Embed Size (px)
Citation preview
Accepted Manuscript
Title: Growth of the fungus Paecilomyces lilacinus withn-hexadecane in submerged and solid-state cultures andrecovery of hydrophobin proteins
Author: Gabriel Vigueras Keiko Shirai MaribelHernandez-Guerrero Marcia Morales Sergio Revah
PII: S1359-5113(14)00343-2DOI: http://dx.doi.org/doi:10.1016/j.procbio.2014.06.015Reference: PRBI 10174
To appear in: Process Biochemistry
Received date: 20-11-2013Revised date: 5-6-2014Accepted date: 7-6-2014
Please cite this article as: Vigueras G, Shirai K, Hernandez-Guerrero M, Morales M,Revah S, Growth of the fungus Paecilomyces lilacinus with n-hexadecane in submergedand solid-state cultures and recovery of hydrophobin proteins, Process Biochemistry(2014), http://dx.doi.org/10.1016/j.procbio.2014.06.015
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
Page 1 of 26
Accep
ted
Man
uscr
ipt
1
Growth of the fungus Paecilomyces lilacinus with n-hexadecane in submerged and 1
solid-state cultures and recovery of hydrophobin proteins2
3
4
Gabriel Vigueras1*, Keiko Shirai2, Maribel Hernández-Guerrero1, Marcia Morales1, 5
Sergio Revah1,*6
7
8
9
1Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-10
Cuajimalpa, Artificios No. 40, 01120 México D.F., México.11
12
2Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Av. 13
San Rafael Atlixco No. 186, 09340 México D.F., México.14
15
16
17
*Corresponding authors: [email protected]; [email protected]
19
20
21
22
23
Page 2 of 26
Accep
ted
Man
uscr
ipt
2
Abstract 24
The filamentous fungus Paecilomyces lilacinus was grown on n-hexadecane in submerged 25
(SmC) and solid-state (SSC) cultures. The maximum CO2 production rate in SmC (Vmax = 26
11.7 mg CO2 Lg-1 day-1) was three times lower than in SSC (Vmax = 40.4 mg CO2 Lg
-1 day-1). 27
The P. lilacinus hydrophobin (PLHYD) yield from the SSC was 1.3 mg PLHYD g protein-28
1, but in SmC, this protein was not detected. The PLHYD showed a critical micelle 29
concentration of 0.45 mg mL-1. In addition, the PLHYD modified the hydrophobicity of 30
Teflon from 130.1 ±2° to 47 ±2°, forming porous structures with some filaments < 1µm and 31
globular aggregates < 0.25 µm diameter. The interfacial studies of this PLHYD could be 32
the basis for the use of the protein to modify surfaces and to stabilize compounds in 33
emulsions. 34
35
Keywords: Paecilomyces lilacinus, n-hexadecane, submerged and solid-state cultures, 36
hydrophobin, surface activity.37
38
Introduction39
Filamentous fungi produce amphipathic proteins, called hydrophobins, having both low 40
molecular weight (~10 kDa) and surface activity. The interfacial activity of these proteins is 41
of great interest for biotechnological and medical applications such as the immobilization 42
of biomolecules in solid surfaces, as surfactants in biphasic solid-liquid systems, in 43
biosensors, etc. [1-3]. Hydrophobins are moderately to strongly hydrophobic and have eight 44
conserved cysteine (Cys) residues, providing high stability. Class I hydrophobins form 45
highly stable monolayers, tolerating 2% sodium dodecyl sulfate (SDS) at 100 °C, and are 46
Page 3 of 26
Accep
ted
Man
uscr
ipt
3
dissolved only with formic (FA) or trifluoroacetic (TFA) acids. On the other hand, protein 47
aggregates formed by class II hydrophobins are less stable and can be dissolved with 60% 48
ethanol or 2% SDS [2]. These differences in stability have been explained by their 49
hydropathy patterns and structure The loops formed in the class I hydrophobins between 50
Cys residues in the beta barrel structure are much larger, which form rodlet structures 51
similar to those of the amyloid fibrils [4]. X-ray crystal structures show that both 52
hydrophobins classes exist as monomers, dimers and tetramers in solution [5, 6]. The 53
ability of hydrophobins to self-assemble at the air-water interface reduces the water surface 54
tension, allowing the emergence of the hyphae from the liquid media to air [2, 7]. 55
Furthermore, these proteins form a hydrophobic coating on the hyphae, protecting them 56
