Universidad Nacional de La Plata – Facultad de Ciencias Naturales y Museo – Biblioteca 

 
 
 
 

 
 
 
 
 
 
 
 
 
 

4ª Latin American Biodeterioration and Biodegradation 
Symposium 

Buenos Aires - AR, 16 al 20 de abril de 2001 
 

 

Disponible en: http://www.bfa.fcnym.unlp.edu.ar/bfa/bases/doc/index.html                      2011 



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40Latin American Biodelerioratwn andBiodegradatlon Symposill1n

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Filamentous soil fungi with enhanced pbenol utllization precultured

in hydrocarbon medium.

M.Ü.istina Romero, Ana M. Bucsinszky, M.Cecilia Cazau, Guillennina Massnccesi,

Gabriela B. In-azabal & Angélica M ArambalTi.

Instituto Spegazzini, Facultad de Ciencias Naturales y Museo - Universidad Nacional de La

Plata - Calle 53 n° 477 - 1900 La Plata - Argentina. (anmabarr@museo.fcnym.unlp. edu. ar).

Abstracl

Phenolic compounds, likc mono-, di-, poly-chlorophenols and nitrophenoIs, have been

c1assifiedas priority pollutants by thc EPA; so the degradation ability of phenol by filamcntous

soil fungi is relevant in the environmental biotcchnology area. Phenol-degrading fungi were

isolated from polluted sediments, and they were cultured on malt extract-agar and on aromatic

hydrocarbon-agar. Aspergillus terreus, Cladosporium herbarum, Cylindrocarpon didymum,

Fusarium oxysporum, Pestalohopisis spp., Penicillium spp., Phoma spp., Talaromyces helicus

and Tric.:-hodemIaharzianum were able to grow on phenol given in vapour phase. In most of

the cases the myeelia developecl on the agar in prcsence of phenol and spores were also

observecl in A. terreus, C. herbarnm, T. harzianum and Penicillium spp. strains. In these

assays an inercase in the phenol biodegradation were observcd when the strains were

preculturcd in aromatic hydroearbon-agar instead of using meclium with conventional carbon

sources. So, the presencc in contaminated habitats of other compounds, may improved the

degradation rate of phenolic polhitants.

Introduction.

The extensive use of phenolic rdatcd

compounds, likc mono-, di-, poly-

xenobiotics into the environment.. Many of

them are rccalcitrant becausc of their

chlorophenols and nitrophenols, as a resu]t

of rapicl industrial developments over the

chemical structw'e and these sites remain

past few decades, had produccd thc

deliberately or accidentally released of the

polluted for long periods (US. EPA, 1991;

Swannell et al., 1996).

Biorcmediation ofthese so11sis a considc-



....

rabIe challenge due to the complex mih'ture

of the pollutants and it must be rcmarkcd

that the standing stock of filamcntous fungí

in these habitats is about tenfold hígher

acclímation, in whích exposure to one

compound increase the metabolic rates of

another of similar structure; bcíng this

~L-1terials and metbods.

Diluted scdiment samples were assayed [or

the prcsence of hydrocarbon-utilizing

species aceording to Romero el al. (1998).

The isolated fungí were cultivate on agar-

plate with mineral basal salts (MB)

amended with pyrene as substrate and on

rnaIt extract-agar, adjusted to pH 7.0 with

HCI, and arnended wíth streptornycin (0.5

rng.rnl"l),theywere ineubatcdat 28 1:1f) e,

during 30 days. Sterile controIs of both

rnedium were sirnultaneously incubat.cd.

The filarnentous fungí werc identified by

morphology and eolony appearanee

through rnieroseopic observation. All the

ísolated fungi were tested for thcir abílity to

grow on phenol and n-hcxadceane as sole

carbon source, given vía vapour phase.

phenornenon attributed to thc broad

specificíty of the select.ed organisms and

the existenee of eommon pathways

eatabolism (Leahy & Colwell, 1990;

Brunsbach & Reineke, 1995). Therefore,

we focused our study on índígenous

filamentous soil fungí of contam inated

sites; and the aims were to isolate mycclíal

[ungí and to ~malyzethe capacity to grow

on phenol as carbon sourec.

The plates werc inoculated with a loop

with mycelial biomass precultured [or 48 h

on malt extract-agar, and on basal medium

plus pyrcne (MB+pyrene).

Then the inoculated agar-plates, without

any carbon put intosouree, were

cylindrical glass vesscls with covers, and a

smaU 25 mI vesscl containing 50 rng

phenol.

The substrate ll-hexadecane (0.4 mI) was

droppcd onto pieces of sterilizcd filter

papel' placcd in the lid of the petri dishes.

