Evaluation of Support Materials for Immobilization of Pycnoporus sanguineus Mycelia for Laccase Production and Biodegradation of Polycyclic Aromatic Hydrocarbons
Jaime Y.S. Low,
The ability of white rot fungus Pycnoporus sanguineus
to colonize support materials and subsequently produce laccase and degradation
of Polycyclic Aromatic Hydrocarbons (PAHs) was compared with free mycelia
culture. Natural support, Ecomat, was found to be the best support material
for P. sanguineus for mycelial colonization and laccase activity
with maximum activity of 39 nkat mL-1 on day nine of incubation.
Coconut husk and grey scouring sponge produces maximum laccase activity
of 9.17 and 6.67 nkat mL-1, respectively on day 15. Pycnoporus
sanguineus immobilized culture exhibited higher PAHs degradation efficiency
compared to the free mycelia culture during the 20 days of incubation.
The immobilized mycelia culture degraded 88% of phenanthrene, 93% of anthracene
and 85% of pyrene within 20 days. The good correlation between the amount
of PAHs degraded and laccase activity produced in the immobilization medium
indicated that laccase was solely responsible for degradation of the three
PAHs tested. In comparison, free mycelia culture rapidly degraded 42%
of phenanthrene, 92% of anthracene and 87% of pyrene at the cessation
of incubation. However, poor correlation between the amount of PAHs degraded
and laccase activity measured in the free cell culture was obtained. This
suggested that intracellular enzymes could be involved in PAHs degradation
in the free cell culture.
to cite this article:
Jaime Y.S. Low, Noorlidah Abdullah and S. Vikineswary, 2009. Evaluation of Support Materials for Immobilization of Pycnoporus sanguineus Mycelia for Laccase Production and Biodegradation of Polycyclic Aromatic Hydrocarbons. Research Journal of Environmental Sciences, 3: 357-366.
Polycyclic Aromatic Hydrocarbons (PAHs), also known as polynuclear aromatic
hydrocarbons or polyarenes, constitute a large class of organic compounds (Gomez
et al., 2006). PAHs are highly water insoluble and are widespread
pollutants in freshwater and seawater, particularly in estuaries and coastal
waters with pollution from petroleum, coal or other heavy industries (Maldonado
et al., 1999; Mitra et al., 1999).
Ligninolytic fungi, which colonize wood and other ligninocellulosic materials,
are abundant in nature and have gained considerable attention for their bioremediation
potential since the enzymes that are involved in lignin breakdown can also degrade
a wide range of pollutants (Bumpus et al., 1985).
These fungi which includes Phanerochaete chrysosporium (Brodkorb
and Legge, 1992), Trametes versicolor (Field et
al., 1992), Bjerkandera sp. strain BOS55 (Field
et al., 1995) and Pleurotus ostreatus (Bezalel
et al., 1997) have been extensively studied for their ability to
degrade PAHs. Lignin peroxidase (LiP), manganese-dependent peroxidase (MnP),
manganese-independent peroxidase and laccase are the enzymes that have been
implicated in the oxidation of PAHs (Brodkorb and Legge,
1992; Bogan and Lamar, 1996; Pickard
et al., 1999). These extracellular enzymes have low substrate specificity
and diffuse into the soil matrix where the PAHs are entrapped.
Immobilization of microorganisms can be defined as any technique that limits
the free migration of cells. There are two types of cell immobilization: entrapment
and attachment. In the cell 45822_ja., 2004). Alginate,
chitosan, chitin and cellulose derivatives have been used as matrix in the entrapment
technique for cell immobilization (Arica et al., 1993).
While in the cell attachment technique, synthetic foams like polyurethane foams
(Yang and Yu, 1996), nylon sponges, natural materials
such as jute, straw and hemp (Shin et al., 2002)
was commonly employed for the attachment procedure. Immobilization by entrapment
in a matrix such as alginate, chitosan and chitin may be too costly while surface
immobilization via attachment on inexpensive materials such as synthetic sponges
and natural materials are cheaper. Rodriguez Couto et
al. (2004) had shown that the entrapment technique was not suitable
for the immobilization of Trametes hirsuta, whereas the attachment technique
using the stainless steel sponge as the support gave better results. Immobilized
cultures tend to have a higher level of enzymes activity and were more resilient
to environmental perturbations such as pH or exposure to toxic chemicals compared
to free cell culture (Shin et al., 2002).
