Advances in Microbiology, 2012, 2, 375-381
http://dx.doi.org/10.4236/aim.2012.23047 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Preliminary Characterizations of a Carbohydrate from the
Concentrated Culture Filtrate from Fusarium solani and
Its Role in Benzo[a]Pyrene Solubilization
Etienne Veignie1,2, Evgeny Vinogradov3, Irina Sadovskaya1,4, Charlène Coulon1,4, Catherine Rafin1,2*
1Univ Lille Nord de France, Lille, France
2Université du Littoral Côte d’Opale, Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV), Dunkerque, France
3Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada
4Université du Littoral Côte d’Opale, Unité Mixte Technologique, Boulogne-sur-Mer, France
Received July 1, 2012; revised August 1, 2012; accepted August 10, 2012
ABSTRACT
In order to investigate the mechanism of benzo[a]pyrene uptake by a filamentous fungus Fusarium solani, a biochemi-
cal characterization of its concentrated culture filtrate has been conducted. The preparation contained approximately
(w/w): 50% of total carbohydrate, 6.5% of uronic acid and 6% protein, as determined by colorimetric tests. Gel filtra-
tion and anion-exchange chromatographic profiles indicated that the main product of the culture filtrate was a glycoprotein,
which contained mannose, glucose and galactose in an approximate molar ratio of 1.5:0.8:1. The polysaccharide frac-
tion of the culture filtrate was prepared by treatment with proteinase K, followed by gel-filtration chromatography. Its
chemical structure was studied by methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and
Nuclear Magnetic Resonance spectroscopy (NMR). The major carbohydrate was a polymer of β-(1→6)-linked galac-
tofuranose units fully branched at positions O-2 by single residues of
 -glucopyranose. The Fusarium concentrated
culture filtrate increased 4-fold the BaP solubilization in comparison with its aqueous solubility and suggested that the
carbohydrate present in this filtrate should probably be involved in this enhancement. Our findings point out the poten-
tial role of fungal glycoproteins in PAH microbial bioavaibility, an important step for PAH biodegradation.
Keywords: Fusarium solani; Benzo[a]Pyrene; Polysaccharides; Glycoprotein; Nuclear Magnetic Resonance
1. Introduction
Polycyclic aromatic hydrocarbons (PAH) continuously
entering the environment from natural sources (biogenic
and geochemical) as well as anthropogenic ones are con-
sidered as priority pollutants due to their toxicity, muta-
genic and carcinogenic properties [1]. Although PAH
may undergo adsorption, volatilization, photolysis and
chemical degradation, microbial degradation is the major
degradation process [2]. The persistence of PAH in the
environment is dependent on a variety of factors, such as
their chemical structure, their concentration and their
bioavailability. In general, the higher the molecular
weight of the PAH, the lower its aqueous solubility,
which limits the interaction of these compounds with
microbial cells which principally use molecules that are
dissolved in the water phase. Substrate bioavailability is
therefore considered as one of the most important factors
in bioremediation.
In a search for indigenous soil filamentous fungi with
potential to degrade PAH with four or more rings, our
laboratory has isolated a collection of telluric fungi from
PAH-contaminated soil [3-5]. We focused our attention
on a Deuteromycete fungus Fusarium solani (Mart.) Sacc.
(1881) [teleomorph: Haematonectria haematococca (Berk.
and Broome) Samuels and Rossman, Ascomycota, Hy-
pocreales, Nectriaceae] that was able to incorporate
benzo[a]pyrene (BaP) into small vesicles observed in
fungal hyphae with high fluorescence due to accumula-
tion of BaP or its metabolites [3,6] before degradation
and mineralisation [7,8]. This cytological observation
underlines that this fungus has developed a strategy to
enhance the bioavailability and gained access to hydro-
phobic compounds such as BaP. While numerous works
concern the uptake of alkanes by fungi, and especially
yeasts [9,10], the mechanisms of PAH solubilization and
transport were rarely studied in fungi [6,11,12]. In order
to better understand the interaction mechanisms between
the fungal cells and PAH, we have undertaken the pre-
sent investigation which reports the partial characteriza-
tion of the concentrated fungal filtrate produced by the
*Corresponding author.