against both excessive cytoplasmic water evaporation and wetting. 57
An important biological role of hydrophobins is to cover conidia allowing their adhesion to 58
hydrophobic surfaces such as the cuticle from insect or nematode hosts [1, 8]. 59
Consequently, these proteins enable entomopathogeneous and nematophagous 60
fungi to grow on the hydrophobic surfaces of the host facilitating the enzymatic 61
degradation of the cuticular hydrocarbons followed by the production of other hydrolytic 62
enzymes such as chitinases and proteases in an antagonistic mechanism. The waxy 63
epicuticle of the insect is composed of a complex mixture of aliphatic hydrocarbons 64
including n-hexadecane [9-10]. Studies show that fungi are capable of using aliphatic and 65
aromatic compounds as the sole carbon source [11, 12]. Several efforts have been 66
conducted to increase the fungal virulence against pests, including alkane growth 67
adaptation. In this regard, Crespo et al. [10] reported an increased virulence of Beauveria 68
bassiana against the insect host when adding n-hexadecane to the culture medium. 69
Page 4 of 26
Accep
ted
Man
uscr
ipt
4
Paecilomyces lilacinus is an ascomycete filamentous fungus used for the biocontrol of 70
phytopathogenic nematodes. This fungus is able to metabolize recalcitrant aromatic 71
compounds such as biphenyl and dibenzofuran by oxidative biotransformation [13-14] and 72
produces hydrophobins during assimilation of toluene in biofilters [15].73
The goal of this study was to assess the effect of a hydrophobic substrate (n-hexadecane) on 74
P. lilacinus growth and hydrophobin production in submerged (SmC) and solid-state 75
cultures (SSC). In addition, the surface activities on air-solid and air-liquid interfaces of 76
produced hydrophobins were evaluated.77
Materials and methods78
Fungal strain79
P. lilacinus CBS 284.3 is a nematophagous fungus used in biological control. The strain 80
was propagated on potato dextrose agar at 28 °C and maintained at 4 °C until needed. 81
Conidia suspensions were obtained by adding few milliliters of a 0.1 % (v/v) Tween 80 82
solution and scrapping off the agar surface with glass beads. 83
Culture medium84
The culture medium composition (g L-1) was NaNO3 6, KH2PO4 1.3, MgSO4·7H2O 0.38, 85
CaSO4·2H2O 0.25, CaCl2 0.055, and 4 mL L-1 of solution of trace elements containing 86
FeSO4·7H2O 0.015, MnSO4·7H2O 0.012, ZnSO4·7H2O 0.013, CuSO4·7H2O 0.0023, and 87
CoCl2·6H2O 0.0015. The medium contained 17 g L-1 n-hexadecane (Sigma-Aldrich, 88
Mexico) as the water-insoluble carbon source. The pH was 5.3.89
Microcosm experiment 90
The fungus was cultivated in 125-mL flasks sealed with inert Teflon valves (VICI Precision 91
Sampling). The SmC contained 10 mL of culture medium. For SSC, 10 mL of culture 92
Page 5 of 26
Accep
ted
Man
uscr
ipt
5
medium were mixed with 4 g of perlite (dry weight) as inert support [16]. Control 93
experiments were prepared as above but without n-hexadecane. All cultures were 94
inoculated with 2×107 conidia per mL and incubated at 28 ºC on a rotary shaker at 180 rpm. 95
CO2 and biomass production rates.96
The CO2 concentrations were monitored by gas chromatography from the gaseous 97
headspace (0.115 Lg for SmC and 0.102 Lg for SSC) and the maximum CO2 production rate98
in the microcosms was calculated with the integrated Gompertz model (Vmax=0.368αk, 99
where α = maximum CO2 concentration/ mg CO2 Lg-1; k = CO2 production rate constant/ 100
days-1) as reported by Acuña et al. [17]. The parameters of the model were calculated using 101
the Origin software (Origin-Lab Corporation version 7.0).102
Biomass (as mg protein L-1) was determined from the total protein as reported by García-103
Peña et al. [18]. The soluble protein was determined according to Bradford [19]. All 104
determinations were performed by triplicate. Both the CO2 and biomass concentrations 105
used in the accumulation profiles were calculated by substracting the values obtained from 106
the control experiments. 107
Gas chromatography analysis108
The CO2 concentration was determined by injecting 200 μL of headspace with a precision 109
