f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 2 6 8e2 7 4

journa l homepage : www.e lsev ier . com/ loca te / funb io

Potential of autochthonous fungal strains isolated fromcontaminated soils for degradation of polychlorinatedbiphenyls

Bello MOUHAMADOUa,*, Mathieu FAUREa, Lucile SAGEa, Johanna MARCAISa,Florence SOUARDb, Roberto A. GEREMIAa

aLaboratoire d’Ecologie Alpine, UMR 5553 CNRS, Universit�e Joseph Fourier, Grenoble 1, BP 53, 38041 Grenoble Cedex 9, FrancebD�epartement de Pharmacochimie Mol�eculaire, UMR 5063 CNRS, Universit�e Joseph Fourier, Grenoble 1, BP 53,

38041 Grenoble Cedex 9, France

a r t i c l e i n f o

Article history:

Received 20 November 2012

Received in revised form

15 February 2013

Accepted 27 February 2013

Available online 14 March 2013

Corresponding Editor:

John Dighton

Keywords:

Ascomycota

Autochthonous fungal strains

Biodegradation

PCBs

Zygomycota

* Corresponding author. Tel.: þ33 476 63 54 4E-mail address: bello.mouhamadou@ujf-g

1878-6146/$ e see front matter ª 2013 The Bhttp://dx.doi.org/10.1016/j.funbio.2013.02.004

a b s t r a c t

Up to now, most studies on polychlorinated biphenyl (PCB) bioremediation have examined

the ability of model fungal strains to biodegrade PCBs. Yet, there is limited information

concerning the potential of autochthonous filamentous fungal strains in the biodegrada-

tion of PCBs and their possible use in the environmental technologies. In this study, we in-

vestigated the capacity of autochthonous fungal strains in the biodegradation of PCBs by

isolating 24 taxa from former industrial sites highly contaminated by PCBs. Microscopic

and molecular analyses using the internal transcribed spacer (ITS) region revealed that

the fungal strains belonged to the phyla Ascomycota (19 strains) and Zygomycota (five

strains). The chromatography gas analysis revealed evidence of degradation of seven

PCB congeners. With the exception of Circinella muscae which presented no degradation po-

tential, the other fungal strains exhibited a rate of biodegradation ranging from 29 to 85 %

after 7 d of incubation in liquid medium. Among these strains, Doratomyces nanus, Dorato-

myces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena,

and Thermoascus crustaceus showed remarkable degradation ability (>70 %) regardless of

the number of chlorine substituents on the biphenyl nucleus and a high tolerance towards

PCBs. To our knowledge, this is the first study that demonstrates the ability of PCB degra-

dation by these species and indicates the potential effectiveness of some autochthonous

fungal strains in bioremediation systems.

ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction hydraulic fluids, plasticizers or fire retardant because of their

Polychlorinated biphenyls (PCBs) are a class of compounds

containing a biphenyl molecule that is chlorinated to form

209 different congeners (Beaudette et al. 1998). These mole-

cules have been largely used in industries with numerous ap-

plications mainly in electrical equipment and were used as

1; fax: þ33 476 51 44 63.renoble.fr (B. Mouhamaritish Mycological Societ

physical properties (Pieper & Seeger 2008; Xing et al. 2010). Be-

cause of this widespread use, a large amount of PCBs has been

spread into environment, accumulated in soils and sedi-

ments, and represents a threat to ecosystem health due to

their low degradability and high rate of toxicity (Pointing

2001; Dercova et al. 2009). Through their lipophilicity, PCBs

dou).y. Published by Elsevier Ltd. All rights reserved.

Table 1 e Concentration of the seven PCB congeners inthe studied soil.

PCB congeners mg g�1 Dry matter

28 (2,4,40-Trichlorobiphenyl) 0.45

52 (2,20,5,50-Tetrachlorobiphenyl) 18.35

101 (2,20,4,5,50-Pentachlorobiphenyl) 51.20

118 (20,3,4,40,50-Pentachlorobiphenyl) 33.00

138 (2,20,3,4,40,50-Hexachlorobiphenyl) 44.98

153 (2,20,4,40,5,50-Hexachlorobiphenyl) 101.77

180 (2,20,3,4,40,5,50-Heptachlorobiphenyl) 70.94

Potential of autochthonous fungal strains for degradation of PCBs 269

are able to bioaccumulate and biomagnify along the food

chain (Bedard et al. 2007). They are readily stored in fatty tis-

sues and confer acute or chronic toxicity to animals and

humans and they are also considered as potentially carcino-

genic (McLachlan 1996; Pointing 2001).