The assays were performed during 20

days, 28 1: 1f)e in darkncss, with weekJy

rcadings; at the end the rcsults wcrc

obtained by comparing the growth with the

negative control plates.

than bacteria (Yanagita, 1990).

N evertheless, numerous studíes had

provided evidence for fungí eross-



Results and discussion.

Twenty mycelial soil [ungi were isolated

from polluted sedlinents that received the

discharge of the Oil Refinery YPF- La

Plata, in aromatic hydrocarbon medium.

They identified as Aspergilluswere

lerreus, Cladosporium herbarum,

Cylindrocarpon didymwn, Fusarium

oxysporum (three strains), Geofrichillm

candidum, Penicillium spp. (two strains),

Peslalotiopisis spp., Phoma spp. (two

strains), Talaromyces helicus (three

strains), Trichoderma harzianum (four

strains); and a sterilia pigmented mycelium

was also cultivated.

Among the isolated [ungi, somc strains

were ablc to grow on phenol given m

vapour phase, and an mercase in the

phenol utilization was observed whcn the

strains were precultured in hy(b'ocarbon-

Trichoderma harzianum strains.

In the positive rcactions, the mycclia

developed onto the agar m presence of

phcnoL and in two Talaromyces heliclls

strains-plates, a red pigmented substance

was observed in the surface agar.

According to Hofrichter el al (1993) a

characteristic red colour was accumulated

in the medium when the phcnol mctabolitc,

4-nitrocatcchol, was detceted in the fungal

experiments.

On the other hand, sporcs germinated in

most of the fungal cultures, as in A.

terreus, C. herbarum, T. harzianum and

Penicillium spp. strains; sporulated eulturcs

were also observed with this substrate, but

with Penicillium spp. (Hofrichter el al.,

1993).

Thc growth bchaviour of the T. harzianum

strains must be stand out, as they

dcvelopccl a strong mycclia and mmlerous

spores on phenoJ, no matter the precuJture

m edium was.

Ansclmo el al. (1989) dcscribed the

growth of Fusarium floccifem,m and

Madhosnight (1958), F. oxysporum with

phenol and dinitrophcnot Seow el al.

(1990) researehed the mincralizarion of

phenol by Penicillium strams. A. niger ami

A. japunicus were studied by Shailubhai el

al. (1984) and Sahasrabudhe el aL (1987),

respectively.

In summary, the results presented in this

study, mentionated filamentous fungi

spccies able to grow on phcnol as carbon

source, not aIready cited by other

rcsearchers. Morcovcr, an inercase in the

adaptation to pollutants whcn somc spccics

agar instead of usrng m(dium with

conventional carbon sources, m the

Talaromyces helicus strains and



I!""

were precultivated in thc presence of

similar chemical structure was observed;

and this can be due to the lack of

specificity of the enzymes involvcd in the

phenol degradation (Hofrichter & Fritsche,

1996; Saparrat et al. 2000).

Altbough additional work is needed to

determine the significancc of thcsc

Acknowledgements

This work was supported by grants from the

Natianal Conncí1 of Scíentific and

Technological Research - CONICET amI from

Reference.~.

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degradation rates of phenolic pollutants.

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de Ciencias Naturales y Museo, UNLP),

Argentina.

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793-798.

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553-567.

by

I.

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~

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4°La/in American Biodeterloration and Biodegradation Symposium

Table 1: Experimetal results.( D negative~ [8] positive~ gm strong positive result; ph: phenoJ,

n-hex: hexadecane].

-lll~¡j¡rj¡rj¡[¡[¡jj

malt-a~ar

,j¡i...¡~¡¡lliL,-
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pbenol culturesFun~i species

Talnromyces lzeIicus (sfrain 1)

Talaromyces lzeIicus (strnin 2)

TalaromyceslteIicus (sfrain 3)

PltOma spp. (strain 1)

red colour mcclium

Phoma spp. (strain 2)

Geotric/zillm caltdidHm

Aspergillus terreus

ClD.dosporulm lterbarlU11
--------

PeJlidlliUI7l SI)P. (strain 1)

Penidlli1l11tspp. (sfrain 2)

Tric/zoderma hnrzianllm (sfrain 1)

Tric/zoderma I,arzialtum (sfrain 2)

TridlOderma harziattum (strain 3)

TridlOder11la ltarzianll11l (strain 4)

FlIsarium oxysporum (sfrain 1)

Fusarium oxysporu11l(strain 2)

Fllsarmm oxysponun. (sfrain 3)

Cy[jmlrocarpon didynmm

Pestalotiopisis spp.

mycella sterllla pigmented

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