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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambie. 26 (3) 193-199, 2010
FILAMENTOUS FUNGI REMOVE WEATHERED HYDROCARBONS FROM POLLUTED
SOIL OF TROPICAL MÉXICO
Beatriz PÉREZ-ARMENDÁRIZ
1,2
*, Daniel MARTÍNEZ-CARRERA
3
, María CALIXTO-MOSQUEDA
1,4
,
Joel ALBA
1
and Refugio RODRÍGUEZ-VÁZQUEZ
1
1
Laboratorio de Compuestos Xenobióticos, Departamento de Biotecnología y Bioingeniería, Centro de Inves-
tigación y de Estudios Avanzados, Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional 2508, San
Pedro Zacatenco, Delegación Gustavo A. Madero, México, D. F. 07360
2
Centro Interdisciplinario de Posgrados, Investigación y Consultoría, Universidad Popular Autónoma del
Estado de Puebla, 21 Sur 1103 Col. Santiago. CP 72160, Puebla, México. (+52) 222-229 94 00 ext 7527.
beatriz_pereztij@yahoo.com.mx
3
Biotecnología de Hongos Comestibles, Colegio de Postgraduados en Ciencias Agrícolas (COLPOS), Campus
Puebla, Apartado Postal 701, Puebla 72001, Puebla, Mexico
4
Departamento de Bioprocesos, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico
Nacional, Av. Acueducto s/n, México, D.F. 07340
*
Postdoctoral position supported by the Consejo Nacional de Ciencia y Tecnología in México and supervised
by D. Martínez-Carrera
(Recibido junio 2009, aceptado enero 2010)
Key words:
Aspergillus
, bioaugmentation,
Cladosporium
, ±lamentous fungi,
Penicillium
, total petroleum
hydrocarbons (TPH) removal, tropical Mexico
ABSTRACT
Weathered hydrocarbons from worldwide petrolic activities become more recalcitrant
over time. The removal of petroleum hydrocarbons from a polluted soil [65,000 mg
total petroleum hydrocarbons (TPH)/kg soil], which had been exposed to tropical en-
vironmental conditions for more than 20 years in southeast Mexico, was studied using
±lamentous fungi. Experiments were carried out in batch reactors (60 mL) containing a
substrate consisting of polluted soil and sugar cane bagasse pith as bulk agent (80:20,
w/w). Sterile and non-sterile batch reactors were inoculated with spore suspensions
from
Aspergillus niger
,
Penicillium glabrum
, and
Cladosporium cladosporioides
.
The TPH were determined at the beginning and at the end of the experiments, and the
CO
2
production and accumulation were monitored by gas chromatography. All fungal
species studied were associated to the removal of TPH, either on sterile or non-sterile
treatments. A bioaugmentation process was observed due to the synergistic effect of
C. cladosporioides
and well-adapted indigenous microbial populations from the con-
taminated soil, as the highest removal of TPH (78.5%) and CO
2
accumulation (14.3%)
were recorded in this non-sterile treatment. By contrast, the lowest TPH removal was
recorded in the same species, but in the sterile treatment (62.3%) showing that the ab-
sence of adapted indigenous microbiota signi±cantly reduced fungal metabolism (CO
2
accumulation: 9.1%), as well as the removal of TPH. Patterns of CO
2
accumulation and
TPH removal in other treatments suggested interspeci±c competence between fungal
species and the adapted indigenous microbiota.
B. Pérez-Armendáriz
et al
.
194
Palabras clave:
Aspergillus
, bioaumentación,
Cladosporium
, hongos flamentosos,
Penicillium
, remoción de
hidrocarburos totales del petroleo (HTP), trópico mexicano.
