Documents
Resources
Learning Center
Upload
Plans & pricing Sign in
Sign Out

Biodeg of Polycylic aromatic hydrocarb in forest and salt marsh soils by white rot fungi

VIEWS: 25 PAGES: 7

									                                                             ARTICLE IN PRESS



                                         International Biodeterioration & Biodegradation 58 (2006) 15–21
                                                                                                                          www.elsevier.com/locate/ibiod




Biodegradation of polycyclic aromatic hydrocarbons in forest and salt
                   marsh soils by white-rot fungi
                                L. Valentı´ na,1, G. Feijooa, M.T. Moreirab,Ã, J.M. Lemab
             a
                 Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, E-15782 Santiago, Spain
             b
                 Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, E-15782 Santiago, Spain
                               Received 15 November 2005; received in revised form 31 March 2006; accepted 11 April 2006
                                                            Available online 26 May 2006



Abstract

   Single strains of nine white-rot fungal species were screened for the ability to degrade polycyclic aromatic hydrocarbons (PAHs) in
forest and salt marsh soils. Of these, Bjerkandera adusta, Irpex lacteus and Lentinus tigrinus were outstanding PAH degraders in both
saline and non-saline conditions. These three strains were tested in parallel to investigate the levels of salinity and PAH concentration
which had negative effects on the ligninolytic activity of the fungi as measured by the anthraquinone dye Poly R-487. Salinity
characteristic of sea water had minimal effect on ligninolytic activity of I. lacteus and L. tigrinus, while in B. adusta, activity was inhibited
by salinity levels of 32%. The results also indicated that the concentration of each single PAH should be o10 mg lÀ1, equivalent to a
concentration of 100 mg kgÀ1 polluted soil. These results were statistically analysed using analyses of covariance (ANCOVA). The
statistical analyses confirmed that the decolourization rate varied with salinity and PAH concentration, and between fungi. In the light of
these results, future research should be focused on the selection of the most appropriate bioreactor configuration for soil detoxification.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Biodegradation; Polycyclic aromatic hydrocarbons (PAHs); Soil-slurry system; White-rot fungi; Marsh soil; Forest soil; Salinity



1. Introduction                                                                 compounds (Bumpus, 1989; Field et al., 1992; Barr and
                                                                                Aust, 1994; Bogan and Lamar, 1996; Pointing, 2001).
   Polycyclic aromatic hydrocarbons (PAHs) are important                           The natural environment of these fungi is wood, in which
pollutants of soil and sediments. Most biological ap-                           they grow, secreting ligninolytic enzymes that depolymerize
proaches considered for the restoration of PAH-contami-                         the lignin. A major drawback arises when these fungi are
nated sites depend on the activity of bacteria. A possible                      intended for remediating polluted soils, where the promo-
alternative studied for degradation of this type of                             tion of their growth and generation of ligninolytic enzymes
compound is the use of ligninolytic fungi. These possess                        have been found to be difficult (Okeke and Agbo, 1996).
an extracellular degradation system which is capable of                         Moreover, PAHs have a strong tendency to sorb onto the
breaking down lignin (Kirk and Farrell, 1987), an                               soil matrix, and thus their desorption from soil limits their
amorphous and complex biopolymer with an aromatic                               availability for fungal biodegradation.
structure similar to the aromatic molecular structure of                           Very few reports have considered the complexity of
some environmental pollutants such as PAHs, pesticides,                         degradation of PAHs in soil-slurry systems by white-rot
polychlorinated biphenyls (PCBs), synthetic dyes, etc. This                     fungal cultures (Brodkorb and Legge, 1992; Zheng and
structural resemblance makes possible the use of white-rot                      Obbard, 2002). Even more, there is still a lack of
fungi to treat sites contaminated with these recalcitrant                       information about fungal growth and PAH oxidation in
                                                                                soil-slurry reactors treating soil from marine areas. Such
  ÃCorresponding author. Fax: +34981528050.                                     information is basic for the successful treatment of oil spills
  E-mail address: tmoreira@usc.es (M.T. Moreira).
                                                                                at shorelines and marsh ecosystems. Essential aspects to be
  1
  Present address: Department of Applied Chemistry and Microbiology,            considered for application of these fungi to detoxification
University of Helsinki, 00014 Helsinki, Finland.                                of marine sites contaminated with PAHs are (a) selection of