Biological technologies using microorganisms or their en-

zymes to promote toxic compounds degradation are widely

investigated because of their low environmental impact and

their economic advantage compared to the physicochemical

methods. Bacteria are the most studied microorganisms for

the biological destruction of PCBs and numerous studies

have shown their capacity to degrade less-chlorinated conge-

ners (Abramowicz 1990; Quensen et al. 1990; Rojas-Avelizapa

et al. 1999). The degradation mechanisms are well known

and involve dehalogenase enzymes which catalyse the selec-

tive removal of meta- and para-chlorine and the dioxygenase

degradative pathway resulting in the degradation or mineral-

ization of low chlorinated congeners (Abramowicz 1990;

Pieper 2005; Pieper & Seeger 2008). However, the efficiency of

bacterial remediation is low in the highly polluted habitats

and negatively affected by the bioavailability of pollutants

making them inaccessible to unicellular organisms (Harms

et al. 2011).

The potential use of fungi able to degrade PCB congeners

can be an attractive alternative compared to the bacterial sys-

tem. Firstly, fungi are ubiquitous microorganisms found in

aquatic sediments, terrestrial habitats, and water surfaces

and their hyphal development promotes access to pollutants

(April et al. 2000). Secondly, many reports have demonstrated

the capacity of fungal species such as Phanerochaete chrysospo-

rium, Lentinus tigrinus, Pleurotus ostreatus, Trametes versicolor or

some filamentous ascomycetous fungi to degrade various PCB

congeners (Kubatova et al. 2001; Ruiz-Aguilar et al. 2002; Kamei

et al. 2006; Tigini et al. 2009; Federici et al. 2012). However, al-

though the nonspecific oxidative exoenzymes such as lignin

peroxidase, manganese-dependent peroxidase, and laccases

have been described as involved in the degradation of numer-

ous organic pollutants, their direct involvement in PCB degra-

dation has not been demonstrated.

Despite the interesting ecological and biochemical proper-

ties of fungal species, their introduction for bioremediation

purposes in environments which differ from their natural

habitats proved to be inefficient (Baldrian 2008; Harms et al.

2011). This phenomenon might be either due to their inability

to compete with indigenousmicrobiota or to their nonadapta-

tion to these new environments. For example, studies on the

degradation of organic pollutants such as fluorene have

shown a greater degradation potential of autochthonous fun-

gal strains isolated from contaminated soils than those from

similar uncontaminated environments (Garon et al. 2004).

Similarly, the role of autochthonous fungi in degradation of

aromatic hydrocarbons has been shown (D’Annibale et al.

2006). Up to now, only few studies have investigated the effec-

tiveness of the autochthonous fungal species in the biodegra-

dation of PCBs (Tigini et al. 2009).

In this study, the potential of fungal strains isolated from

PCB contaminated soils in PCBs biodegradation was evalu-

ated. Fungal strains were isolated from former industrial sites

and identified bymorphological andmolecular methods, with

the latter relying on the nuclear ribosomal internal

transcribed spacer (ITS) sequences. Their capacity to degrade

seven PCB congeners was assessed in liquid cultures of the

identified isolates by gas chromatographic analysis.

Materials and methods

Materials

PCB 28, 52, 101, 118, 138, 153, and 180 that are the most abun-

dant in the environmental and biological matrices, were ob-

tained from SigmaeAldrich Corp. (St. Louis, MI, USA). A

stock solution containing 15 mg of each PCB in 105 ml of iso-

octane was prepared and stored in a freezer (�20 �C). Thecontaminated soil was sampled from a former industrial

site located in Metz (France). The concentrations of the

seven PCB congeners investigated in this study are shown

(Table 1).

Fungal isolation and morphological identification

For the isolation of autochthonous filamentous fungal strains,

1 g of contaminated soil was added to 9ml of sterilewater con-

taining 0.05 % of sodium dodecyl sulphate (w/v). After stirring,

aliquots of the suspension were serially diluted and spread

onto Petri dishes containing malt extract agar (1.5 % w/v) sup-

plemented with 0.05 % chloramphenicol. Cultures were incu-

bated at 25 �C or 37 �C to optimize the isolation of strains

possessing different optimal growth temperatures. The iso-

lated strains were characterized by morphological criteria

and identified according to the general principle of fungal

classification (Domsch et al. 1980; Arx 1981).