Mycelial immobilization has been used for the production of ligninolytic enzymes
and bioremediation of pollutants such as chlorophenols (Sedarati
et al., 2003), textile dyes (Ziegenhagen and Hofrichter,
2000) and mercury (Arica et al., 1993), but
has not been applied to the biodegradation of PAHs. Thus, in this study, the
immobilization of Pycnoporus sanguineus (Linn. Ex Fr.) Murrill strain
CY788 to produce high laccase activity for PAHs degradation was evaluated.
MATERIALS AND METHODS
The study of the effect of various types of support materials for P.
sanguineus immobilization, laccase production and PAHs degradation
was conducted in 2004.
Pycnoporus sanguineus (Linn. ex Fr.) Murrill strainCY788 isolated
in Thailand was maintained in 30% (v/v) glycerol at 4°C. The stock
was inoculated and re-stimulated by growing on sterilized wood chip placed
on Potato Dextrose Agar (PDA) plates.
The synthetic sponges and natural support materials were shown in Fig.
1. The chemical structures of the support materials were not studied. Selection
of support materials was made based on literature (Rodriguez
Cuoto et al., 2004) and the texture and porosity of the material,
which encouraged colonization of mycelium onto the support. Synthetic sponges
such as grey scouring sponge (Crocodile), white sponge (Scotch Brite), green
scouring pad (Scotch Brite), yellow nylon pad (Tono), soft sponge (Scotch Brite)
and nylon mesh (Scotch Brite) were selected. The synthetic supports pre-treatment
method was as described in Rodriguez Cuoto et al.
(2004). The natural support materials evaluated in this study were Ecomat
(made of ligninocellulolytic fibres of empty fruit bunches of oil palm) obtained
from Ecofibre Technology and coconut husk. Ecomat was cut into cubes and irregular
sized shreds of coconut husk were dried overnight at 65°C in the oven and
cooled in the desiccator before the dry weights were taken.
|| Synthetic and natural support materials selected for the
immobilization of P. sanguineus; (A) Grey scouring sponge, (B) White
sponge, (C) Green scouring pad, (D) Yellow nylon pad, (E) Soft sponge, (F)
Nylon mesh, (G) Ecomat and (H) Coconut husk
|| Ten days old fungal mycelial of immobilized P. sanguineus
in GYMP medium (control) and medium exposed to 10 ppm PAHs
The culture medium was Glucose-Yeast-Malt-Peptone (GYMP), containing,
gram per litre, MgSO4.7H2O, 0.5; KH2PO4,
0.46; K2HPO4, 1.0; glucose, 20.0; peptone, 2.0;
yeast extract, 2.0 and malt extract, 2.0. Two milliliters of 2% (v/v)
P. sanguineus mycelial suspension was inoculated into 250 mL Erlenmeyer
flask containing sterile 100 mL GYMP medium and the support material under
aseptic condition. The flasks were incubated at 150 rpm at 29±2°C
for 15 days. Mycelia of P. sanguineus was found to colonize the
surface and within the porous matrix of Ecomat (Fig. 2).
Immobilized cultures were filtered through Whatman filter paper. The
immobilized mycelia were washed twice with distilled water to remove mycelium
which was not colonized on the surface of the support material and oven
dried at 65oC until a constant weight was obtained (3-4 days).
Laccase Activity Assay
Laccase activity in the culture medium during immobilization of P. sanguineus
on the selected support materials and free mycelia culture were assayed at intervals
of three days for 15 days. Laccase activity was measured spectrophotometrically
at 525 nm (Harkin and Obst, 1973). One unit (U) of enzyme
activity was defined as the amount of enzyme producing 1OD unit in 1 mL of the
culture medium after one minute. For conversion, 1U is equivalent to 16.67 nkat
Biodegradation of PAHs
The culture medium of 3-day-old immobilized and free cell culture
was decanted and incubated with 100 mL sterile GYMP medium containing
PAHs (anthracene, phenanthrene and pyrene) at 10 ppm final concentrations
of each PAH. Cultures were incubated for 20 days.
Extraction of PAHs from Liquid Cultures
Residual PAHs in the medium was extracted with 20 mL of dichloromethane
and shaken vigorously for 5 min in a 250 mL-separatory flask. The lower
dichloromethane layer was collected and evaporated to dryness. The dried
extract was then re-dissolved in 1 mL of acetonitrile and analyzed.
