hydrocarbons followed by
the production of other hydrolytic enzymes such as chitinases
and proteases in an antagonistic mechanism. The waxy epicuti-
cle of the insect is composed of a complex mixture of aliphatic
hydrocarbons including n-hexadecane [9,10]. Studies show that
fungi are capable of using aliphatic and aromatic compounds as
the sole carbon source [11,12]. Several efforts have been con-
ducted to increase the fungal virulence against pests, including
alkane growth adaptation. In this regard, Crespo et al. [10] reported
an increased virulence of Beauveria bassiana against the insect
host when adding n-hexadecane to the culture medium. Pae-
cilomyces lilacinus is an ascomycete filamentous fungus used for the

http://dx.doi.org/10.1016/j.procbio.2014.06.015
1359-5113/© 2014 Elsevier Ltd. All rights reserved.
 G. Vigueras et al. / Process Biochemistry 49 (2014) 1606–1611
biocontrol of phytopathogenic nematodes. This fungus is able to
metabolize recalcitrant aromatic compounds such as biphenyl and
dibenzofuran by oxidative biotransformation [13,14] and produces
hydrophobins during assimilation of toluene in biofilters [15].
The goal of this study was to assess the effect of a hydrophobic
substrate (n-hexadecane) on P. lilacinus growth and hydrophobin
production in submerged (SmC) and solid-state cultures (SSC). In
addition, the surface activities on air-solid and air-liquid interfaces
of produced hydrophobins were evaluated.
2. Materials and methods
2.1. Fungal strain
P. lilacinus CBS 284.3 is a nematophagous fungus used in bio-
logical control. The strain was propagated on potato dextrose agar
at 28 ◦ C and maintained at 4 ◦ C until needed. Conidia suspensions
were obtained by adding few milliliters of a 0.1% (v/v) Tween 80
solution and scrapping off the agar surface with glass beads.
2.2. Culture medium
The culture medium composition (g L−1 ) was NaNO3 6, KH2 PO4
1.3, MgSO4 ·7H2 O 0.38, CaSO4 ·2H2 O 0.25, CaCl2 0.055, and 4 mL L−1
of solution of trace elements containing FeSO4 ·7H2 O 0.015,
MnSO4 ·7H2 O 0.012, ZnSO4 ·7H2 O 0.013, CuSO4 ·7H2 O 0.0023, and
CoCl2 ·6H2 O 0.0015. The medium contained 17 g L−1 n-hexadecane
(Sigma–Aldrich, Mexico) as the water-insoluble carbon source. The
pH was 5.3.
2.3. Microcosm experiment
The fungus was cultivated in 125-mL flasks sealed with inert
Teflon valves (VICI Precision Sampling). The SmC contained 10 mL
of culture medium. For SSC, 10 mL of culture medium were mixed
with 4 g of perlite (dry weight) as inert support [16]. Control exper-
iments were prepared as above but without n-hexadecane. All
cultures were inoculated with 2 × 107 conidia per mL and incubated
at 28 ◦ C on a rotary shaker at 180 rpm.
2.4. CO2 and biomass production rates
The CO2 concentrations were monitored by gas chromatogra-
phy from the gaseous headspace (0.115 Lg for SmC and 0.102 Lg for
SSC) and the maximum CO2 production rate in the microcosms was
calculated with the integrated Gompertz model (Vmax = 0.368˛k,
where ˛ = maximum CO2 concentration/mg CO2 Lg −1 ; k = CO2 pro-
duction rate constant/days−1 ) as reported by Acuña et al. [17]. The
parameters of the model were calculated using the Origin software
(Origin-Lab Corporation version 7.0).
Biomass (as mg protein L−1 ) was determined from the total pro-
tein as reported by García-Peña et al. [18]. The soluble protein was
determined according to Bradford [19]. All determinations were
performed by triplicate. Both the CO2 and biomass concentrations
used in the accumulation profiles were calculated by substracting
the values obtained from the control experiments.