DNA extraction and PCR

For themolecular analysis of the isolated strains, total DNA of

each strain was extracted using a Fast DNA Spin Kit (QBIO-

gene, Germany) according to the manufacturer’s recommen-

dations. The PCRs were carried out according to

conventional protocols using Ampli Taq Gold DNA polymer-

ase (Applied Biosystems, USA) and the primers ITS4 and

ITS5 (White et al. 1990) synthesized by Eurogentec (Seraing,

Belgium) were used to amplify the fungal ITS regions. The

PCRs were performed in a programmable thermal cycler

GeneAmps 2720 (Applied Biosystems). Amplifications were

carried out in 50 ml reaction mixtures as described by Molitor

et al. (2010).

270 B. Mouhamadou et al.

Sequencing and sequence analysis

The PCR product of each strain was sequenced by Beckman

Coulter Genomics (United Kingdom). Comparisons with se-

quences from the GenBank databases were made using the

BLAST search algorithm (Altschul et al. 1990). Alignments of

nucleotide sequences were carried out with the Clustal W

software (Thompson et al. 1994). Phylogenetic analyses were

carried out using the neighbour-joining method based on

Clustal W alignments and the robustness of tree topologies

was evaluated by performing bootstrap analysis of 1000 data

sets using MEGA 3.1 (Tamura et al. 2007). For the alignment

and phylogeny, only the 5.8S ribosomal DNA sequences and

the partial sequences of ITS1 and ITS2 were used because of

the high polymorphism of the ITS regions across the phyloge-

netically distant strains.

Biodegradation test in liquid medium

The mycelium of each isolated strain, cultivated in malt ex-

tract (1.5 % w/v), was scraped with a scalpel and ground

with an Ultra Turrax homogenizer (IKA T8, Germany) in 3 ml

of sterile water. 400 ml aliquots were introduced into 100 ml

Erlenmeyer flasks containing 20 ml of modified GS liquid me-

dium (Galzy & Slonimski 1957) supplemented with glucose

(5 g l�1) and incubated at 25 �C or 37 �C for the thermophilic

strains on a rotary shaker (180 rpm). After 48 h incubation,

the culture of each strain was spiked with 400 ml of a PCB

mix (seven PCB congeners in isooctane) giving a final concen-

tration of 3 mg of each congener per ml. All Erlenmeyer flasks

were sealed with Teflon stoppers (VWR, Fontenay-sous-Bois,

France) to prevent evaporation of PCBs. Each experiment con-

taining each strain was performed in triplicate and included

cell-free flasks for stability assessment. After 7 d incubation

on a rotary shaker at 120 rpm, the cultures were stopped

and the mycelium was separated from the culture medium

by filtering through Whatman 40 filter.

PCB extraction and Gaz chromatography (GC) analyses

Culture medium and fungal mycelium were extracted by us-

ing hexane. Culture medium was extracted three times with

20 ml of hexane with agitation (250 rpm, room temperature)

for 30min. Myceliumwas first homogenized in the serum bot-

tles with a POTTER homogenizer in 20 ml of hexane and incu-

bated 24 h at room temperature with agitation (250 rpm) then

extracted three times with 20 ml of hexane with agitation

(250 rpm, room temperature, 30 min). The organic phases

from culture medium and mycelium were evaporated under

vacuum using a rotary evaporator and adjusted to a volume

of 5 ml.

For the extraction of PCBs from the contaminated soil, 5 g

of dry soil were extracted using hexane acetone (1:1, v/v)

with the extractor ASE (Accelerated Solvent Extraction) (Agi-

lent Technologies, USA) according to the manufacturer’s

recommendations.

For the PCB analyses, aliquots of 200 ml were analysed using

a gas chromatograph (7890A), equipped with a DB-XLB capil-

lary column (ID: 0.25 mm, Length: 60 m, Film: 0.25 mm) and

an electron capture detector (mECD) (Agilent Technologies,

USA). The carrier gas was hydrogen. The injector temperature

was 250 �C. The detector temperature was 310 �C. The column

temperature was 50 �C. PCB 30 (2,4,6-trichlorobiphenyl) was

used as an internal standard.

The PCB recovery yield was determined from the cell-free

controls at initial time. The abiotic losses were determined

from the cell-free controls incubated in parallel with fungal

cultures.

Since the ultimate aim of this study was to provide novel

strains for bioaugmentation applications, the selection of

the fungal strains that we considered as the most effective is

based on two criteria: high rate of degradation and nonpatho-

genicity of the strain.

Statistical analysis

A statistical method to partition the sums of squares of Y

among experimental treatments (Anderson 2001) was

used. Two experimental factors were analysed, namely

strains (A) and PCB congeners (B). The generic form of the

model was thus: Y ¼ A þ B þ ε, where ε is an error term.