syringe (VICI Precision Sampling) into a gas chromatograph (GOW MAC series 580) 110
equipped with a thermal conductivity detector and a Poropack column. The operating 111
conditions were injector 50 °C, oven 40 °C, detector 115 °C, and flow rate of 4.4 mL min-1.112
Extraction of PLHYD proteins 113
The PLHYD proteins from the mycelium, produced in both SmC and SSC, were extracted 114
according to a modified version of the procedure described by Lunkenbein et al. [20]. 115
Page 6 of 26
Accep
ted
Man
uscr
ipt
6
Initially, the residual n-hexadecane from SmC and SSC was eliminated with one hexane 116
extraction (1:10). An extraction with 1% w/v SDS in 100 mM Tris–HCl buffer, pH 8.0 was 117
performed for 10 min at 90 °C, followed by centrifugation (8,000×g for 10 min at 4 °C). 118
The pellet was washed six times with water and suspended in concentrated FA at 4 °C, 119
followed by centrifugation as above. The supernatant was neutralized as described by 120
Vigueras et al. [15], followed by centrifugation as above, and the pellet was suspended in 121
100 mM Tris–HCl buffer, pH 8.0. The PLHYD were concentrated and desalinated by 122
ultrafiltration with a Vivaspin PES membrane with a 3 kDa cut-off (Sartorius). The 123
total protein content was determined by direct spectrophotometry at 260/280 nm using 124
a Nano Drop ND-1000.125
Analysis of proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)126
Protein profiles were analyzed based on molecular mass by SDS-PAGE using the technique 127
of Laemmli on 4% stacking gel and 15% resolving gel at 150 V, using broad range standard 128
proteins (Bio-Rad). Gels were stained with Coomassie Blue G-250 (Bio-Rad). 129
Purification by reversed phase high performance liquid chromatography (RP-HPLC)130
The PLHYD protein was purified by RP-HPLC using a 5 m Supelcosil LC 304 column 131
(25 cm x 4.6 mm ID) protected by a 5 m Supelguard LC 304 guard column (2 cm x 4.6 132
mm ID; Supelco). The volume injection was 100 L. The mobile phase A consisted of 133
0.1% (w/v) trifluoracetic acid (TFA) and mobile phase B of 0.1% (w/v) TFA in acetonitrile 134
with a pH of 3.0. The proteins were eluted following the gradient: 20–40% in 15 min, 40–135
80% 20 min, 80–90% 2 min and 90% 3 min. The gradient was returned to 20% in 5 min. 136
The flow rate was 1.0 mL min-1 and the detection was performed at 210-400 nm. The 137
Page 7 of 26
Accep
ted
Man
uscr
ipt
7
fractions of three injections were collected, the mobile phase evaporated, and the samples 138
were kept at -20 °C until analyzed.139
PLHYD analysis by size exclusion high performance liquid chromatography (SEC-HPLC)140
SEC-HPLC was performed using a gel filtration column of 5 m Bio-Silect 250 SEC (30 141
cm x 7.8 mm ID; Bio-Rad). The injection volume was 20 L. The mobile phase contained 142
NaH2PO4 0.05 M, Na2HPO4 0.05 M and NaCl 0.15 M, pH 6.8. The flow rate was 1.0 mL 143
min-1 and detection was performed at 280 nm. Molecular weight standard (Bio-Rad) was 144
used to calibrate the system.145
Surface activity of PLHYD determined by goniometry 146
The surface tension of PLHYD aqueous solutions (0 to 1.47 mg protein mL-1) was 147
determined with a Theta KSV optical tensiometer system (KSV Instruments), the results 148
were analyzed through Young-Laplace model. The control samples consisted of Milli-Q 149
grade pure water and two solutions; namely bovine serum albumin protein (BSA) (2 mg 150
mL-1) and a rhamnolipid (~1 mg mL-1) extracted from Pseudomonas aeruginosa [21]. 151
Additionally, the surface modification of PLHYD on the hydrophobic surface of Teflon 152
was evaluated. The Teflon surface was rinsed three times with ethanol then three times with 153
water and finally dried for 12 h. Then 100L of the hydrophobin solution (0.45 mg protein 154
mL-1) were drop-cast onto the surface and dried for 24 h. Two washings were done for 1 155
minute with water followed by 16 h of drying. Control samples were identically prepared 156
without PLHYD solution. Surface hydrophobicity was determined by measuring the 157