2.5. Gas chromatography analysis
The CO2 concentration was determined by injecting 200 ␮L of
headspace with a precision syringe (VICI Precision Sampling) into
a gas chromatograph (GOW MAC series 580) equipped with a ther-
mal conductivity detector and a Poropack column. The operating
conditions were injector 50 ◦ C, oven 40 ◦ C, detector 115 ◦ C, and flow
rate of 4.4 mL min−1 .
1607
2.6. Extraction of PLHYD proteins
The PLHYD proteins from the mycelium, produced in both SmC
and SSC, were extracted according to a modified version of the pro-
cedure described by Lunkenbein et al. [20]. Initially, the residual
n-hexadecane from SmC and SSC was eliminated with one hex-
ane extraction (1:10). An extraction with 1% (w/v) SDS in 100 mM
Tris–HCl buffer, pH 8.0 was performed for 10 min at 90 ◦ C, fol-
lowed by centrifugation (8000 × g for 10 min at 4 ◦ C). The pellet
was washed six times with water and suspended in concentrated
FA at 4 ◦ C, followed by centrifugation as above. The supernatant
was neutralized as described by Vigueras et al. [15], followed by
centrifugation as above, and the pellet was suspended in 100 mM
Tris–HCl buffer, pH 8.0. The PLHYD were concentrated and desali-
nated by ultrafiltration with a Vivaspin PES membrane with a 3 kDa
cut-off (Sartorius). The total protein content was determined by
direct spectrophotometry at 260/280 nm using a Nano Drop ND-
1000.
2.7. Analysis of proteins by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE)
Protein profiles were analyzed based on molecular mass by SDS-
PAGE using the technique of Laemmli on 4% stacking gel and 15%
resolving gel at 150 V, using broad range standard proteins (Bio-
Rad). Gels were stained with Coomassie Blue G-250 (Bio-Rad).
2.8. Purification by reversed phase high performance liquid
chromatography (RP-HPLC)
The PLHYD protein was purified by RP-HPLC using a 5 ␮m Supel-
cosil LC 304 column (25 cm × 4.6 mm ID) protected by a 5 ␮m
Supelguard LC 304 guard column (2 cm × 4.6 mm ID; Supelco). The
volume injection was 100 ␮L. The mobile phase A consisted of 0.1%
(w/v) trifluoroacetic acid (TFA) and mobile phase B of 0.1% (w/v) TFA
in acetonitrile with a pH of 3.0. The proteins were eluted following
the gradient: 20–40% in 15 min, 40–80% 20 min, 80–90% 2 min and
90% 3 min. The gradient was returned to 20% in 5 min. The flow rate
was 1.0 mL min−1 and the detection was performed at 210–400 nm.
The fractions of three injections were collected, the mobile phase
evaporated, and the samples were kept at −20 ◦ C until analyzed.
2.9. PLHYD analysis by size exclusion high performance liquid
chromatography (SEC-HPLC)
SEC-HPLC was performed using a gel filtration column of 5 ␮m
Bio-Silect 250 SEC (30 cm × 7.8 mm ID; Bio-Rad). The injection vol-
ume was 20 ␮L. The mobile phase contained NaH2 PO4 0.05 M,
Na2 HPO4 0.05 M and NaCl 0.15 M, pH 6.8. The flow rate was
1.0 mL min−1 and detection was performed at 280 nm. Molecular
weight standard (Bio-Rad) was used to calibrate the system.
2.10. Surface activity of PLHYD determined by goniometry
The surface tension of PLHYD aqueous solutions
(0–1.47 mg protein mL−1 ) was determined with a Theta KSV
optical tensiometer system (KSV Instruments), the results were
analyzed through Young-Laplace model. The control samples
consisted of Milli-Q grade pure water and two solutions; namely
bovine serum albumin protein (BSA) (2 mg mL−1 ) and a rham-
nolipid (∼1 mg mL−1 ) extracted from Pseudomonas aeruginosa
[21]. Additionally, the surface modification of PLHYD on the
hydrophobic surface of Teflon was evaluated. The Teflon surface
was rinsed three times with ethanol then three times with water
and finally dried for 12 h. Then 100 ␮L of the hydrophobin solution
(0.45 mg protein mL−1 ) were drop-cast onto the surface and dried
1608 G. Vigueras et al. / Process Biochemistry 49 (2014) 1606–1611
Fig. 1. CO2 concentration (closed symbols), and biomass as total protein (open sym-
bols) evolution in SmC (circles) and SCC (squares) and biomass as total protein (open
symbols) produced with P. lilacinus. Lines correspond to the Gompertz model applied
for CO2 .