0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2006.04.002
                                                              ARTICLE IN PRESS
16                                          ´
                                   L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21


fungal strains with high degradative ability and (b)                            Table 1
evaluation of the effects of the high salinity associated                       Physico-chemical characterization of forest and marsh soils
with coastal areas on fungal growth, production of                                                                      Forest soil                 Marsh soil
enzymes and ligninolytic activity. The ligninolytic potential
of these fungi is related to the secretion of oxidative                         pH                                      4.04                        5.57
enzymes, which permit the effective decolourization of an                       Carbon concentration                    7.85%                       2.12%
                                                                                Nitrogen concentration                  0.55%                       0.55%
anthraquinone dye, Poly R-478 (Glenn and Gold, 1983).
                                                                                Organic matter                          9.8                         1.6–2.2
   The main purpose of this study was to screen nine well-                      CEC (cmolc kgÀ1)a                       4.98                        115.53
known lignin-degrading white-rot fungi suitable for treat-                      Mg2+ (cmol kgÀ1)                        0.73                        98.40
ing salt marsh soil polluted with PAHs, focusing on four                        Na+ (cmol kgÀ1)                         0.04                        13.05
different PAHs with three (phenanthrene (PHEN)) or four                         Ca2+ (cmol kgÀ1)                        2.5                         2.91
                                                                                K+ (cmol kgÀ1)                          0.2                         1.17
benzene rings (fluoranthene (FLU), pyrene (PYR) and                              Al3+ (cmol kgÀ1)                        1.51                        0.11
chrysene (CHRY)). An initial screening assay was con-                           Fe (g kgÀ1)                             15.9                        1.71
ducted on forest and marsh soils under non-saline and                           Mn (g kgÀ1)                             30                          184
saline conditions, respectively, with the aim of selecting                      Zn (g kgÀ1)                             149                         80
those strains with better degradative potential in both                           a
                                                                                   CEC: cationic exchange capacity, defined as the amount of exchange-
cases. Thereafter, three fungi selected on the basis of these                   able cations that can be adsorbed onto the soil at pH 7.
assays were studied in more detail to assess levels of sea
water content and PAH concentration having minimal
effect on dye decolourizing capacity as a measurement of                        Table 2
ligninolytic activity. This work is part of a wider project for                 Properties and initial concentration of PAHs in forest and marsh soils
development of remediation technology based on the use of                       (based on dried weight of soil)
a soil-slurry reactor for the degradation of PAHs in marine                     PAH compound Number            Solubility     Ionization      Concentration
areas, in order to assess its applicability in treating oil                                  of rings          (mg lÀ1)a      potential (eV)b (mg kgÀ1)
spills.
                                                                                Phenanthrene        3          1.29           8.03             50
                                                                                Fluoranthene        4          0.26           7.95             50
2. Materials and methods                                                        Pyrene              4          0.14           7.41             50
                                                                                Chrysene            4          0.002          7.60             50
2.1. Microorganisms                                                               a
                                                                                      Sims and Overcash (1983).
                                                                                  b
                                                                                      Levin and Lias (1982).
   The fungal strains screened were Phanerochaete chrysosporium BKM-
F-1767, Phanerochaete sordida YK-624, Polyporus ciliatus ONO94-1,
Stereum hirsutum PW93-4, Lentinus tigrinus PW94-2, Bjerkandera adusta
                                                                                coast (Spain). Each soil was sieved and the fraction passing a 0.5-mm mesh
BOS55, Irpex lacteus Fr. 238 617/93, Pleurotus eryngii CBS 613.91 and           stored at 4 1C. Prior to use, this soil was autoclaved (121 1C, 20 min) and
Phlebia radiata WIJSTER94-6. All strains were from the culture collection       spiked with PAH in acetone. The solution contained PHEN, FLU, PYR
of the Chemical Engineering Department, University of Santiago de               and CHRY to achieve a final individual PAH concentration in the soil of
Compostela (Spain). All were transferred from culture flasks (maintained
                                                                                50 mg kgÀ1 soil (Table 2). The soil was then held for 24 h under a fume
at 4 1C) to malt extract agar plates comprising (lÀ1) 15.0 g agar, 3.5 g malt   hood for the solvent to evaporate.
extract and 10.0 g glucose, and incubated at 30 1C for 5–7 days before
being stored at 4 1C and subcultured every 6 months.
                                                                                2.4. Screening assays
2.2. Preparation of fungal inoculum
                                                                                   Small-scale screening was performed in 100-ml Erlenmeyer flasks
   Fernsbach flasks (1 l) containing 100 ml modified Kirk medium (Tien            containing a slurry of 2 g (dry weight) soil in 16 ml culture medium
and Kirk, 1988) were each inoculated with five 5-mm plugs of active              prepared with distilled water and BIII mineral medium for the forest soil,
mycelium cut from malt extract agar plate cultures, and incubated at            and with marsh water for the marsh soil (Table 3), to which 4 ml inoculum
30 1C. The culture medium contained (lÀ1) 10 g glucose as carbon source,        was added to give an initial fungal biomass of 1.4 g lÀ1 (dry weight). The
2 g ammonium tartrate or peptone as nitrogen source and 100 ml BIII             flasks were closed with a cotton plug and incubated at 30 1C in total
mineral medium. Prior to sterilization by autoclaving, the medium was           darkness for 30 days in an orbital shaker operating at 120 rpm to gently
adjusted to pH 4.5 with 20 mM acetate buffer. After autoclaving, filter-         agitate the soil. Six replicates of abiotic controls were harvested after 1 and
sterilized thiamine stock solution (200 mg lÀ1) was added at the rate of        30 days, and four replicates for each fungus harvested after 30 days in
1 ml lÀ1 medium. After 7 days, the contents of each culture flask were           order to measure the residual PAHs in the soil.
homogenized in a sterilized blender for 1 min, providing inoculum for the
screening assays.                                                               2.5. Effect of salinity and PAH concentration on dye decolourizing
                                                                                rate
2.3. Soil preparation
                                                                                   The effects of salinity and PAH concentration on decolourization of
   Forest soil and marsh soil with characteristics determined by standard       Poly R-478 dye, a polyanthraquinone (MW ¼ 220–250 kDa) which has a
methods (ASTM, 1999) (Table 1) were used for the screening assays. The          structural resemblance to some PAHs, were analysed using B. adusta, I.
forest soil was collected at a depth of 20 cm from a grassy area in an oak      lacteus and L. tigrinus. The decolourization capacity served as an indirect
grove and the marsh soil was harvested from a salt marsh on the Galician        measurement of the enzyme activity of each fungus (Gold et al., 1988).
                                                             ARTICLE IN PRESS
                                            ´
                                   L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21                               17