contact angle of a water drop (2 L) with a Theta KSV goniometer. All analyses were 158
replicated six times, in three different points of each sample.159
Page 8 of 26
Accep
ted
Man
uscr
ipt
8
Scanning electron microscopy (SEM)160
SEM images of the hydrophobin coated surfaces were acquired through secondary electron 161
imaging mode in a JSM 5900 LV (Jeol) Microscope. The samples were mounted onto SEM 162
stubs using conductive carbon adhesive tabs. Prior to the microscopy analysis, the samples 163
were dried and sputtered coated with gold with a Denton Vacuum, LLC sputter coater. 164
Results and discussion 165
Growth and CO2 production in SmC and SSC with n-hexadecane166
Dense fungal growth was observed in SmC and SSC supplemented with n-hexadecane. In 167
SmC, the fungus grew forming pellets, which adhered to the bottle wall, while in SSC the 168
fungal growth was visible over the solid support. Fig. 1 shows the kinetics of CO2 169
production, where the maximum production rate of CO2 in SmC (Vmax 11.7 mg CO2 Lg-1170
day-1) was three times lower than that for SSC (Vmax 40.4 mg CO2 Lg-1 day-1). The 171
maximum CO2 concentration of 90.3 and 344.6 mg CO2 Lg-1 were reached on day 20 for 172
both SmC and SSC. The final biomass contents, produced per microcosm, were 5.0 ± 1.0 173
and 15.3 ± 1.0 mg protein L-1 for SmC and SSC, respectively. Despite the differences in 174
both cultivation methods, the specific CO2 yields were similar (208 mg CO2 mg protein-1 175
for SmC and 229 mg CO2 mg protein-1 for SSC). The results showed that the growth of P. 176
lilacinus with n-hexadecane in SSC was up to three times faster than in SmC after 21 days 177
with an initial n-hexadecane content of 17 g L-1. Furthermore, they confirm previous 178
findings supporting the positive effect the reduced water content of solid state cultivation 179
for the utilization of hydrophobic substrates [22, 23]. P. lilacinus has shown the ability to 180
consume or transform complex and hydrophobic molecules including biphenyl, 181
dibenzofuran and benzo [α] pyrene in liquid medium using P. lilacinus [13, 14, 24]. 182
Page 9 of 26
Accep
ted
Man
uscr
ipt
9
Recently, a strain of P. lilacinus isolated from soil was associated to the removal of 183
phenanthrene, fluoranthene and pyrene [25]. In a previous study, we reported the 184
assimilation of toluene in a gas phase biofilter with the same strain [15]. These reports and 185
the results from this study show the wide metabolic diversity of P. lilacinus, which could 186
have a potential application in bioremediation. 187
188
Recovery of PLHYD proteins189
The protein analysis by SDS-PAGE, Fig. 2, showed one band corresponding to a 190
denatured protein ca. 7 kDa only in SSC, in contrast to the SmC where no low 191
molecular weight proteins were detected. The chromatogram obtained by RP-192
HPLC of proteins extracted from the mycelium produced in SSC shows two intense 193
and well-defined peaks detected at 7.4 and 26.0 min. The first peak eluted with a 194
low concentration of acetonitrile (29.8%), while the second peak had a greater 195
intensity and required 62.7% of the organic phase to elute (see Fig. 1SI). The Fig. 196
3a shows SEC-HPLC analysis of the fraction corresponding to the peak eluted at 197
26.0 min in the RP-HPLC had a retention time of 10.8 min, which corresponds to a 198
molecular weight of ca. 12 kDa under native conditions; while the same fraction analyzed 199
under denaturing conditions in a SDS-PAGE, Fig. 3b, shows a band of ca. 7 kDa. The 200
fraction collected at 7.4 min, showed a weak signal after 14 minutes in SEC-HPLC, 201
corresponding to peptides lower than 1.35 kDa. These results correspond to the 202
reported molecular weight of PLHYD, previously identified by MALDI-TOF, produced by 203
the fungus growing on gaseous toluene in a biofilter [15]. The RP-HPLC analysis shows 204
that PLHYD has hydrophobic characteristics requiring over 60% acetonitrile to elute from 205
Page 10 of 26
Accep
ted
Man
uscr
ipt
10
the column. Tagu et al. [26] reported that the HYDPt-1 hydrophobin eluted with 43% 206