C
opyright © 2012 SciRes. AiM

E. VEIGNIE ET AL. 376
same Fusarium solani strain in liquid culture provid-
ing information on its chemical composition and on its
properties to solubilize BaP.
2. Materials and Methods
2.1. Microorganism and Growth Conditions
Fusarium solani previously isolated from petroleum-con-
taminated soil [3] was supplied from our UCEIV mycol-
ogy collection (Dunkerque, France). Cultures were con-
ducted in 300 ml mineral salts medium (MM medium)
containing 20 g·l–1 of glucose in 1 l Erlenmeyer flasks.
The standard mineral salts medium (MM) consisted of
(g·l–1): KCl, 0.25; NaH2PO4 2H2O, 3.235; Na2HPO4
2H2O, 5.205; MgSO4, 0.244; NH4NO3, 2; and trace-
element solution consisting of (mg·l–1): ZnSO4·7H2O, 1;
MnCl2·4H2O, 0.1; FeSO4·7H2O, 1; CuSO4·5H2O, 0.5;
CaCl2·2H2O, 0.1; MoO3, 0.2. The culture medium was
adjusted to pH 7. After sterilization (121˚C for 20 min),
inoculation was performed by adding a spore suspension
of F. solani (aged 7 days), prepared as described previ-
ously [3], so as to obtain a final concentration of 104
spores ml–1. After inoculation, Erlenmeyer flasks were
incubated for 5 days at room temperature with shaking
on a reciprocating shaker (Infors, Massy, France, 90 min–1).
2.2. Preparation and Analysis of the Culture
Filtrate
After 5 days, hyphal fragments were removed by two
filtrations through filter paper (45 µm and then 2.5 µm
filter). The culture filtrate was then lyophilized. The dry
extract was dissolved in 25 ml of deionized water, fil-
tered through 2.5 µm filter and dialyzed through a mem-
brane (Difco, 12,000 to 14,000 Daltons) against deionized
water at 4˚C for 3 days (with changing water every 12 h)
and lyophilized, giving the concentrated culture filtrate.
This crude culture filtrate was further fractionated by gel-
filtration chromatography on Sephadex G-50 and Sephacryl
S-200 columns, by ion-exchange chromatography on Q-
Sepharose, and analyzed by SDS-PAGE.
2.3. Preparation and Structural Elucidation of
the Polysaccharide
The concentrated culture filtrate was dissolved in water
and digested with proteinase K (Sigma, 2 mg·ml–1) at
40˚C for 24 h. The digest was then fractionated by gel-
filtration chromatography on a Sephadex G-50 column
(2.5 × 60 cm). The main peaks were collected and ana-
lyzed by composition analysis, methylation analysis and
NMR spectroscopy.
2.4. General and Analytical Methods
Alditol acetates and partially methylated alditol acetates
were analyzed by GC-MS on Varian Saturn 2000 system,
equipped with DB-17 (30 m × 0.25 mm) fused-silica
column using a temperature gradient of 180 (2 min)
→240˚C at 2˚C/min, with ion-trap mass spectral detec-
tor. Prior to analysis, samples were hydrolyzed with 4 M
TFA (120˚C, 3 h) and converted to alditol acetates by
conventional methods. Methylation analysis was per-
formed using the method previously described [13].
Gel-permeation chromatography was carried out on
Sephadex G-50 columns (1.6 × 100 cm and 2.5 × 60 cm;
GE Helthcare), irrigated with 1% acetic acid - 0.4%
pyridine buffer. Ion-exchange chromatography was per-
formed on a Q-Sepharose fast flow column (1 × 10 cm,
GE Healthcare) eluted with water followed by a 60 ml
linear gradient of aqueous NaCl (0 - 0.5 M). An aliquot
of each fraction was assayed colorimetrically for aldose
[14], uronic acid [15], and protein. Protein content was
assayed by screening the fractions at OD280 or by BioRad
colorimetric assay. D-Glucose (Glc, Sigma), D-glucuro-
nic acid (GlcA, Sigma) and bovine serum albumin (BSA,
Acros Organic) were used as standards.