RESUMEN
Los hidrocarburos intemperizados de las actividades petroleras son más recalcitrantes
a medida que transcurre el tiempo. Se estudió la remoción por hongos flamentosos de
los hidrocarburos de petróleo de un suelo contaminado [65,000 mg de hidrocarburos
totales de petróleo (HTPs)/kg de suelo], el cual había estado expuesto a condiciones
ambientales tropicales por más de 20 años en el sureste de México. Los experimentos
se llevaron a cabo en reactores de lote (60 mL), los cuales contenían un sustrato a base
de suelo contaminado y bagacillo de caña de azúcar como agente texturizante (pro-
porción 80:20, peso/peso). Se inocularon reactores de lote, estériles y no estériles, con
suspensiones de esporas de
Aspergillus niger
,
Penicillium glabrum
, y
Cladosporium
cladosporioides
. Los HTP se determinaron al principio y al fnal de los experimentos,
mientras que la producción y acumulación de CO
2
se determinó por cromatograFía
de gases. Todas las especies Fúngicas estudiadas estuvieron asociadas a la remoción
de HTP, tanto en tratamientos estériles como no estériles. Se observó un proceso de
bioaumentación debido al eFecto sinérgico entre
C. cladosporioides
y la poblaciones
microbianas nativas bien adaptadas del suelo contaminado, ya que la remoción más
alta de HTP (78.5%) y mayor acumulación de CO
2
(14.3%) se registraron en este tra-
tamiento no estéril. En cambio, la remoción más baja de HTP se observó en la misma
especie, pero en el tratamiento estéril (62.3%), demostrando que la ausencia de micro-
biota nativa adaptada redujo signifcativamente el metabolismo Fúngico (acumulación
de CO
2
: 9.1%), así como la remoción de HTP. Los patrones de acumulación de CO
2
y la remoción de HTP en los otros tratamientos pueden relacionarse con competencia
interespecífca entre el hongo flamentoso y la microbiota nativa adaptada.
INTRODUCTION
Petrolic activities have generated extensive pol-
lution oF soils worldwide, mainly in those regions
where petroleum is explored, extracted, and refned.
The composition oF hydrocarbons on polluted soil
varies according to environmental conditions and
natural degradation processes. Weathered hydro-
carbons, which are predominantly saturated and
aromatic, become more recalcitrant iF polluted soils
are not remediated, aFFecting underground water,
Food chains, and diverse human activities (e.g.,
agriculture, cattle raising, silviculture) (Atlas and
Bartha 1998).
Bacteria and Fungi have been used For degradation
and removal oF hydrocarbons, as they are capable oF
producing signifcant amounts oF eFfcient enzymes
(Saraswathy and Hallberg 2002, Chávez-Gómez et
al. 2003, Moody et al. 2004, Genovese et al. 2008).
±ilamentous Fungi, commonly Found on lignocel-
lulosic substrates, produce extracellular enzymes oF
low specifcity, which can degrade diFFering recalci-
trant compounds, such as hydrocarbons, resins and
asphaltens (Tortella et al. 2005). Important enzymes
used For bioremediation are lignin peroxidase, man-
ganese-dependent peroxidase, and laccase (Evans
and Hedger 2001).
Most Fungal research work on bioremediation
has been Focused on the degradation oF specifc
hydrocarbons using a variety oF substrates, showing
diFFerent levels oF pollutant removal (Hammel et
al. 1986, Gramss et al. 1999, Novotny et al. 1999,
Cortés-Espinosa et al. 2006, Leonardi et al. 2007,
Genovese et al. 2008). Mineralization rates by Fungal
enzymes ranging From 25-93% have been reported
For polycyclic aromatic hydrocarbons (PAH) dur-
ing a period oF 8-15 weeks. In a previous research
work, it was shown that the removal oF weathered
hydrocarbons From polluted soil can be improved
synergistically by microorganisms From sugar cane
bagasse pith, reaching up to 60% removal oF total
petroleum hydrocarbons (TPH) (Pérez-Armendáriz
et al. 2004). In this study, it was assessed the eFFect oF
bioaugmentation using three species oF flamentous
Fungi on the removal oF hydrocarbons From real pol-
luted soil exposed to tropical environmental condi-
tions For more than 20 years in the State oF Tabasco,
southeast México.
FUNGAL BIOAUGMENTATION REMOVES HYDROCARBONS FROM TROPICAL SOIL
195
MATERIALS AND METHODS
Fungal strains
The flamentous ±ungi studied were
Cladosporium
cladosporioides
(Fresen.) G.A. de Vries,
Penicillium
glabrum
(Wehmer) Westling, and
Aspergillus niger
Tiegh. These species were previously isolated ±rom
sugar cane bagasse pith, and identifed by molecu-
lar analysis (Cortés-Espinosa et al
.
2006). Selected
strains were grown on minimal medium: g/L: 7,
(NH
4
)
2
SO
4
; 5.7, K
2
HPO
4
; 2, KHPO
4
; 2, MgSO
4
;
15, agar; 1,000 ml distilled water, and preadapted to
a petroleum environment using Mayan petroleum
provided by the Mexican State company (PEMEX).
Mayan petroleum was added on sterilized flter paper
(3 cm
2
;
ca.
2 g petroleum) to every lid in order to de-
velop an atmosphere o± volatile hydrocarbons inside
the petri dish. Bacterial growth was inhibited adding
3.5 ml/L o± rose Bengal (Sigma-Aldrich, USA) and
40
m
g/mL o± streptomycin (Sigma-Aldrich, USA).