acetonitrile in similar chromatographic conditions. The final hydrophobin yield from SSC, 207
1.3 mg PLHYD g total protein−1, was also similar to the previous work with toluene, 1.7 208
mg PLHYD g total protein−1 in Vigueras et al. [15]. The fact that the PLHYD was not 209
found in SmC confirms previous reports suggesting the protein is an important factor when 210
the fungus grows forming aerial mycelium. Peñas et al. [27] observed that both 211
hydrophobins expression and metabolism in SSC differ respect to SmC. Additionally, 212
Vergara-Fernández et al. [23] reported the increase in surface hydrophobicity of the F. 213
solani mycelium when grown on solid media with hydrophobic substrates suggesting that 214
this effect is directly related to the presence of hydrophobins. Boualem et al. [28] reported 215
that P. camemberti grown in solid culture increased the expression of the rodA gene, which 216
correlated with the excretion of the protein and increased drastically the mycelial 217
hydrophobicity; on the other hand, no RodA production occurred in liquid cultures and the 218
mycelium remained hydrophilic, and no conidiation was detected. Likewise, Vigueras et al.219
[29] showed that surface hydrophobicity of R. similis and expression of hydrophobin-like 220
proteins was modified in solid culture when using compounds with different polarities such 221
as ethanol or n-hexane. Rocha-Pino et al. [30] showed that the production of chitinases and 222
hydrophobins from Lecanicillium lecanii was influenced by the cultivation method and 223
type of carbon source. Zajc et al. [31], reported recently a significant enrichment of 224
hydrophobins from Wallemia ichthyophaga in response to the drought stress produced by 225
hypersaline environments, as indicated by genomic and transcriptomic analysis. 226
227
Surface activity of the PLHYD228
Page 11 of 26
Accep
ted
Man
uscr
ipt
11
A solution with 1.47 mg PLHYD mL-1 decreased the surface tension of water to 34.8 229
mN m-1, whereas the BSA (2 mg mL-1) decreased to 49.8 mN and Rhamnolipid (1 230
mg mL-1) reached 28.3 mN. When increasing the PLHYD concentration, the surface 231
tension of water gradually decreased as observed in Fig. 4a. A transition zone marked232
by a change in slope is distinguished between PLHYD concentrations of 0.2 up to 233
0.45 mg mL-1 and therefore, the critical micelle concentration of PLHYD was determined 234
as 0.45 mg mL-1 with a decrease in surface tension of 36.7 mN m-1(see Fig. 4a, b and 235
c).236
The concentration needed to lower the surface tension of water to around 35 mN m-1 (1.2 237
mg mL-1) is similar to the values reported for conventional surfactants such as SDS (1 mg 238
mL-1) and ESO-derived surfactants (0.7 mg mL-1) measured with the axisymmetric drop 239
shape analysis method [32-33]. This concentration was higher to that reported for the most 240
studied hydrophobins, SC3 and HFBII were ca. 32 mNm-1, 5.7x10-3 mg SC3 mL-1 or 241
2.7x10-3 mg HFBII mL-1, as well as other biosurfactants, 0.04 mg rhamnolipid mL-1 [34, 242
35]. Kisko et al. [36] reported the formation of tetramers of HFBII with a concentration of 243
10 mg mL-1.244
The surface activity, analyzed as the ability of molecules to modify the degree of 245
hydrophobicity (or wettability) of a hydrophobic surface (see Fig. 4d, e), showed 246
that PLHYD modified the hydrophobicity by decreasing the contact angle of Teflon from 247
130.1 (±2°) to 47 (±2°). These results agree with the work reported by Wösten et al. [2] 248
where a coat of SC3 hydrophobin on Teflon decreased the contact angle from 108 ± 2° to 249
62 ± 8°, and Misra et al. [3] reported for the adsorbed protein coatings on polystyrene 250
reduced the contact angle from 80° to 30°. 251
Page 12 of 26
Accep
ted
Man
uscr
ipt
12
The SEM images show the Teflon surface (Fig. 5, white region) and the protein-coated 252
surface (Fig. 5, dark region). As observed from the SEM images, the PLHYD film 253
presented an ordered structure with pores (< 1 µm) and zones with filaments (ca. 0.1 to 1 254