SDS-PAGE was performed using a Bio-Rad Protean I
system according to the method previously described [16]
with 12% separating gel and 4% stacking gel. Gels were
visualized with Coomassie blue stain for proteins and
alcian blue/silver stain [17] for glycoproteins.
1H and 13C NMR spectra were recorded using a Varian
Inova 500 MHz spectrometer for samples in D2O solu-
tions at 25˚C - 45˚C with acetone internal reference (2.23
ppm for 1H and 31.5 ppm for 13C) using standard pulse
sequences DQCOSY, TOCSY (mixing time 120 ms),
NOESY (mixing time 400 ms), HSQC and HMBC (100
ms long range transfer delay). 1H-31P HMQC and HMQC-
TOCSY were run with 1H-31P coupling set to 11 Hz,
TOCSY mixing time 100 ms.
2.5. BaP Solubilization
BaP was initially dissolved in methanol MeOH (40
mg· l–1), then deposited into a haemolysis tube by addi-
tion of 375 µl of BaP solution and allowing MeOH sol-
vent to evaporate. 3 ml of concentrated culture filtrate
previously obtained was added (at working concentra-
tions: 1, 2.5, 5, 7.5 and 10 mg·ml–1) into the haemolysis
tube. Tubes were incubated in the dark for 24 hours. BaP
fluorescence in filtrate solution was analyzed on a Perkin
Elmer LS B50 spectrofluorimeter (excitation 295 nm,
emission 406 nm, time integration 10 s [18]). Blanks
were set up similarly in water with no filtrate added. The
same experiment was also conducted at the concentration
of 1 mg·ml–1 in water with standard yeast mannan (Sigma,
St Quentin Fallavier, France) and with hydroxypropyl-β-
cyclodextrin (HPBCD) kindly donated from Roquette
Frères (Lestrem, France) as references. For each treat-

E. VEIGNIE ET AL. 377
ment, triplicates were realized. Results were expressed as
solubilization mean value ± standard error for triplicates.
3. Results
3.1. BaP Solubilization
Figure 1 showed a linear relationship between culture
filtrate concentration and BaP solubilization (measured
by relative fluorescence intensity and expressed by cal-
culus in µg·l–1) with a high correlation factor R2 = 0.95.
For comparison, the same assessment of BaP solubiliza-
tion was also conducted at one chosen concentration (1
mg· ml –1) in the presence of culture filtrate, water, stan-
dard yeast mannan and a well known cyclic oligosaccha-
ride HPBCD as references. Concerning the BaP solubili-
zation, the results indicated that, in our experimental
conditions, the culture filtrate significantly increased the
solubility of BaP. Indeed, solubility of BaP was 11.8
µg·l–1 with culture filtrate, a 4-fold increase compared to
the BaP aqueous solubility of 2.7 µg·l–1. This solubility
enhancement was comparable to the one obtained with
the yeast mannan. In the presence of a well known cyclic
oligosaccharide HPBCD as reference, the BaP solubili-
zation was about 28.4 µg·l–1 (Table 1).
3.2. Preparation and Analysis of the Culture
Filtrate from Fusarium solani
The culture filtrate was prepared from the culture me-
Benzo[a]pyrene solubilization (µg l
-1
)
y = 0.1464 x + 5.0127
R2 = 0.95
Culture filtrate (mg ml
-1
)
0246810
0
12
14
Culture filtrate (mg·ml
–1
)
(μg·l
–1
)
Figure 1. Relationship between culture filtrate concentra-
tion and BaP solubilization.
Table 1. Effect of tested compounds (at 1 mg·ml–1) on benzo
[a]pyrene solubilization.