Sterile petri dishes containing potato dextrose agar
(PDA, Bioxon, México) as culture medium were
inoculated with strains preadapted, and incubated
±or 5 days at 28 ºC ±or mycelial growth and spore
production.
Batch reactors
A soil sample was obtained ±rom a petroleum well
located in the tropical State o± Tabasco, southeast o±
México. The soil sample (ca. 20 kg) was placed in a
dark glass container and kept at 4 ºC ±or transporta-
tion. In the laboratory, the soil was exposed to the sun
±or a ±ew hours, homogenized manually ±or 15 min,
and characterized as loamy sand (silt 4 %, sand 96 %)
having a moisture content o± 2 %, an organic matter
content o± 8.3 %, and a pH o± 7.3. The concentration
o± total petroleum hydrocarbons (TPH) in the polluted
soil was o± 65 000 mg/kg, as the well ±rom which the
sample was taken had been exposed to diesel contami-
nation ±or more than 20 years. Sugar cane bagasse
pith (a lignocellulosic byproduct ±rom the local sugar
industry) was sterilized at 121 ºC ±or 20 min and it
was used as solid support and carbon source in all
experiments. This pith was characterized as ±ollows:
pH 4.5, density 0.8 g/cm
3
, moisture content 80 %,
total sugars 2.23 mg glucose/g (Rodríguez-Vázquez
et al
.
1999). Six di±±erent treatments were carried out
as shown in
table I
. A non-sterile soil sample was
used directly ±or part o± the experiment, whereas
the soil sample used in the other experimental part
was autoclaved three times (every 24 h) at 121 ºC
±or 15 min, according to previous research (Pérez-
Armendáriz et al. 2004, Cortés-Espinosa et al. 2006).
Two controls, involving either sterile or non-sterile
soil, were included. The batch reactors were glass
serum bottles (60 ml) containing 16 g o± soil, either
sterile or non sterile, and 4 g o± sterile sugar cane
bagasse pith. The moisture content was adjusted to
60 %, using sterile distilled water. The C/N/P ratio
was 100/10/1 using a sterile water solution o± 1 N
NH
4
SO
4
(Sigma-Aldrich, USA) and 1 N K
2
HPO
4
(Sigma-Aldrich, USA) as nitrogen and phosphorus
sources. Fungal inocula ±rom strains studied were
prepared on PDA plates. Spores were harvested
adding 2.5 ml o± tween 80 (Sigma-Aldrich, USA)
in order to obtain a sterile solution at a standardized
concentration. Every reactor was inoculated with 1
mL o± the spore suspension (inoculum at 2
x
10
7
spores/mL). Each bottle was capped with a Te²on
septum. The atmosphere o± reactors was exchanged
every two days, injecting an air ²ow through a sterile
flter (millipore 0.2 mm) ±or 15 min. All experiments
were carried out in triplicate. Residual TPH were
determined a±ter 25 days o± incubation at 25 ºC
(Chávez-Gómez et al. 2003, Pérez-Armendáriz et
al. 2004, Cortés-Espinosa et al. 2006).
Sample analysis
Microbial activity
. It was monitored every two
days, be±ore each atmosphere exchange, taking an
TABLE I.
EXPERIMENTAL DESIGN AND TREATMENTS STUDIED
Code
Soil treatment
Fungal species
Substrate (ratio 80:20, w/w)
T1
Non sterile
Aspergillus niger
Polluted soil + pith*
T2
Sterile
A. niger
Polluted soil + pith
T3
Non sterile
Penicillium glabrum
Polluted soil + pith
T4
Sterile
P. glabrum
Polluted soil + pith
T5
Non sterile
Cladosporium cladosporioides
Polluted soil + pith
T6
Sterile
C. cladosporioides
Polluted soil + pith
T7
Control 1 (non sterile)
-
Polluted soil + pith
T8
Control 2 (sterile)
-
Polluted soil + pith
* The sugar cane bagasse pith was sterile in all treatments and controls.
B. Pérez-Armendáriz
et al
.
196
air sample of 5 µL from the headspace of batch
reactors using a sterile syringe, which was injected
into the gas chromatograph (Gow-Mac 580, USA)
having a thermal conductivity detector. Data were
expressed as percent of CO
2
produced and accumu-
lated (Rodríguez-Vázquez et al. 1999).
Total petroleum hydrocarbons (TPH)
. The con-
tent of TPHs was determined in three samples at
the beginning and at the end of the experiments, as
previously described (Pérez-Armendáriz et al
.
2004).