Analysis of PAHs
Samples were analyzed by high performance liquid chromatography (Spectra-Physics
HPLC system). The column (150x4.6 mm ID) packed with Prosphere 300A PAH
(5 μ particles) and coupled with guard column was used for PAHs
separation. The flow rate was set at 1.5 mL min-1. Samples
were eluted isocratically with an eluent consisting 38/62 (% v/v) water/acetonitrile.
The PAHs were detected at 254 nm with a UV detector. Product inhibition
studies were not conducted as degradation of PAHs was rapid and laccase
activity was maintained at high levels throughout the whole incubation
period, which suggested the by-products were not inhibitory to P.
sanguineus and inhibit the biodegradation rate.
Growth of Free Mycelia and Immobilized Mycelia Culture
Figure 3 shows the dry weight increase in mycelial
biomass of free mycelia culture and immobilized mycelia culture on different
support materials. The growth in free mycelia culture of P. sanguineus
was visible two days after inoculation. Fungal biomass as tiny pellets
at day three was 188 mg and steadily increased to a maximum of 250 mg
at day nine of incubation.
All support materials, except the Scotch Brite nylon mesh, facilitated
mycelia colonization during the 15 days incubation. Pycnoporus sanguineus
grew well on the synthetic supports without colour leaching from the supports
and there was no loss in the integrity of the supports over a two-week
period. The dry weight of biomass increased rapidly and maximum biomass
concentration was obtained on day nine, after which the biomass level
declined. Among the synthetic support materials selected in this study,
there was good mycelial growth on grey sponge with highest dry mycelial
biomass of 298 mg on day nine of incubation. This was followed by soft
sponge, yellow nylon pad, white sponge, green scouring pad and antibiotic
|| Comparison of mycelial biomass of P. Sanguineus
in immobilized and free cell cultures. Results are expressed as the mean
of triplicate values
In contrast, natural support materials showed better colonization by
P. sanguineus compared to synthetic support materials. The mycelial
biomass was well attached to the support materials and the supports stayed
intact throughout the incubation period. The culture grown on Ecomat and
coconut husk gave higher mycelial biomass dry weights of 497 and 554 mg,
respectively on day nine of incubation.
Laccase Activity of Free Mycelia and Immobilized Mycelia Cultures
on Selected Support Materials
Previously, higher biomass was obtained in cultures immobilized on
grey scouring sponge, Ecomat and coconut husk. Immobilization of mycelia
on these support materials were further evaluated for the production of
laccase and compared with free cell cultures. Table 1
shows the laccase activity of the free mycelia and immobilized mycelia
cultures measured during the 15 days of incubation. Laccase activity was
assayed on free mycelia culture and immobilized mycelia on grey sponge,
Ecomat and coconut husk. The onset of laccase activity occurred after
three days of incubation in free cell culture. Rapid laccase production
only occurred after day six and attained its maximum activity of 7.63
nkat mL-1 on day nine of incubation. Comparatively, it was
shown that optimum laccase production in free mycelia culture at day nine
was 27 folds and 1.4 fold higher than cultures immobilized on grey sponge
and coconut husk, respectively. In contrast, laccase activity obtained
in free mycelia culture at day nine was five folds lower than immobilized
culture on Ecomat. Thus, Ecomat was selected as the immobilization support
for P. sanguineus for further studies of PAHs degradation.
Biodegradation of PAHs by P. sanguineus
All three PAHs studied in this study registered sharp exponential
decline in concentration in the free mycelia culture after two days of
incubation (Fig. 4). It was found that phenanthrene,
anthracene and pyrene were efficiently degraded, with relative removals
of over 13, 64 and 58%, to give final concentrations of 8.62, 3.55 and
4.15 ppm, respectively after two days of incubation. Anthracene was degraded
most rapidly by the free mycelia culture of P. sanguineus with
relative removal of over 92%, closely followed by pyrene and phenanthrene,
with relative removal of over 87 and 42%, respectively, within 20 days
of incubation. Their corresponding final concentrations were 5.74, 0.74
and 1.28 ppm for phenanthrene, anthracene and pyrene, respectively, at
the cessation of incubation. Concomitant to the rapid PAHs degradation,
laccase activity in free mycelia culture increased during the whole incubation
period. However, the increase in degradation of PAHs by P. sanguineus
free cell culture was not proportionate with the increase of laccase
activity. Therefore, there were relatively low correlations between degradation
of these PAHs and laccase activity (r2<0.67). This indicated
that laccase may not be solely responsible for PAHs degradation in free
|| Comparison of laccase production by P. sanguineus
immobilized on grey scouring sponge, Ecomat, coconut husk and free cell
|Means of triplicates±SD
||Percentage of PAHs degraded during 20 days of incubation with
P. sanguineus free cell culture. Results are expressed as the mean of triplicate
|| Percentage of phenanthrene degraded during the 20 days of
incubation with immobilized culture P. sanguineus. Results are expressed
as the mean of triplicate values
The immobilized P. sanguineus culture proved to be more efficient
in PAHs degradation as the degradation rate was more rapid compared to
free cell culture. The PAHs degraded by immobilized culture on day two
was over 65, 89 and 80%, with corresponding concentrations of 6.57, 8.91
and 8.03 ppm for phenanthrene, anthracene and pyrene, respectively (Fig.