for 24 h. Two washings were done for 1 min with water followed
by 16 h of drying. Control samples were identically prepared
without PLHYD solution. Surface hydrophobicity was determined
by measuring the contact angle of a water drop (2 ␮L) with a Theta
KSV goniometer. All analyses were replicated six times, in three
different points of each sample.
2.11. Scanning electron microscopy (SEM)
SEM images of the hydrophobin coated surfaces were acquired
through secondary electron imaging mode in a JSM 5900 LV (Jeol)
Microscope. The samples were mounted onto SEM stubs using con-
ductive carbon adhesive tabs. Prior to the microscopy analysis, the
samples were dried and sputtered coated with gold with a Denton
Vacuum, LLC sputter coater.
3. Results and discussion
3.1. Growth and CO2 production in SmC and SSC with
n-hexadecane
Dense fungal growth was observed in SmC and SSC supple-
mented with n-hexadecane. In SmC, the fungus grew forming
pellets, which adhered to the bottle wall, while in SSC the fungal
growth was visible over the solid support. Fig. 1 shows the kinetics
of CO2 production, where the maximum production rate of CO2 in
SmC (Vmax 11.7 mg CO2 Lg −1 day−1 ) was three times lower than that
for SSC (Vmax 40.4 mg CO2 Lg −1 day−1 ). The maximum CO2 concen-
tration of 90.3 and 344.6 mg CO2 Lg −1 were reached on day 20 for
both SmC and SSC. The final biomass contents, produced per micro-
cosm, were 5.0 ± 1.0 and 15.3 ± 1.0 mg protein L−1 for SmC and SSC,
respectively. Despite the differences in both cultivation methods,
the specific CO2 yields were similar (208 mg CO2 mg protein−1 for
SmC and 229 mg CO2 mg protein−1 for SSC). The results showed
that the growth of P. lilacinus with n-hexadecane in SSC was up
to three times faster than in SmC after 21 days with an initial
n-hexadecane content of 17 g L−1 . Furthermore, they confirm pre-
vious findings supporting the positive effect the reduced water
content of solid state cultivation for the utilization of hydrophobic
substrates [22,23]. P. lilacinus has shown the ability to consume or
transform complex and hydrophobic molecules including biphenyl,
dibenzofuran and benzo [␣] pyrene in liquid medium using P. lilac-
inus [13,14,24]. Recently, a strain of P. lilacinus isolated from soil
Fig. 2. Protein analyses by SDS-PAGE. Lane M: molecular weight standard; lanes A
and B: proteins extracted from SmC and SSC respectively.
was associated to the removal of phenanthrene, fluoranthene and
pyrene [25]. In a previous study, we reported the assimilation of
toluene in a gas phase biofilter with the same strain [15]. These
reports and the results from this study show the wide metabolic
diversity of P. lilacinus, which could have a potential application in
bioremediation.