µm) and globular aggregates (< 0.25 µm) at the edge of the coating. (Fig. 5 inset). The 255
observed structures are similar to the porous film and thin filaments (rodlets ca. 0.05 and 256
0.3 µm) obtained by Kirkland and Keyhani [37] with a class I hydrophobin. The presence 257
of rodlet-like structures on the continuous hydrophobin film is common as reported in 258
another work by Janseen et al. [38]. It is also common that proteins form aggregates and 259
these structures, with size ranging from 1 up to 15 µm have been described for soy protein 260
[39]. The globular aggregates shown in the SEM of the PLHYD coating are in this size 261
range. However, in this case, only a few globular aggregates and filaments were observed.262
The interfacial activity that PLHYD presented is an important parameter showing its 263
potential for biotechnological applications such as the stabilization of hydrophobic 264
compounds in liquids or solid surface modification for immobilization of biosensors, with 265
advantages in biodegradability and biocompatibility [1, 2, 7, 40]. Finally, according to the 266
best of our knowledge, this is the first study on the growth of P. lilacinus with n-267
hexadecane as carbon source, and the recovery of valuable products such as hydrophobins. 268
The interfacial properties of the PLHYD hydrophobins extracted in this work are of interest 269
in the area of materials for the control of the wettability of surfaces and for the stabilization 270
of emulsions. The work presents interesting possibilities as new methods of growing fungi 271
on hydrophobic substrates, either solid, liquid or gaseous, are continually been proposed 272
which can foster both higher cell and hydrophobin production.273
274
Page 13 of 26
Accep
ted
Man
uscr
ipt
13
Acknowledgements275
The authors wish to thank CONACYT and PROMEP No. 47410256 for financing 276
this work. In addition, we thank the technical support of Sergio Hernández, José 277
Campos-Terán, Jorge Gracida and the experimental assistance of Irving Jiménez 278
García.279
280
References281
[1] Linder M, Szilvay G, Nakari-Setälä T, Penttilä M. Hydrophobins: the protein-282
amphiphiles of filamentous fungi. FEMS Microb Rev 2005; 29:877-896 283
[2] Wösten H. Hydrophobins: multipurpose proteins. Annu Rev Microbiol 2001; 55:625-284
646285
[3] Misra R, Li J, Cannon GC, Morgan SE. Nanoscale reduction in surface friction of 286
polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune. 287
Biomacromolecules 2006; 7:1463-1470288
[4] Linder M. Hydrophobins: proteins that self assemble at interfaces. Curr Opin Colloid 289
Interface Sci 2009 14; 356-363. 290
[5]Kisko K, Szilvay G, Vainio U, Linder M, Serimaa R. Interactions of hydrophobin 291
proteins in solution studied by small-angle X-ray scattering. Biophys J 2008; 94: 198-206.292
[6] Wang X, Graveland-Bikker J, De Kruif C, Robillard G. Oligomerization of 293
hydrophobin SC3 in solution: From soluble state to self-assembly. Protein Sci 2004; 294
13:810-821.295
[7] Wessels J. Hydrophobins: proteins that change the nature of the fungal surface. Adv 296
Microb Physiol 1997; 38:1-45 297
Page 14 of 26
Accep
ted
Man
uscr
ipt
14
[8] Wessels J. Developmental regulation of fungal cell wall formation. Annu Rev 298
Phytopathol 1994; 32:413-437299
[9] Napolitano R and Juárez M. Entomopathogenous fungi degrade epicuticular 300
hydrocarbons of Triatoma infestans. Arch Biochem Biophys 1997; 344:208-214301
[10] Crespo R, Juarez M, Dal Bello G, Padín S, Calderón G, Pedrini N. Increased mortality 302
of Acanthoscelides obtectus by alkane-grown Beauveria bassiana. Biocontrol 2002; 303
47:685-698304
[11] García-Peña E, Hernández S, Auria R, Revah S. Correlation between biological 305
activity and reactor performance in the biofiltration of toluene with the fungus 306
Paecilomyces variotii CBS 115145. Appl Environ Microbiol 2005; 71:4280-4285307
[12] Prenafeta-Boldu FX, Summerbell R, Sybren de Hoog G. Fungi growing on aromatic 308
hydrocarbons: biotechnology’s unexpected encounter with biohazard? FEMS Microbiol 309