This multivariate analysis of variance was carried out using

the function adonis of the package vegan (Anderson 2001;

Oksanen et al. 2009), a method similar to a redundancy anal-

ysis (McArdle & Anderson 2001). Sums of squares and

resulting F-tests from permutations of the raw data were

calculated to test for the significance of experimental fac-

tors on Y. We used the KruskaleWallis tests (Kruskal &

Wallis 1952) to test for each selected strain, significant dif-

ferences between presence and absence of PCB. All statisti-

cal analyses were performed using R package vegan

(Oksanen et al. 2009).

Results

Characterization of species isolated from the contaminatedsoil

Strains isolated from PCB contaminated soil were identified by

their morphological criteria. A total of 24 strains were found

and all these strains belonged to Ascomycota and Zygomycota

phyla. A phylogenetic analysis was performed by adding the

reference sequences available in the GenBank database. The

phylogram on Fig 1 confirmed the morphological identifica-

tion of the strains and showed that the 24 strainswere divided

into 15 and four genera belonging to the Ascomycota (19

strains) and Zygomycota (five strains) phyla respectively.

Most genera were represented by a single strain with the ex-

ception of Trichoderma (two strains), Doratomyces (three

strains), Penicillium (two strains), and Mucor (two strains)

(Fig 1).

Analysis of PCB reduction

In our experimental procedure, the PCB recovery yield was

78 % and the abiotic losses were 7 %. Considering these data,

we determined the percentage of depletion of PCBs by taking

into account the quantity of residual PCBs in the culture me-

dium and in themycelium (biosorption) of each strain. Results

Doratomyces nanus (S2-12): JX537957

D. purpureofuscus FJ914688.1

D. purpureofuscus (S2-32): JX537967

D. verrucisporus (S2-33): JX537968

D. stemonitis JN104543.1

Pseudallescheria boydii (S2-23): JX537963

Gliomastix roseogrisea (S2-17): JX537960

Fusarium solani (S2-27): JX537966

Trichoderma spirale JF439515.1

Trichoderma harzianum (S2-18): JX537961

T. harzianum (S2-19): JX537962

Myceliophthora thermophila (S2-1): JX537951

Chaetomium murorum JQ946413.1

C. piluliferum AB625587

C. piluliferum (S2-2): JX537970

Thielavia sp (S2-24): JX537964

Geomyces pannorum (S2-11): JX537957

Geomyces pannorum HQ533810.1

Thermoascus crustaceus (S2-7): JX537956

T. crustaceus U18353.1

Aspergillus terreus FJ011538.1

Neosartorya pseudofischeri (S2-14): JX537959

A. fumigatus (S2-9): JX537971

Penicillium chrysogenum (S2-20): JX537974

P. aurantiogriseum (S2-34): JX537969

Phoma eupyrena (S2-26): JX537965

Mortierella elongata (S2-6): JX537954

M. elongata JF439485.1

Galactomyces geotrichum (S2-13): JX537958

G. geotrichum HE799669.1

Mucor circinelloides (S2-3): JX537952

M. plumbeus (S2-5): JX537955

Lichtheimia corymbifera (S2-4): JX537953

Circinella muscae (S2-10): JX537972

100

100

100

100

96

5087

98

60

99

9894

95

90

100

97

95

9390

52

94

99

54

0.05

Fig 1 e Consensus trees obtained using the neighbour-joining method based on aligned ITS sequences. Bootstrap values are

shown on the branches. The accession numbers of reference sequences which have been recovered from GenBank are in-

dicated. The strains isolated in this study and their accession numbers are indicated in bold.

Potential of autochthonous fungal strains for degradation of PCBs 271

showed that the reduction of PCBs was variable according to

the strains (Table 2). For instance, Circinella muscae showed

the lowest reduction of PCBs (9.76 %). Fifteen strains showed

intermediate rates of PCB reduction (20e70 %). Finally the

cultures of eight strains (Thermoascus crustaceus, Neosartorya

pseudofischeri, Doratomyces purpureofuscus, Myceliophthora

thermophila, Aspergillus fumigatus, Doratomyces nanus, Dorato-

myces verrucisporus, and Phoma eupyrena) led to an important

loss of PCB (>70 %). Because A. fumigatus and the related spe-

cies N. pseudofischeri are pathogenic fungi that cannot be used

in the process of bioremediation by bioaugmentation, we used

the six other strains only for further analysis.

Tolerance and efficiency of the selected strains in disruption ofPCB congeners

The tolerance of each of the six selected strains towards PCBs

was assessed by comparing the mycelial dry weight of each

strain incubated in presence or in the absence of PCBs. There

were no statistically significant differences in growth of any

fungal isolate when grown in the presence or absence of

PCBs, as determined by KruskaleWallis analysis (Fig 2).