3.2. Recovery of PLHYD proteins
The protein analysis by SDS-PAGE, Fig. 2 showed one band
corresponding to a denatured protein ca. 7 kDa only in SSC, in
contrast to the SmC where no low molecular weight proteins
were detected. The chromatogram obtained by RP-HPLC of pro-
teins extracted from the mycelium produced in SSC shows two
intense and well-defined peaks detected at 7.4 and 26.0 min. The
first peak eluted with a low concentration of acetonitrile (29.8%),
while the second peak had a greater intensity and required 62.7%
of the organic phase to elute (see Fig. 1SI). Fig. 3a shows SEC-
HPLC analysis of the fraction corresponding to the peak eluted at
26.0 min in the RP-HPLC had a retention time of 10.8 min, which
corresponds to a molecular weight of ca. 12 kDa under native
conditions; while the same fraction analyzed under denaturing
conditions in a SDS-PAGE, Fig. 3b shows a band of ca. 7 kDa. The
fraction collected at 7.4 min, showed a weak signal after 14 min in
SEC-HPLC, corresponding to peptides lower than 1.35 kDa. These
results correspond to the reported molecular weight of PLHYD,
previously identified by MALDI-TOF, produced by the fungus
growing on gaseous toluene in a biofilter [15]. The RP-HPLC anal-
ysis shows that PLHYD has hydrophobic characteristics requiring
over 60% acetonitrile to elute from the column. Tagu et al. [26]
reported that the HYDPt-1 hydrophobin eluted with 43% acetoni-
trile in similar chromatographic conditions. The final hydrophobin
yield from SSC, 1.3 mg PLHYD g total protein−1 , was also similar to
the previous work with toluene, 1.7 mg PLHYD g total protein−1 in
Vigueras et al. [15]. The fact that the PLHYD was not found in SmC
 G. Vigueras et al. / Process Biochemistry 49 (2014) 1606–1611 1609

Fig. 3. (a) Separation by SEC-HPLC of the fraction of PLHYD purified by RP-HPLC. The molecular weights of standards are indicated at the top. (b) Protein profile analysis by
SDS-PAGE of the same fraction. Molecular weight standard in Lane M and fraction of PLHYD in Lane 1.

confirms previous reports suggesting the protein is an important
factor when the fungus grows forming aerial mycelium. Peñas et al.
[27] observed that both hydrophobins expression and metabolism
in SSC differ respect to SmC. Additionally, Vergara-Fernández et al.
[23] reported the increase in surface hydrophobicity of the Fusarium
solani mycelium when grown on solid media with hydrophobic
substrates suggesting that this effect is directly related to the pres-
ence of hydrophobins. Boualem et al. [28] reported that Penicillium
camemberti grown in solid culture increased the expression of
the rodA gene, which correlated with the excretion of the pro-
tein and increased drastically the mycelial hydrophobicity; on the
other hand, no RodA production occurred in liquid cultures and the
mycelium remained hydrophilic, and no conidiation was detected.
Likewise, Vigueras et al. [29] showed that surface hydrophobicity of
Rhinocladiella similis and expression of hydrophobin-like proteins
was modified in solid culture when using compounds with differ-
ent polarities such as ethanol or n-hexane. Rocha-Pino et al. [30]
showed that the production of chitinases and hydrophobins from
Lecanicillium lecanii was influenced by the cultivation method and
type of carbon source. Zajc et al. [31], reported recently a signifi-
cant enrichment of hydrophobins from Wallemia ichthyophaga in
response to the drought stress produced by hypersaline environ-
ments, as indicated by genomic and transcriptomic analysis.
Supplementary material related to this article can be found, in
the online version, at doi:10.1016/j.procbio.2014.06.015.
3.3. Surface activity of the PLHYD
A solution with 1.47 mg PLHYD mL−1 decreased the surface
tension of water to 34.8 mN m−1 , whereas the BSA (2 mg mL−1 )
decreased to 49.8 mN and Rhamnolipid (1 mg mL−1 ) reached
28.3 mN. When increasing the PLHYD concentration, the surface
tension of water gradually decreased as observed in Fig. 4a. A
transition zone marked by a change in slope is distinguished
between PLHYD concentrations of 0.2 up to 0.45 mg mL−1 and
therefore, the critical micelle concentration of PLHYD was deter-
mined as 0.45 mg mL−1 with a decrease in surface tension of
36.7 mN m−1 (see Fig. 4a–c).