Samples were taken for analysis at day 0 (initial soil
sample) and at day 25. The total content of each batch
reactor was homogenized by thorough manual mixing
for 10 min. A sample (2 g) was taken from the mix,
and dried at room temperature for 24 h. Hydrocarbons
were extracted for 8 h with 80 mL of dichloromethane
(Merck, USA) using Soxhlet. Dichloromethane phase
was performed in a rotary evaporator, adding 2 g
of anhydrous Na
2
SO
4
(Sigma-Aldrich, USA). Dry
samples were dissolved in 10 mL of dichlorometh-
ane, taking an aliquot of 500 µL to be dried at room
temperature and dissolved in 5 mL of CCl
4
(Merck,
USA). Residual hydrocarbons were determined fol-
lowing the modiFed EPA 418.1 method (USEPA
1979). Total petroleum hydrocarbons were assessed
by infrared spectroscopy (Buck ScientiFc, model HC-
404, USA), using 2930 cm
-
1
absorbance, speciFc for
C-H bonds. Samples were analyzed by interpolation
in a calibration plot.
Statistical analysis
Data were expressed as means ± standard devia-
tions. One way analysis of variance and ² test, at a
level of signiFcance of P<0.05, were carried out in
order to determine signiFcant differences among
the means. Multiple comparisons among treatment
means were made using the Tukey’s test. All data
were analyzed on Minitab Ltd. statistic software.
RESULTS AND DISCUSSION
The non-sterile control T7 showed the highest
CO
2
production and accumulation (24.0 %) of all
treatments, followed by the non-sterile treatment
T5 (14.3 %) (
Table II
,
Fig. 1
). This indicated the
presence of well-adapted indigenous microbial popu-
lations in the contaminated soil of T7, which were
even biostimulated by the addition of nitrogen and
phosphorus sources and thus they were responsible
for the removal (61.5%) of TPH (
Table II
). In T5,
it can be seen a bioaugmentation process due to the
synergistic effect of the Flamentous fungus
Clado-
sporium cladosporioides
and the adapted indigenous
microbiota. This was conFrmed by the highest TPH
removal (78.5 %) of all treatments recorded in T5
(
Table II
). It is possible that a longer incubation of T5
would have led to further development of
C. cladospo-
rioides
and the indigenous microbiota, and accordingly
to a higher production of CO
2
, biomass, and enzymes
(D’Annibale et al
.
2006). The lowest TPH removal of
all treatments was recorded in the same species, but
on the sterile treatment (T6, 62.3 %) showing that the
absence of adapted indigenous microbiota signiFcantly
reduced CO
2
production and accumulation (9.1 %),
as well as the removal of TPH. The bioaugmentation
process is important considering the high level of
initial pollution found in the soil studied (65 000 mg
TPH/kg soil), which had been weathered for more
than 20 years. The possible effect of soil sterilization
on these results will be matter for further research.
Very low levels of CO
2
production and accumula-
tion (1.5 %), as well as TPH removal (16.0 %), in the
sterile control T8 may be associated to the presence
of reproductive structures from adapted indigenous
microbiota capable of resisting sterilization pro-
cesses, which also affect substrate (polluted soil +
sugar cane bagasse pith) structure and composition
(Sylvia et al
.
1999).
The comparison of sterilized and non-sterilized
treatments showed variation according to each spe-
cies (
Table II
,
Fig. 1
). In the case of
Aspergillus
niger
, the CO
2
accumulation (T2, 9.9 %) and the
TPH removal (T2, 76.3 %) on the sterile treatment
were higher than those on the non-sterile treatment
containing adapted indigenous microbiota (T1: CO
2
,
3.4 %; TPH, 64.6 %). Similar results were obtained
in
Penicillium glabrum
for CO
2
accumulation and
TPHs removal, which were higher on the sterile
treatment (T4: CO
2
, 8.9 %; TPH, 72.5 %). Higher
values for CO
2
accumulation and TPH removal
on sterile treatments suggested some degree of in-
terspeciFc competence between Flamentous fungi
studied (
A. niger
,
P. glabrum
) and adapted indigenous
microbiota. Competence has also been reported
for
Phanerochaete chrysosporium
and indigenous
microorganisms (²ernández-Sánchez et al. 2001).
By contrast, the removal of TPHs on the non-sterile
treatment containing
Cladosporium cladosporioides
and adapted indigenous microbiota (T5, 78.5 %)
was higher than that on the sterile treatment (T6,
62.3 %). In this case, the CO
2
production and ac-
cumulation were also higher on the non-sterile treat-
ment (T5, 14.3 %) than on the sterile treatment (T6,
9.1 %), which indicated the presence of interspeciFc
FUNGAL BIOAUGMENTATION REMOVES HYDROCARBONS FROM TROPICAL SOIL
197
TABLE II.