The effectiveness of the selected strains in the biodegrada-

tion of each PCB congener was determined by calculating the

equivalent of the consumption of each PCB per mg of mycelial

Table 2 e Comparison of PCB removal by fungal strains isolated from a contaminated soil.

Strains % PCBs in the liquid medium % PCBs in the mycelium % PCBs removal

Myceliophthora thermophila S2-1 7.85 � 0.32 17.19 � 1.35 74.96 � 1.10

Chaetomium piluliferum S2-2 46.04 � 2.54 9.89 � 0.13 44.07 � 2.18

Mucor circinelloides S2-3 45.00 � 2.80 1.40 � 0.31 53.60 � 3.11

Lichtheimia corymbifera S2-4 21.90 � 2.20 16.76 � 3.06 61.34 � 3.91

Mucor plumbeus S2-5 45.90 � 10.55 10.56 � 2.98 43.54 � 8.23

Mortierella elongata S2-6 9.48 � 2.86 40.70 � 7.75 49.82 � 10.59

Thermoascus crustaceus S2-7 11.31 � 1.09 10.28 � 0.51 78.41 � 0.74

Aspergillus fumigatus S2-9 2.53 � 0.25 12.48 � 1.34 84.99 � 0.89

Circinella muscae S2-10 52.06 � 8.41 38.18 � 4.31 9.76 � 12.03

Geomyces pannorum S2-11 31.49 � 6.73 1.07 � 0.12 67.44 � 6.84

Doratomyces nanus S2-12 18.62 � 1.96 7.57 � 1.02 73.81 � 2.78

Galactomyces geotrichum S2-13 57.24 � 4.33 2.55 � 0.43 40.20 � 4.37

Neosartorya pseudofischeri S2-14 11.11 � 2.72 15.22 � 3.11 73.67 � 3.31

Gliomastix roseogrisea S2-17 33.40 � 4.59 29.46 � 3.71 37.14 � 2.57

Trichoderma harzianum S2-18 27.05 � 2.43 10.55 � 1.83 62.39 � 1.67

Trichoderma harzianum S2-19 66.12 � 6.41 4.35 � 0.54 29.52 � 4.74

Penicillium chrysogenum S2-20 8.88 � 3.36 56.03 � 2.70 35.09 � 0.54

Pseudallescheria boydii S2-23 49.70 � 4.30 6.92 � 0.15 43.38 � 4.27

Thielavia sp. S2-24 5.30 � 0.10 55.51 � 14.15 39.19 � 11.71

Phoma eupyrena S2-26 4.37 � 0.39 14.05 � 2.06 81.59 � 1.81

Fusarium solani S2-27 31.23 � 3.68 4.65 � 0.83 64.12 � 4.51

Doratomyces purpureofuscus S2-32 10.20 � 2.60 7.79 � 1.51 82.01 � 3.97

Doratomyces verrucisporus S2-33 20.53 � 6.61 9.45 � 0.52 70.02 � 7.08

Penicillium aurantiogriseum S2-34 20.87 � 3.21 10.18 � 3.04 68.95 � 0.94

The results are expressed as means � standard deviation.

These values were determined by taking into account the PCB recovery yield (78 %) and the abiotic losses (7 %).

272 B. Mouhamadou et al.

dry weight. Doratomyces verrucisporus showed the highest rate

of consumption of PCBs, between 52 and 62 mg of each PCB and

there was no significant variation of the removal rate accord-

ing to the congeners used. Doratomyces nanus also removed

equivalently all the seven congeners but with relatively low

efficiency (30e36 mg). The four other fungal strains were sim-

ilar and showeda lower consumption of eachPCB (<15 mgmg�1

of mycelium). Statistical test (Table 3) indicated significant

Myc

elia

l dry

wei

ght

(g)

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35-PCBs

+PCBs

Strains

MT TC DN PE DP DV

Fig 2 e Growth of mycelium (dry mass) of the six selected

strains from the culture incubated in absence or presence of

seven PCB congeners. Significant differences between

presence and absence of PCB have been tested by Krus-

kaleWallis tests. MT: Myceliophthora thermophila, TC: Ther-

moascus crustaceus, DN: Doratomyces nanus, PE: Phoma

eupyrena, DP: Doratomyces purpureofuscus, DV: Doratomyces

verrucisporus.

differences between fungal strains and confirmed the absence

of variation according to the PCB congeners (Fig 3).