5). At the cessation of the incubation, only 11.7, 6.3 and 14.9% of
phenanthrene, anthracene and pyrene, respectively was recovered from the
culture medium. Their corresponding final concentrations were 0.12, 0.06
and 0.15 ppm for phenanthrene, anthracene and pyrene, respectively. In
this study, the high correlation coefficient (r2>0.96) between
degradation of PAHs and laccase activity indicated that the increase in
PAHs degradation was proportionate with the increase in laccase activity
of P. sanguineus. Thus, laccase activity of the immobilized culture
highly correlated with degradation of the PAH compounds.
Initially, removal of PAHs from the liquid culture was more rapid in
the immobilized mycelia culture compared to the free mycelia culture.
Degradation efficiency of phenanthrene, anthracene and pyrene in immobilized
culture was 4.8, 1.4 and 1.4 fold higher than the free mycelia culture
on day two of incubation, respectively. Subsequently, degradation efficiency
of PAHs was reduced in the immobilized mycelia culture. Degradation efficiency
of phenanthrene in immobilized mycelia culture was over 2 fold higher
than the free mycelia culture at the cessation of incubation. Degradation
efficiency of anthracene was comparable between immobilized mycelia and
free mycelia cultures of P. sanguineus at the end of the incubation.
Degradation efficiency of pyrene in immobilized mycelia culture was 2.4%
lower than the free mycelia culture at the cessation of incubation.
This study had shown that the natural material such as Ecomat was able to maintain
high levels of laccase production compared to free mycelia. The advantages of
selecting natural materials as an immobilization support as these were compostable
and inexpensive. Reports had shown that natural materials such as wheat straw
(Zafar et al., 1996) and barley bran (Rodriguez
Cuoto et al., 2003) had supported biofilms and sustained high enzyme
activity. Shin et al. (2002) had also reported
that natural supports such as jute, straw and hemp fiber were shown to provide
the carbon source to the culture of Trametes versicolor and high levels
of degrading enzymes were sustained during growth. In this study, immobilization
of P. sanguineus on natural support materials supported better mycelial
growth compared to the free mycelia culture. This could be due to these natural
materials provide exogenous nutrients (carbon source from the breakdown of cellulose
and lignin) to encourage biomass proliferation. Ecomat was found to be the best
support for immobilization of P. sanguineus as the mycelium adhered to
it readily and the biomass did not slough off easily. Ecomat made from 100%
natural oil palm residues contained a higher proportion of easily accessible
and biodegradable plant material such as cellulose or hemicellulose. Furthermore,
immobilized culture on Ecomat was able to sustain high laccase productivity
for longer incubation period compared to other supports. This could be due to
the ability of P. sanguineus to metabolize the carbohydrates in Ecomat,
making the whole process much more economical. Comparatively, the laccase activity
of immobilized mycelia of P. sanguineus on Ecomat was higher than other
species of white rot fungi cultivated in submerged liquid culture such as Pleurotus
ostreatus (Medeiros et al., 1999) and Trametes
spp. (Rodriguez Cuoto et al., 2003,
2004; Trupkin et al., 2003).Thus,
this revealed the high potential of Ecomat to improve laccase production by
The degradation of each PAH by P. sanguineus in both free mycelia and
immobilized culture in descending order was anthracene>pyrene>phenanthrene.