Compound BaP solubilization [µg·l–1]
Water 2.8 ± 0.3
Culture filtrate 11.8 ± 0.5
Yeast mannan 9.4 ± 0.3
HPBCD 28.4 ± 0.6
dium after several filtration steps, dialysis and lyophili-
zation. As assessed by colorimetric tests, the culture fil-
trate contained approximately (w/w): 50% of total car-
bohydrate, 6.5% of uronic acid and 6% protein when
respectively Glc, GlcA and BSA were used as standards.
Upon gel-filtration chromatography, the culture filtrate
gave the main fraction which eluted at the void volume
on a Sephadex G-50 column, and as a broad peak with
Kav ~ 0.4 - 0.75 on Sepharose S-200. In both cases, pro-
tein and carbohydrate eluted simultaneously (data not
shown) indicating that the major component of the cul-
ture filtrate was a glycoprotein.
The preparation was analyzed by SDS-PAGE with the
periodate-silver staining, enhanced with alcian blue [17].
It showed a heterogeneous pattern with the major bands
around 15 - 20 kDa and other smaller bands ranging from
30 to 150 kDa (Figure 2). On anion-exchange-column,
the preparation eluted as a single sharp peak with simul-
taneous elution of total carbohydrate, protein and uronic
acid at a NaCl concentration of ~0.15 M (Figure 3). This
could indicate that despite the MW heterogeneity, the
glycoprotein preparation was homogeneous in charge.
3.3. Preparation and Structural Elucidation of
the Polysaccharide
1H NMR spectrum of the sample contained two intense
anomeric signals and several anomeric signals of smaller
1 2
200
150
100
75
37
50
25
20
15
Mw, kDa
Figure 2. SDS-PAGE profile of the Fusarium solani culture
filtrate (lane 1) along with the Precision plus protein stan-
dards (Bio-Rad). The gel was stained with alcian blue fol-
lowed by periodate oxidation-silver staining.

E. VEIGNIE ET AL. 378
and 2D-NMR techniques.
μg/ml
0.5
0.4
0.3
0.2
0.1
NaCl, M
0 2 4 6 8 10 12 14 16 18
Tube number
200
180
160
140
120
100
80
60
40
20
0
Monosaccharide composition analysis led to identifi-
cation of mannose (Man), glucose (Glc) and galactose
(Gal) as main components in an approximate molar ratio
of 1.5: 0.8: 1 and small amount of glucosamine (GlcN).
2D NMR spectra (DQCOSY, TOCSY, NOESY,
1H-13C HSQC and HMBC, 1H-31P HMQC) were re-
corded and interpreted. Two major sugar spin systems, A
and B, were identified (Figure 4). Residue A had 13C
NMR signals in the low field region (Table 2), which
indicated furanoside form. Comparison of the 13C chemi-
cal shifts with known values [19] indicated that it had
β-galacto-configuration, and it was therefore identified as
a  -galactofuranose. Residue B was identified as a  -
glucopyranose based on the characteristic vicinal proton
coupling constants.
Figure 3. Elution profile of the Fusarium solani culture fil-
trate on an anion-exchange Q- Sepharose fast flow column,
eluted with 0 - 0.5 M gradient NaCl. Aliquots of each frac-
tion were assayed for total sugars (●), protein (○) and
uronic acid (▲) and expressed in µg ml-1 of Glc, BSA and
GlcA, accordingly. The linkages between sugars were identified as A1 →
A6 and B1 → A2 on the basis of NOE correlations:
A1:A6, B1:A2, and confirmed by observation of HMBC
cross peaks A H-1:A C-6; B H-1:A C-2. Thus, the major
carbohydrate corresponded to a linear chain of β-(1,6)-
linked Galf residues to which Glcp is attached via α-(1,2)-
linkage as side chains (Figure 5).
intensity. Wobbling baseline and broad signals at 1 - 3
ppm indicated the presence of protein. Attempts to purify
the major component by gel and anion exchange chro-
matography were not successful (data not shown), indi-
cating that protein and carbohydrate moieties could be
covalently linked. Sample was treated with proteinase K
and products were separated by gel chromatography on
Sephadex G-50. Polysaccharide containing fractions,
eluted close to void volume, were collected and analyzed
by composition analysis, methylation analysis, and 1-
The variant of
 -Glc (B’ on Figure 4) also had NOE
from H-1 to the A H-2, and strongly shifted H-4 signal at
4.22 ppm. This was due to phosphorylation of its O-4;
H-4 gave 1H-31P correlation (31P signal at 2.02 ppm).