Table 3                                                                        3. Results and discussion
Chemical composition of marsh and sea water

Compounds                        Marsh water                     Sea water
                                                                               3.1. Screening in forest soil

Mg2+ (mg lÀ1)                    525                             1270             The biodegradative and abiotic losses of PAHs with time
Na+ (mg lÀ1)                     4000                            10710         of incubation for the forest soil are summarized in Table 4.
Ca2+ (mg lÀ1)                    188.4                           400
                                                                               The data presented, corresponding to the average value of
K+ (mg lÀ1)                      253.4                           390
Mn+ (mg lÀ1)                     0.09                            0.05          six parallel controls taken at zero time and day 30 and
NO3À (mg lÀ1)                    0.7                             1.24          samples in triplicate at day 30, show that all fungi except
PO3(mg lÀ1)
   4                             0.062                           0.088         Phlebia radiata were capable of degrading the mixture of
Salinity (%)                     10                              32            PAHs to some extent, although Pleurotus eryngii, B. adusta
                                                                               and I. lacteus were outstanding strains with average
                                                                               degradation of the four PAHs being 67%, 55% and
Decolourization assays were performed statically in 100-ml Erlenmeyer          53%, respectively, by the three strains.
flasks containing 15 ml culture medium comprising (lÀ1) 10 g glucose,              The recovery of the PAHs from incubated biologically
100 ml BIII mineral medium and 0.2 g Poly R, and adjusted to pH 4.5 and
amended with thiamine as described earlier. For examination of the effect      inactive control soils was different for each PAH. The
of salinity, the medium was made up in distilled water and 25%, 50%,           coefficients of variation between controls at zero time and
75% and 100% sea water (Table 3), and for the effect of PAH, it was made       day 30 did not exceed a maximum of 20%, except for
up in distilled water with each individual PAH added concentrations of         PHEN for which degradation was somewhat obscured by
zero, 1, 5, 10 and 50 mg lÀ1. These were inoculated with two 5-mm mycelial
                                                                               strong abiotic losses, primarily by volatilization during
plugs and incubated under air at 30 1C for 22 days. The dye was added to
give a concentration of 0.2 g lÀ1 culture medium immediately before
                                                                               incubation. This effect was evident for samples taken at
incubation (zero time). Decolourization of the dye was monitored as            day 30, when the loss in PAH concentration ranged from
percentage reduction in the absorbance ratio at 520 and 350 nm in              35% to 45%. However, it was still possible to detect
comparison with the dye control. Before reading absorbance, 0.2 ml             significant divergence between controls and biologically
centrifuged sample (20,000 Â g, 10 min) was diluted with 0.8 ml distilled      active samples, with an average difference around 30–50%.
water and measured spectrophotometrically at 30 1C in a 1-cm cuvette.
Experimental assays and controls were run in triplicate.                          All strains screened were able to degrade FLU, with
                                                                               degradation ranging from 40% (Phanerochaete chrysospor-
                                                                               ium) to 71% (Pleurotus eryngii), except for Phlebia radiata,
2.6. Extraction and analyses
                                                                               which degraded o12%. More than 54% of FLU was
                                                                               degraded by fungi such as I. lacteus, S. hirsutum and B.
   All culture flasks were weighed before and after the experiment to