THE REMOVAL OF TOTAL PETROLEUM HYDROCARBONS (TPH) AND CO
2
ACCUMULATION FROM DIFFE-
RENT TREATMENTS STUDIED, USING POLLUTED SOIL AND SUGAR CANE BAGASSE PITH AS SUBSTRATE.
THE INITIAL TPHs CONCENTRATION OF THE POLLUTED SOIL SAMPLE WAS 65 000 mg/kg
Code
Soil treatment
Fungal species
Residual
TPH
1,2
(mg/kg)
SD
(mg/kg)
Proportion of
TPH removal
(%)
Final
CO
2
3
(%)
SD
(%)
T1
Non sterile
Aspergillus niger
22,966.6
b
±2,282.7
64.6
3.4
e
±0.58
T2
Sterile
A. niger
15,383.3
a
±2,089.4
76.3
9.9
c
±0.15
T3
Non sterile
Penicillium glabrum
22,750.0
b
±650.0
65.0
5.4
d
±0.24
T4
Sterile
P. glabrum
17,875.0
a
±2,298.1
72.5
8.9
c
±0.14
T5
Non sterile
Cladosporium cladosporioides
13,975.0
a
±1,378.8
78.5
14.3
b
±0.03
T6
Sterile
C. cladosporioides
24,483.3
b
±1,635.8
62.3
9.1
c
±0.24
T7
Control 1 (non sterile)
-
25,025.0
b
±459.6
61.5
24.0
a
±0.19
T8
Control 2 (sterile)
-
54,600.0
c
±919.2
16.0
1.5
f
±0.49
1
Residual TPH were assessed after 25 days of incubation
2
TPH mean value (n=3). Means with different letters are signi²cantly different (P<0.05)
3
Final value for the CO
2
produced and accumulated (n=3). Means with different letters are signi²cantly different (P<0.05)
SD= Standard deviation
Fig. 1
. Metabolic activity as CO
2
production and accumulation (%) in soil treatments studied, including non-sterile (T7) and sterile (T8)
controls. A:
Aspergillus niger
on non-sterile soil (T1) and
A. niger
on sterilized soil (T2). B:
Penicillium glabrum
on non-sterile
soil (T3) and
P. glabrum
on sterilized soil (T4). C:
Cladosporium cladosporioides
on non-sterile soil (T5) and
C. cladosporioides
on sterilized soil (T6)
30
a)
b)
c)
25
20
15
5
5
CO
2
produced and accumulated (%)
0
0
10
10
Time (days)
T1
T2
T7
T8
15
20
25
30
30
25
20
15
5
5
CO
2
produced and accumulated (%)
0
0
10
10
Time (days)
T3
T4
T7
T8
15
20
25
30
30
25
20
15
5
5
CO
2
produced and accumulated (%)
0
0
10
10
Time (days)
T5
T6
T7
T8
15
20
25
30
B. Pérez-Armendáriz
et al
.
198
co-metabolism between the flamentous Fungus and
adapted indigenous microbiota. Previous research has
shown that a closely related species,
Cladosporium
sphaerospermum
, which was isolated From aged
soil oF a gas manuFacturing plant, was capable oF
degrading several PAH in Four weeks using a non-
sterile soil model (Potin et al
.
2004). This ability to
degrade hydrocarbons is due to the concerted action
oF extracellular and metabolic enzymes produced
during
Cladosporium
growth.
An advantage oF using experimental models
based on real polluted soil is that the eFfciency oF
a Fungal species, such as
A. niger
,
P. glabrum
and
C. cladosporioides
, is directly tested against well-
adapted indigenous microbial populations. This is
in contrast with those species which are useFul For
bioremediation under laboratory conditions, but not
eFfcient and poorly competitive under natural condi-
tions where population development is aFFected by
many Factors, e.g.,
Phanerochaete chrysosporium
(Lindley and Heydeman 1985, Meulenberg et al
.
1997, ±ernández-Sánchez
et al
.
2001, Potin et al
.
2004). This research showed that
A. niger
,
P. gla-
brum
and
C. cladosporioides
are flamentous Fungi
with good potential For bioremediation processes in
tropical regions oF Latin America. They are capable
oF removing and degrading recalcitrant hydrocarbons
through bioaugmentation and biostimulation, using
sugar cane bagasse pith as bulking agent. However,
they show diFFering responses when conFronted with
indigenous microbiota. ±urther studies are needed
to understand the biological interactions between
flamentous Fungi and indigenous microbiota.
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