Discussion

A crucial step in the development of mycoremediation tech-

nologies is the availability of fungal strains with biodegrada-

tion potential. Although the use of autochthonous strains

has proven to be a satisfactory approach for the cleanup of

contaminated sites (Garon et al. 2004; Potin et al. 2004;

D’Annibale et al. 2006), most studies on mycoremediation of

PCBs are based on the allochthonous fungi often from the cul-

ture collection. We investigated the capacity of native species

in the biodegradation of PCBs by isolating 24 taxa from former

industrial site highly contaminated by PCBs. A consortium of

fungal species was obtained with the dominance of filamen-

tous ascomycetes consistent with studies carried out on soils

(Tigini et al. 2009). Althoughwe observed a low fungal diversity

Table 3 e Differences between fungal strains andbetween PCB congeners.

Variables Df Sums of sqs Mean sqs F model Pr (>F )

Strain 5 50 319.00 10 064.00 198.63 2 � 10�16

PCB 6 137.00 23.00 0.45 0.84

Strain � PCB 30 155.00 5.00 0.10 1.00

Residuals 84 4256.00 51.00 e e

Total 125 50 319.00 e e e

Summary analysis of variance of mycelia consumption of seven

PCB congeners (see Fig 3).

0

10

20

30

40

50

60

70

802852101118153138180

Qua

ntit

y of

eac

h P

CB

con

gene

r (µ

g / m

g of

myc

elia

l dry

wei

ght)

MT TC DN PE DP DV

a

b

c

cddd

Fig 3 e Consumption of each PCB congener by the six se-

lected strains. Lower-case letters indicate significant dif-

ferences between strains but not between congeners within

strains (see Table 3).

Potential of autochthonous fungal strains for degradation of PCBs 273

in comparison to that estimated in unpolluted soils (Thorn

1997), our result is consistent with previous studies

(D’Annibale et al. 2006; Tigini et al. 2009) and suggests that

the high quantity of pollutants (>300mg g�1 of soil dry weight)

may exert a selective effect on soil fungi. This effect could re-

sult from the selection of strains tolerant to pollutants as

shown by Martino et al. (2000) or strains potentially able to de-

grade PCBs by the process of cometabolism. Interestingly,

some isolated strains, including those belonging to the genera

Aspergillus, Penicillium or Pseudallescheria have often been iso-

lated from polluted soils and their tolerance and their remark-

able degradative ability towards different organopollutants

have been reported (Pant & Adholeya 2007; Junghanns et al.

2008; Chang 2008; Tigini et al. 2009; Pinedo-Rilla et al. 2009).

The capacity of the isolated strains to degrade PCBswas de-

termined by taking into account both the remaining PCBs in

the culture medium and those extracted from the mycelium

corresponding to the fungal biosorption. Due to the strong

ability of PCBs to bind to the fungal biomass, the extraction

procedure of PCBs from the mycelium was modified from

that of PCBs present in the liquid medium by incubating the

mycelium of each strain 24 h in hexane with vigorous agita-

tion. We concluded that the substantial losses of PCBs were

essentially due to the fungal biodegradation. With the excep-

tion of Circinella muscae, the other strains exhibited variable

biodegradation capacity, from 29 to 85 %, comparable to the

result obtained by Ruiz-Aguilar et al. (2002) when using the ba-

sidiomycete ligninolytic fungi. This result confirms that most

fungi isolated from the highly polluted substrates are effective

in the pollutant biodegradation and is also consistent with the

reported high capacity for the ascomycetes to metabolize var-

ious organic chemicals (Harms et al. 2011).

We considered that efficient strains which could be used

for bioaugmentation applications should be nonpathogenic

and present at least 70 % of degrading rate. This criterion

was fulfilled by six strains among which three strains belong-

ing to the genus Doratomyces, two thermophilic strains

Myceliophthora thermophila and Thermoascus crustaceus, and

one strain belonging to the genus Phoma. The six selected

strains showed a high tolerance towards PCBs and a remark-

able ability to degrade different PCB congeners. In addition,

there was no significant selective degradation regarding the

number of chlorine on the biphenyl nucleus. This contrasts

with studies on the bacterial systems in which the extent of

degradation seemed to be affected by the degree or the posi-

tion of chlorine (Abramowicz 1990; Ohtsubo et al. 2000), but

is consistent with the study conducted by Federici et al.

(2012) which showed that different chlorinated PCBs were de-

graded to the same extent by the basidiomycetous fungus Len-

tinus tigrinus.

Conclusion

Our results based on GC analyses showed that the strains iso-

lated from a soil heavily contaminated by PCBs are tolerant

and likely to degrade these xenobiotics. The high rates of dis-

appearance of PCBs observed in the cultures of six strains sug-

gested a metabolization of PCBs by these fungi and their

ability to be used in bioremediation through bioaugmentation.