correct for concentration effects resulting from evaporation of water. The     adusta. Less active species such as Phanerochaete sordida,
whole assays were extracted in order to analyse the residual PAH in the        Polyporus ciliatus, Phanerochaete chrysosporium and L.
soil. Hexane–acetone (1:1, 40 ml) was added to each flask and agitated for      tigrinus achieved degration rates up to 48%. PYR was the
2 h at 300 rpm in a horizontal shaker in order to achieve the transport of     most extensively degraded compound with 85%, 83% and
PAHs from water or soil to the organic solvent phase. Following this, if an
                                                                               81% being degraded by, respectively, I. lacteus, Phaner-
emulsion formed during agitation, the contents were sonicated for a
further 30 min for separation of the organic solvent and aqueous phases. A     ochaete sordida and B. adusta. However, Pleurotus eryngii
1.5-ml aliquot of the liquid was filtered through nylon to remove small soil
particles and collected in an amber vial. The average extraction efficiencies
                                                                               Table 4
for forest soil were 88.92712.14% (PHEN), 95.2777.75% (FLU),
                                                                               Residual PAH concentration (mg kgÀ1) after incubation of nine strains of
93.5477.59% (PYR) and 90.6876.78% (CHRY). The corresponding
                                                                               white-rot fungi for 30 days under non-saline conditions (forest soil)
average efficiencies for marsh soil were 93.4374.92%, 97.1474.47%,
75.7973.29% and 81.9373.55%. The contents of the vials were analysed
                                                                                                   Phenanthrene Fluoranthene Pyrene        Chrysene
on a Hewlett-Packard HP 1090 HPLC equipped with a diode array
detector monitoring the absorbance at 235 nm for PHEN and FLU,                 Control, zero       45.076.1     47.473.9      46.873.8     45.373.4
240 nm for PYR and 254 nm for CHRY. A 4.6 Â 200 mm Spherisorb                  time
ODS2 reverse-phase column (5 mm; Waters) and a HP ChemStation data             Control, 30 days    30.472.9     36.673.7      35.173.4     42.2710
processor were used for determining the PAH concentration. The injection       Phanerochaete       20.371.7     22.071.6      14.172.5     23.570.2
volume was set at 10 ml and the isocratic eluent (acetonitrile–water, 80:20)   chrysosporium
was delivered at a rate of 0.4 ml minÀ1.                                       Phanerochaete       19.972.8     19.273.0       6.072.1     32.371.8
                                                                               sordida
                                                                               Polyporus           20.471.6     20.372.5      12.270.9     32.071.6
2.7. Statistical analysis
                                                                               ciliatus
                                                                               Stereum             17.171.4     16.272.4      13.470.1     31.972.1
   The effects of salinity and PAH on the decolourization of Poly R-478        hirsutum
were evaluated by covariance analysis (ANCOVA). Each of the two                Lentinus tigrinus   17.873.1     21.871.7      14.172.7     31.171.0
experiments was considered as a factorial design consisting of four            Bjerkandera         14.470.9     16.971.2       6.570.9     28.772.3
treatments (the three fungal species and the control) with three replicates    adusta
per treatment and two covariates, time and either PAH or salinity to           Irpex lacteus       18.871.3     15.771.7       5.171.6     29.271.2
define the covariation of either salinity or PAH and species with time (i.e.    Pleurotus eryngii    2.070.3     10.577.3       9.678.3     29.072.4
the rate of decolourization). All analyses were carried out in statistical     Phlebia radiata     25.771.6     32.473.9      30.073.2     34.073.4
package R.
                                                          ARTICLE IN PRESS
18                                        ´
                                 L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21