The concentration needed to lower the surface tension of
water to around 35 mN m−1 (1.2 mg mL−1 ) is similar to the val-
ues reported for conventional surfactants such as SDS (1 mg mL−1 )
and ESO-derived surfactants (0.7 mg mL−1 ) measured with the

Fig. 4. Surface activity of the PLHYD determined by the pendant drop method: (a)  (mg mL−1 ) respect to protein concentration (mg mL−1 ), (b) pure water (control)
(72 mN m−1 ) (c) critical micelle concentration (0.45 mg PLHYD mL−1 ), and wettability determined by contact angle, (d) pristine Teflon surface (control, 130.1 ± 2◦ ) and (e)
PLHYD coated Teflon (47 ± 2◦ ).
1610 G. Vigueras et al. / Process Biochemistry 49 (2014) 1606–1611
Fig. 5. Scanning electron micrographs (500×) of a PLHYD film deposited on Teflon;
the white area represents the Teflon surface. The inset 2500× shows a porous pattern
and a zone with thin filaments and globular aggregates.
axisymmetric drop shape analysis method [32,33]. This concentra-
tion was higher to that reported for the most studied hydrophobins,
SC3 and HFBII were ca. 32 mN m−1 , 5.7 × 10−3 mg SC3 mL−1
or 2.7 × 10−3 mg HFBII mL−1 , as well as other biosurfactants,
0.04 mg rhamnolipid mL−1 [34,35]. Kisko et al. [36] reported the
formation of tetramers of HFBII with a concentration of 10 mg mL−1 .
The surface activity, analyzed as the ability of molecules to
modify the degree of hydrophobicity (or wettability) of a hydropho-
bic surface (see Fig. 4d and e), showed that PLHYD modified the
hydrophobicity by decreasing the contact angle of Teflon from
130.1 (±2◦ ) to 47 (±2◦ ). These results agree with the work reported
by Wösten et al. [2] where a coat of SC3 hydrophobin on Teflon
decreased the contact angle from 108 ± 2◦ to 62 ± 8◦ , and Misra
et al. [3] reported for the adsorbed protein coatings on polystyrene
reduced the contact angle from 80◦ to 30◦ .
The SEM images show the Teflon surface (Fig. 5, white region)
and the protein-coated surface (Fig. 5, dark region). As observed
from the SEM images, the PLHYD film presented an ordered struc-
ture with pores (<1 ␮m) and zones with filaments (ca. 0.1–1 ␮m)
and globular aggregates (<0.25 ␮m) at the edge of the coating (Fig. 5
inset). The observed structures are similar to the porous film and
thin filaments (rodlets ca. 0.05 and 0.3 ␮m) obtained by Kirkland
and Keyhani [37] with a class I hydrophobin. The presence of rodlet-
like structures on the continuous hydrophobin film is common as
reported in another work by Janseen et al. [38]. It is also common
that proteins form aggregates and these structures, with size ran-
ging from 1 up to 15 ␮m have been described for soy protein [39].
The globular aggregates shown in the SEM of the PLHYD coating
are in this size range. However, in this case, only a few globular
aggregates and filaments were observed.
The interfacial activity that PLHYD presented is an important
parameter showing its potential for biotechnological applications
such as the stabilization of hydrophobic compounds in liquids or
solid surface modification for immobilization of biosensors, with
advantages in biodegradability and biocompatibility [1,2,7,40].
Finally, according to the best of our knowledge, this is the first study
on the growth of P. lilacinus with n-hexadecane as carbon source,
and the recovery of valuable products such as hydrophobins. The
interfacial properties of the PLHYD hydrophobins extracted in this
work are of interest in the area of materials for the control of the
wettability of surfaces and for the stabilization of emulsions. The
work presents interesting possibilities as new methods of growing
fungi on hydrophobic substrates, either solid, liquid or gaseous, are
continually been proposed which can foster both higher cell and
hydrophobin production.
Acknowledgements
The authors wish to thank CONACYT and PROMEP No. 47410256
for financing this work. In addition, we thank the technical sup-
port of Sergio Hernández, José Campos-Terán, Jorge Gracida and
the experimental assistance of Irving Jiménez García.
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