Our results are in line with other previous studies which have

reported a high potential of the autochthonous microflora in

bioremediation processes.

Acknowledgements

This research was financed by the Cluster Axelera (Pole de

comp�etitivit�e Chimie-Environnement Lyon & Rhone-Alpes).

The authors would like to thankMohamed Abdelghafour, Car-

ole Gaignaire, and JacquesMehu for their help on the GC-mass

analysis. We wish to thank Sophie P�erigon for her help con-

cerning the statistical analysis. We also really appreciated

the critical reading of the manuscript by Viviane Barbreau

and address our special thanks to Nael Mouhamadou for his

help.

r e f e r e n c e s

Abramowicz DA, 1990. Aerobic and anaerobic biodegradation ofPCB: a review. Critical Reviews in Biotechnology 10: 241e250.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990. Basiclocal alignment search tool. Journal of Molecular Biology 215:403e410.

Anderson MJ, 2001. A new method for non-parametric multivar-iate analysis of variance. Austral Ecology 26: 32e46.

April TM, Foght JM, Currah RS, 2000. Hydrocarbon-degrading fil-amentous fungi isolated from flare pit soils in northern andwestern Canada. Canadian Journal of Microbiology 46: 38e49.

Arx JA Von, 1981. The Genera of Fungi Sporulating in Pure Culture, 3rdedn. Cramer, Vaduz.

Baldrian P, 2008. Wood-inhabiting ligninolytic basidiomycetes insoils: ecology and constraints for applicability in bioremedia-tion. Fungal Ecology 1: 4e12.

Beaudette LA, Davies S, Fedorak PM, Ward OP, Pickard PA, 1998.Comparison of gas chromatography and mineralization ex-periments for measuring loss selected polychlorinated

274 B. Mouhamadou et al.

biphenyl congeners in cultures of white rot fungi. Applied andEnvironmental Microbiology 64: 2020e2025.

Bedard DL, Ritalahti KM, L€offler FE, 2007. The Dehalococcoidespopulation in sediment-free mixed cultures metabolicallydechlorinates the commercial polychlorinated biphenyl mix-ture aroclor 1260. Applied and Environmental Microbiology 73:2513e2521.

Chang YS, 2008. Recent developments in microbial biotransfor-mation and biodegradation of dioxins. Journal of Molecular Mi-crobiology and Biotechnology 15: 152e171.

D’Annibale A, Rosetto F, Leonardi V, Federici F, Petruccioli M,2006. Role of autochthonous filamentous fungi in bioremedi-ation of a soil historically contaminated with aromatic hy-drocarbons. Applied and Environmental Microbiology 72: 28e36.

Dercova K, Seligova J, Dudasova H, Mikul�a�sov�a M, �Silh�arov�a K,T�othov�a L, Hucko P, 2009. Characterization of the bottomsediments contaminated with polychlorinated biphenyls:evaluation of ecotoxicity and biodegradability. InternationalBiodeterioration and Biodegradation 63: 440e449.

Domsch KH, Gams W, Anderson TH, 1980. Compendium of SoilFungi. Academic Press, London.

Federici E, Giubilei M, Santi G, Zanaroli G, Negroni A, Fava F,Petruccioli M, D’Annibale A, 2012. Bioaugmentation of a his-torically contaminated soil by polychlorinated biphenyls withLentinus tigrinus. Microbial Cell Factories 23: 11e35.

Galzy P, Slonimski P, 1957. Variations physiologiques de la levureau cours de la croissance sur l’acide lactique comme seulesource de carbone. Comptes Rendus de l’Acad�emie des Sciences245D: 2423e2426.

Garon D, Sage L, Siegle-Murandi F, 2004. Effects of fungal bio-augmentation and cyclodextrin amendment on fluorine deg-radation in soil slurry. Biodegradation 15: 1e8.

Harms H, Schlosser D, Wick LY, 2011. Untapped potential: ex-ploiting fungi in bioremediation of hazardous chemicals. Na-ture Reviews Microbiology 9: 177e192.

Junghanns C, Krauss G, Schlosser D, 2008. Potential of aquaticfungi derived from diverse freshwater environments to de-colourise synthetic azo and anthraquinone dyes. BioresourceTechnology 99: 1225e1235.

Kamei I, Kogura R, Kondo R, 2006. Metabolism of 4,40-dichlorobi-phenyl by white-rot fungi Phanerochaete chrysosporium andPhanerochaete sp. MZ142. Applied Microbiology and Biotechnology72: 566e575.