Minor signals in the spectra were identified as belonging
ppm
55
60
65
70
75
80
85
90
95
100
105
B6
5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4
A1
ppm
A2
A3
A4
A5 B4
A6
B2
B5 B3
B
′
1
B1
Figure 4. 1H-13C HSQC spectrum of the main carbohydrate, obtained after digestion of Fusarium solani culture filtrate with
proteinase K. Unlabeled signals belong to mannan.

E. VEIGNIE ET AL. 379
Table 2. 1H and 13C chemical shifts of the polysaccharide isolated from Fusarium solani culture filtrate following proteinase
K treatment.
Unit Atom 1 2 3 4 5 6a/6b
1H 5.18 4.17 4.24 4.03 4.00 3.66/3.91
 -Galf A 13C 106.8 87.1 75.9 82.9 70.0 69.7
 -Glc B 1H 5.08 3.58 3.71 3.44 3.78 3.78/3.89
13C 98.4 71.6 73.2 70.0 72.8 61.1
1H 5.10 3.68 3.85 4.20 3.78
 -Glc B’
13C 98.4 71.2 71.8 71.9 73.7
A
[-6)-  -Galf-(1- ]
|
 -Glcp-(1-2)
B
Figure 5. Suggested schematic structure of the main carbo-
hydrate chain of the glycoprotein of Fusarium solani cul-
ture filtrate.
to a α- and β-mannose (data not shown). In addition,
NMR spectra of all fractions contained a sharp peak at
3.25/54 ppm, which was higher in lower molecular mass
fractions, and thus did not seem to belong to the main
polysaccharide chain structure.
Methylation analysis afforded terminal glucose and
2,6-substituted galactofuranose, in agreement with the
proposed structure. Additionally, peaks of 2- and 2,6-
substituted mannose were observed.
4. Discussion
In the present work, we prepared an extracellular extract
from a strain of Fusarium solani previously isolated from
petroleum-contaminated soil and showed that this prepa-
ration was able to solubilize a model organic pollutant
BaP. We present evidence that the major part of the ex-
tract contains an acidic glycoprotein, heterogeneous in
size and containing Man, Glc, Gal and uronic acid. We
showed that the major carbohydrate chain of the glyco-
protein corresponded to a linear chain of β-(1,6)-linked
Galf residues, to which Glcp is attached via α-(1,2)-
linkage as side chains.
Knowledge on extracellular and cell-wall polysaccha-
rides from Fusarium spp. and in particular in Fusarium
solani is quite limited. Siddiqui and Adams were first to
report the presence of an extracellular galactofuranose-
containing glycoprotein in Gibberella fujikuroi (Fusa-
rium moniliforme) [20]; only few structural elements
were then presented. To our knowledge, mycelium gly-
coproteins of only one Fusarium sp., designated M7-1,
were elucidated in details [21-23]. Fusarium sp. M7-1
was found to produce acidic polysaccharides as compo-
nents of the cell wall, and these polysaccharides were
O-glycosidically linked to a protein moiety. The main
structure of the polysaccharide consisted of a linear chain
of β-(1,6)-linked Galf residues with various substitutions.