Rev 2006; 30:109-130310
[13] Gesell M, Hammer E, Specht M, Francke W. Biotransformation of biphenyl by 311
Paecilomyces lilacinus and characterization of ring cleavage products. Appl Environ 312
Microbiol 2001; 67:1551-1557313
[14] Gesell M, Hammer E, Mikolasch A, Schauer F. Oxidation and ring cleavage of 314
dibenzofuran by the filamentous fungus Paecilomyces lilacinus. Arch Microbiol 2004; 315
182:51-59316
[15] Vigueras G, Shirai K, Martins D, Franco T, Fleuri L, Revah S. Toluene gas phase 317
biofiltration by Paecilomyces lilacinus and isolation and identification of a hydrophobin 318
protein produced thereof. Appl Microbiol Biotechnol 2008; 80:147-154319
Page 15 of 26
Accep
ted
Man
uscr
ipt
15
[16] Prenafeta-Boldú F, Kuhn A, Luykx D, Anke H, van Groenestijn J, de Bont J. Isolation 320
and characterisation of fungi growing on volatile aromatic hydrocarbons as their sole 321
carbon and energy source. Mycol Res 2001; 105:477-484.322
[17] Acuña M, Pérez F, Auria R, Revah S. Microbiological and kinetic aspects of a biofilter 323
for the removal of toluene from waste gases. Biotechnol Bioeng 1999; 63:175-184324
[18] García-Peña E, Hernandez S, Favela-Torres E, Auria R, Revah S. Toluene biofiltration 325
by the fungus Scedosporium apiospermum TB1. Biotechnol Bioeng 2001; 76:61-69326
[19] Bradford MM. A rapid and sensitive method for the quantification of microgram 327
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 328
72:248-254329
[20] Lunkenbein S, Takenberg M, Nimtz M, Berger R. Characterization of a hydrophobin 330
of the ascomycete Paecilomyces farinosus. J Basic Microbiol 2011; 51:404-414331
[21] Medina-Moreno A, Jiménez-Islas D, Gracida-Rodríguez J, Gutiérrez-Rojas M, Díaz-332
Ramírez I. Modeling rhamnolipids production by Pseudomonas aeruginosa from 333
immiscible carbon source in a batch system. Int J Environ Sci Tech 2011; 8:471-482.334
[22] Volke-Sepúlveda T, Gutiérrez-Rojas M, Favela-Torres E. Biodegradation of 335
hexadecane in liquid and solid-state fermentations by Aspergillus niger. Bioresour Technol 336
2003; 87:81-86337
[23] Vergara-Fernandez A, Van Haaren B, Revah S. Phase partitioning of gaseous surface 338
hydrophobicity of Fusarium solani when grown in liquid and solid media with hexanol and 339
hexane. Biotechnol Lett 2006; 28:2011–2017340
[24] Rafin C, Foucault B, Veignie E. Exploring micromycetes biodiversity for screening 341
benzo[a]pyrene degrading potential. Environ Sci Pollut Res 2012; 20:3280-3289 342
Page 16 of 26
Accep
ted
Man
uscr
ipt
16
[25] Wang S, Nomura N, Nakajima T, Uchiyama H. Case study of the relationship between 343
fungi and bacteria associated with high-molecular-weight polycyclic aromatic hydrocarbon 344
degradation. J Biosci Bioeng 2012; 113:624-630.345
[26] Tagu D, De Bellis R, Balestrini R, De Vries O, Piccoli G, Stocchi V, Bonfante P, 346
Martin F. Immunolocalization of hydrophobin HYDPt-1 from the ectomycorrhizal 347
basidiomycete Pisolithus tinctorius during colonization of Eucalyptus globulus roots. New 348
Phytol 2000; 149:127-135349
[27] Peñas M, Rust B, Larraya L, Ramírez L, Pisabarro A. Differentially regulated, 350
vegetative-mycelium-specific hydrophobins of the edible basidiomycete Pleurotus 351
ostreatus. Appl Environ Microbiol 2002; 68:3891-3898352
[28] Boualem K, Wache Y, Garmyn D, Karbowiak T, Durand A, Gervais P, Cavin J. 353
Cloning and expression of genes involved in conidiation and surface properties of 354
Penicillium camemberti grown in liquid and solid cultures. Res Microbiol 2008; 159:110-355
117356
[29] Vigueras G, Arriaga S, Shirai K, Morales M, Revah S. Hydrophobic response of the 357
fungus Rhinocladiella similis in the biofiltration with volatile organic compounds with 358
different polarity. Biotechnol Lett 2009; 31:1203-1209359
[30] Rocha-Pino Z, Vigueras G, Shirai K. Production and activities of chitinases and 360
hydrophobins from Lecanicillium lecanii. Bioprocess Biosyst Eng 2011; 34:681-686361
[31] Zajc J, Liu Y, Dai W, Yang Z, Hu J, Gostincar C, Gunde-Cimerman N. Genome and 362
transcriptome sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations 363