Although phenanthrene and anthracene shared the same molecular weight (178.23
g mol-1), the structure of phenanthrene is more compact and angular
compared to anthracene. This may be a contributory factor in its higher resistance
to fungal enzyme attack. Furthermore, the Ionization Potential (IP) of anthracene
(7.44 eV) is lower than phenanthrene (7.90 eV), indicating its higher susceptibility
to enzymatic reactions (Dasbetani and Ivanov, 1999).
Pyrene, on the other hand, was readily degraded by P. sanguineus
free mycelia culture and immobilized culture compared to phenanthrene during
the 20 days incubation period. Based on the results, there was no direct
correlation between the number of aromatic rings and the metabolism of PAHs
as pyrene is a four-ring PAH compound. The IP of pyrene (7.42 eV) was lower
than both anthracene (7.44 eV) and phenanthrene (7.90 eV), which renders it
liable to attack by ligninolytic enzymes (Hammel et al.,
1986). Collins et al. (1996) had reported
on laccases of Trametes versicolor, which were able to oxidize PAHs with
IP lower than 7.45 eV and thus, was proven by the rapid degradation of anthracene
and pyrene by laccase from P. sanguineus compared to phenanthrene in
this study. The degradation of PAHs was rapid while laccase activity of the
free mycelia culture was found to be low in the early stage of incubation. Results
showed that correlation between PAHs degradation and laccase activity in free
mycelia culture was relatively low. It was deduced that laccase was not responsible
for PAHs degradation in free mycelia culture. Thus, this suggests the involvement
of intracellular degradation of PAHs where intracellular enzymes like cytochrome
P450 monooxygenases oxidized PAH compounds that were adsorbed to the biomass.
Cytochrome P450 monooxygenase has been reported to cause the degradation of
phenanthrene by Phanerochaete chrysosporium (Sutherland
et al., 1991) and Pleurotus ostreatus (Bezalel
et al., 1997). A preliminary study done by Verdin
et al. (2004) assessing crude enzymatic extract of Fusarium solani
grown on synthetic medium containing benzo[a]pyrene showed the presence of cytochrome
P450 monooxygense and low levels of laccase activity. An assumption that intracellular
enzymes of P. sanguineus were likely involved in the oxidation of PAHs,
in which the degradation of PAH compounds are attacked by intracellular enzymes
and extracellular laccase.
The capacity of P. sanguineus immobilized on Ecomat to remove anthracene,
phenanthrene and pyrene exceeded the efficiency of the free mycelia culture.
The rapid degradation of these compounds correlated with the increasing laccase
activity during the whole incubation period. The high correlation between percentages
of PAHs degraded and laccase activity observed could have resulted from a direct
relationship, in which, laccase could be solely responsible for PAHs degradation
in P. sanguineus. According to Braun-Lullemann et
al. (1999), the removal of PAHs was probably due to the extracellular
oxidative enzymes produced by the fungi. The direct involvement of laccase in
PAHs degradation was further confirmed by Gramss et al.
(1999), where PAHs degradation was correlated with laccase activity in wood-degrading
fungi with correlation coefficient equivalent to 0.85.
Pycnoporus sanguineus tested in this study was proven to be an efficient
PAHs degrader compared to some wood-degrading fungi included Bjerkandera
adusta, Hypholoma frowardii and Pleurotus ostreatus reported
by Gramss et al. (1999). They tested 58 fungal
strains, consisting of wood-degrading basidiomycetes isolated from the soil.
They had reported the ability of these basidiomycetes to remove 40-60% of phenanthrene,
72-82% of anthracene and 58-70% of pyrene in a mixture of five PAHs (total 10
ppm PAHs) in 14 days of incubation. However, P. sanguineus in both free
cell and immobilized culture were able to remove 51-81% of phenanthrene, 93-94%
of anthracene and 83-85% of pyrene in 15 days of incubation. While anthracene
removal (total 10 ppm) were comparable with cultures inoculated with Phanerochaete
chrysosporium and Bjerkandera sp. strain Bos55 as reported by Field
et al. (1992). Therefore, P. sanguineus proved to be an efficient
PAHs degrader comparable to other white rot fungi species. Furthermore, the
involvement of laccase in the immobilized culture that played an important role
in PAHs degradation was confirmed. This study also revealed the advantages of
immobilization of P. sanguineus, particularly on Ecomat, for rapid PAHs
The authors gratefully acknowledge the financial support of the University
of Malaya for the Vote F grant F0144/2003C and Ministry of Science and
Environment Grant IRPA 01-02-03-1002.
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