Other minor oligosaccharide chains, released from the
glycoprotein by mild alkaline treatment, were also cha-
racterized. They were composed mainly of Man residues
with α-(1,2)-linkages but also contained GlcNAc, Rha,
Man-6-phosphate [24], Man-6-phosphoethanolamine [21],
and Man-6-phosphocholine [25]. The same authors de-
scribed the production of extracellular acidic glycopro-
teins by several Fusarium species, and characterized the
structure of a glycoprotein from F. oxysporum. Simi-
larly to Fusar ium sp. M7-1, the sugar moiety of the gly-
copeptide contained a linear chain of β-(1,6)-linked Galf
residues with short side chains containing Glc, GlcA and
Man [26]. Da Silva, Ribeiro, Sassaki, Gorin and Barreto-
Bergter [27] analyzed the glycopeptides from mycelia
of F. oxysporum using partial hydrolysis, methylation
analysis and NMR. They concluded that
 -Galf were
substituted at O-6 with terminal
 -Man residues.
Most of studies of polysaccharides of Fusa rium and
related species were aimed at the research of taxonomic
and phylogenetic markers, and were often limited to
monosaccharide composition and linkage type analysis
of alkali-extractable water-soluble cell wall polysaccha-
rides [28]. Ahrazem et al. [29] performed such analysis
of several Fusarium and Gibberella species, but detailed
chemical structures of these polysaccharides were not
presented.
We further studied the properties of the concentrated
fungal filtrate for BaP solubilization, a prerequisite to
permit its incorporation into fungal cells which is one of
the most limiting factors for BaP degradation. The con-
centrated fungal filtrate clearly showed a capacity to
solubilize BaP similar to the yeast mannan standard
(4-fold increase in comparison with the BaP aqueous
solubility). We hypothesized that the enhancement of
BaP solubilization could be partially due to the Fusarium
solani extracellular glycoprotein and more particularly to

E. VEIGNIE ET AL. 380
its carbohydrate part. Indeed, some other carbohydrate
compounds are well known to enhance aqueous solubility
of organic molecules. Thus, cyclic oligosaccharides cyclo-
dextrins possess apolar cavities which enable them to
form inclusion complexes with hydrophobic molecules
[18,30]. We have recently shown by molecular modelling
that the polysaccharide amylopectin present in starch also
presented several hydrophobic sites suitable for BAP
complexation [31]. Glycoproteins could play a role of
solubilizing compounds, possibly due to their amphi-
philic nature. Masuoka and Hazen [32] demonstrated a
relationship between modifications in the acid-labile
β-1,2-oligomannoside chain of a cell wall N-glycosylated
protein and the cell surface hydrophicity status in Can-
dida albicans, indicating the effect of fine structural dif-
ferences in the carbohydrate part of a glycoprotein on its
hydrophobic/hydrophilic properties. At last, a bioemulsi-
fier liposan produced by Candida lipolytica was charac-
terized as a glycoprotein with its carbohydrate moiety
presenting a similar composition to that of Fusarium
solani [33].
Fungal heteromannans are well known as glycosidic
moieties of fungal wall glycopeptides and glycoproteins
[28]. As shown by immunofluorescence for Fusarium
javanicum, they are located in the surface of fungal walls
[29]. Most studies on these polysaccharides were con-
ducted for searching taxonomical and phylogenetical
markers; and the classical extraction process by alkali
treatments hydrolyses the linkage between the protein
and glycosidic moieties. Our findings showing the capacity
of an extracellular glycoprotein from an important soil
fungus F. solani to solubilize a model PAH BaP point
out another important role that can be played by these
molecules: their potential involvement in microbial bio-
avaibility of PAH, an important step in PAH biodegrada-
tion.
5. Conclusion
To summarize, our results show that the concentrated fil-
trate produced by Fusarium solani stimulated BaP solu-
bilization. The glycoprotein present in this filtrate should
probably be involved in this enhancement. The chemical
structure of its main glycosidic chain was established.
Further research should be conducted in order to precise
the role of protein and carbohydrate parts of the glyco-
protein in the solubilization and uptake of BaP by this
strain of F. solani. Our findings point out the potential
role of fungal glycoproteins in microbial degradation of
PAH.
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