Kruskal W, Wallis A, 1952. Use of ranks in one-criterion varianceanalysis. Journal of the American Statistical Association 47:583e621.

Kubatova A, Erbanova P, Eichlerova I, Homolka L, Nerud F,Sasek V, 2001. PCB congener selective biodegradation by thewhite rot fungi Pleurotus ostreatus in contaminated soil. Che-mosphere 43: 207e215.

Martino E, Turnau K, Girlanda M, Bonfante P, Perotto S, 2000. Er-icoid mycorrhizal fungi from heavy metal polluted soils: theiridentification and growth in the presence of zinc ions. Myco-logical Research 104: 338e344.

McArdle BH, Anderson MJ, 2001. Fitting multivariate models tocommunity data: a comment on distance-based redundancyanalysis. Ecology 82: 290e297.

McLachlan MS, 1996. Bioaccumulation of hydrophobic chemicalsin agricultural food chains. Environmental Science and Technol-ogy 30: 252e259.

Molitor C, Inthavong B, Sage L, Geremia RA, Mouhamadou B, 2010.Potentiality of the cox1 gene in the taxonomic resolution ofsoil fungi. FEMS Microbiology Letters 302: 76e84.

Ohtsubo Y, Nagata Y, Kimbara K, Takagi M, Ohta A, 2000. Ex-pression of the bph genes involved in biphenyl/PCB degrada-tion in Pseudomonas sp. KKS102 induced by the biphenyldegradation intermediate, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. Gene 256: 223e228.

Oksanen J, Kindt R, Legendre P, O’Hara B, Gavin L, 2009. vegan:Community Ecology Package. R Package Version 1.15-4.

Pant D, Adholeya A, 2007. Identification, ligninolytic enzyme ac-tivity and decolorization potential of two fungi isolated froma distillery effluent contaminated site. Water Air and Soil Pol-lution 183: 165e176.

Pieper DH, 2005. Aerobic degradation of polychlorinated biphe-nyls. Applied Microbiology and Biotechnology 67: 170e191.

Pieper DH, Seeger MJ, 2008. Bacterial metabolism of polychlori-nated biphenyls. Journal of Molecular Microbiology and Biotech-nology 15: 121e138.

Pinedo-Rilla C, Aleu J, Collado IG, 2009. Pollutants biodegradationby fungi. Current Organic Chemistry 13: 1194e1214.

Pointing SB, 2001. Feasibility of bioremediation by white rot fungi.Applied Microbiology and Biotechnology 57: 20e33.

Potin O, Rafin C, Veignie E, 2004. Bioremediation of an agedpolycyclic aromatic hydrocarbons (PAHs)-contaminated soilby filamentous fungi isolated from the soil. International Bio-deterioration and Biodegradation 54: 45e52.

Quensen III JF, Boyd SA, Tiedje JM, 1990. Dechlorination of fourcommercial polychlorinated biphenyl mixtures (Aroclors) byanaerobic microorganisms from sediments. Applied and Envi-ronmental Microbiology 56: 2360e2369.

Rojas-Avelizapa NG, Rodriguez-Vazquez R, Enriquez-Villanueva F, Martinez-Cruz J, Poggi-Varaldo HM, 1999.Transformer oil degradation by an indigenous microflora iso-lated from a contaminated soil. Resources, Conservation andRecycling 27: 15e26.

Ruiz-Aguilar GML, Fernandez-Sanchez JM, Rodriguez-Vazquez R,Poggio-Varaldo H, 2002. Degradation by white-rot fungi of highconcentrations of PCB extracted from a contaminated soil.Advances in Environmental Research 6: 559e568.

Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: molecularevolutionary genetics analysis (MEGA) software version 4.0.Molecular Biology and Evolution 24: 1596e1599.

Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: im-proving the sensitivity of progressive multiple sequencealignment through sequence weighting, position specific gappenalties and weight matrix choice. Nucleic Acids Research 22:4673e4680.

Thorn G, 1997. The fungi in soil. In: Modern Soil Microbiology.Marcel Dekker, pp. 63e127.

Tigini V, Prigione V, Di Toro S, Fava F, Varese GC, 2009. Isolationand characterization of polychlorinated biphenyl (PCB) de-grading fungi from a historically contaminated soil. MicrobialCell Factories 8: 5.

White TJ, Bruns T, Lee STJ, 1990. PCR Protocols: A Guide to Methodsand Applications.

Xing GH, Wu SH, Wong MH, 2010. Dietary exposure to PCBs basedon food consumption survey and food basket analysis atTaizhou, China e the World’s major site for recycling trans-formers. Chemosphere 81: 1239e1244.