present and absent. BMC Genomics 2013; 14:1-21.364
Page 17 of 26
Accep
ted
Man
uscr
ipt
17
[32] Biresaw G, Liu Z, Erhan S. Investigation of the surface properties of polymeric soaps 365
obtained by ring-opening polymerization of epoxidized soybean oil. J Appl Polym Sci 366
2008; 108:1976-1985367
[33] Xu Q, Liu Z, Nakajima M, Ichikawa S, Nakamura N, Roy P, Okadome H, Shiina T. 368
Characterization of a soybean oil-based biosurfactant and evaluation of its ability to form 369
microbubbles. Bioresour Technol 2010; 101:3711-3717370
[34] Askolin S, Linder M, Scholtmeijer K, Tenkanen M, Penttilä M, de Vocht M Wösten 371
H. Interaction and comparison of a class I hydrophobin from Schizophyllum commune and 372
class II hydrophobins from Trichoderma reesei. Biomacromolecules 2006; 7:1295-1301373
[35] Lee SC, Lee SJ, Kim SH, Park IH, LeeYS, Chung SY, Choi YL. Characterization of 374
new biosurfactant produced by Klebsiella sp. Y6–1 isolated from waste soybean oil. 375
Bioresour Technol 2008; 99:2288-2292376
[36] Kisko K, Szilvay GR, Vourimaa E, Lemmetyinen H, Linder MB, Torkelli M, Serimaa 377
R. Self-assembled films of hydrophobin proteins HFBI and HFBII studied in situ at the 378
air/water interface. Langmuir 2009; 25:1612-1619379
[37] Kirkland B H and Keryhani, N O. Expression and purification of a functionally active380
class I fungal hydrophobin from the entomopathogenic fungus Beauveria bassania in E. 381
coli. J Ind Microbiol Biot 2011; 33:327-335382
[38] Janssen M I, van Leeuwen, M B M, van Kooten, T G, de Vries, J, Dijkhuizen L, 383
Wösten H A B. Promotion of fibroplast activity by coating with Hydrophobins in the β-384
sheet end state. Biomaterials 2004; 25:2731-2739385
[39] Jong, L. Effect of soy protein concentrate in elastomer composites. Compos Appl Sci 386
Manuf 2006; 37:438-446387
Page 18 of 26
Accep
ted
Man
uscr
ipt
18
[40] Scholtmeijer K, Wessels J, Wösten H. Fungal hydrophobins in medical and technical 388
applications. Appl Microbiol Biotechnol 2001; 56:1-8389
Page 19 of 26
Accep
ted
Man
uscr
ipt
19
390
Captions to figures391
Fig. 1 CO2 concentration (closed symbols), and biomass as total protein (open symbols) 392
evolution in SmC (circles) and SCC (squares) and biomass as total protein (open symbols) 393
produced with P. lilacinus. Lines correspond to the Gompertz model applied for CO2.394
Fig. 2 Protein analyses by SDS-PAGE. Lane M: Molecular weight standard; lanes A and395
B: proteins extracted from SmC and SSC respectively.396
Fig. 3 a) Separation by SEC-HPLC of the fraction of PLHYD purified by RP-HPLC. The 397
molecular weights of standards are indicated at the top. b) Protein profile analysis by SDS-398
PAGE of the same fraction. Molecular weight standard in Lane M and fraction of PLHYD 399
in Lane 1 400
Fig. 4 Surface activity of the PLHYD determined by the pendant drop method, a) (mg 401
mL-1) respect to protein concentration (mg mL-1), b) pure water (control) (72 mNm-1) c) 402
critical micelle concentration (0.45 mg PLHYD mL-1), and wettability determined by 403
contact angle, d) pristine Teflon surface (control, 130.1 ± 2°) and e) PLHYD coated 404
Teflon (47 ± 2°). 405
Fig. 5 Scanning electron micrographs (x500) of a PLHYD film deposited on Teflon; the 406
white area represents the Teflon surface. The inset x2500 shows a porous pattern and a 407
zone with thin filaments and globular aggregates.408
409
Page 20 of 26
Accep
ted
Man
uscr
ipt
20
Highlights409
410
The fungus Paecilomyces lilacinus grows on n-hexadecane producing hydrophobins.411
Hydrophobins were only produced by P lilacinus in solid-state cultures.412
The hydrophobins modified the hydrophobicity of Teflon from 130.1±2° to 47 ±2°413
The hydrophobins formed films having pores smaller than 1µm414
415
416
Page 21 of 26
Accep
ted
Man
uscr
ipt
Figure(s)
Page 22 of 26
Accep
ted
Man
uscr
ipt
Figure 2
Page 23 of 26
Accep
ted
Man
uscr
ipt
Figure 3
Page 24 of 26
Accep
ted
Man
uscr
ipt
Figure 4
Page 25 of 26
Accep
ted
Man
uscr
ipt
Figure 5
Page 26 of 26
Accep
ted
Man
uscr
ipt
Graphical abstract