(73%) was also an outstanding PYR degrader, and except                       Table 6
for Phlebia radiata (15%), the remaining fungi were able to                  Average net PAH degradation (%) for Bjerkandera adusta, Irpex lacteus
                                                                             and Lentinus tigrinus under saline (marsh soil) and non-saline conditions
convert 460% of the PYR. CHRY was degraded least, the
                                                                             (forest soil)
average net degradation rate being 28%, with Phaner-
ochaete chrysosporium degrading 45% and B. adusta, I.                                        Phenanthrene      Fluoranthene      Pyrene     Chrysene
lacteus and Pleurotus eryngii 31–32%. The expectation of
                                                                             Marsh soil      48                51                51         49
an inverse correlation between the number of PAH rings
                                                                             Forest soil     44                50                76         30
and the loss from soils was supported by the results, with
the loss for the 3-ring PAH, PHEN, from the forest soil
(and also the marsh soil) being somewhat less than for the
4-ring PAHs, FLU, PYR and CHRY.                                                 Comparatively, the results presented here show greater
                                                                             PAH degradation than obtained for other well-known
3.2. Screening in marsh soil                                                 white-rot strains, such as Phanerochaete chrysosporium
                                                                             cultured in soil-slurry systems (Zheng and Obbard, 2002).
   In the parallel assays performed for salt marsh soil using                This strain showed lower percentage degradation of PHEN
marsh water in the culture medium (Table 5), a strong                        (29%), FLU (16.4%) and PYR (21.9%), and no CHRY
inhibitory effect on PAH degradation was evident except                      degradation, in the presence of 11 mM Mn2+. The results
for three fungi, viz. I. lacteus, B. adusta and L. tigrinus,                 from the present screening tests revealed that B. adusta, I.
which on average degraded the four PAHs by 57%, 49%                          lacteus and L. tigrinus were outstanding PAH degraders, in
and 42%, respectively. Four species (L. tigrinus, B.adusta,                  both saline and non-saline conditions, with average
I. lacteus and Pleurotus eryngii) degraded 440% of the                       degradation of 48% and 44% (PHEN), 51% and 50%
PHEN, o22% was degraded by Phanerochaete chrysos-                            (FLU), 51% and 76% (PYR) and 49% and 30% (CHRY)
porium, Polyporus ciliatus and S. hirsutum. The greatest                     in the presence of 1.36 (marsh) and 33 mM Mn2+ (forest),
FLU and PYR degrader was I. lacteus, which degraded,                         respectively (Table 6).
respectively, 60% and 63% in 30 days under saline                               An important criterion for the selection of PAHs which
conditions. B. adusta and L. tigrinus also notable,                          may be effectively degraded by the ligninolytic enzymes is
degrading 54% and 39% of FLU and 49% and 40% of                              the ionization potential, IP (Table 2). Among ligninolytic
PYR, respectively. These three fungi also showed the                         enzymes, lignin peroxidase (LIP) can degrade PAHs with
greatest ability to degrade CHRY (56%, 48% and 43%,                          IP o7.55 eV (Hammel et al., 1985), and manganese
respectively). In contrast, low FLU and PYR degradation                      peroxidase (MnP) with IP o7.8 eV (Cavalieri and Rogan,
rates were shown by Phlebia radiata (5% and 3%,                              1985). Theoretically, the enzymatic system should be
respectively) and Phanerochaete chrysosporium (8% and                        efficient provided that the oxidation potential of a
12%, respectively). No CHRY degradation was detected                         particular compound is lower than that supplied by the
for S. hirsutum and only negligible amonts removed by                        enzymic cycle. Among the tetracyclic PAHs, the most
Pleurotus eryngii (9%) and Phanerochaete chrysosporium                       condensed, PYR (IP ¼ 7.41 eV), which lacks a bay region,
(12%).                                                                       was subject to the most rapid biodegradation. Moreover,
                                                                             the soil-slurry system proved to oxidize PHEN and FLU,
Table 5
                                                                             which have IP values 47.8 eV. Therefore, it is worthwhile
Residual PAH concentration (mg kgÀ1) after incubation of nine strains of     mentioning the very marked ability of these fungi and their
white-rot fungi for 30 days under saline conditions (marsh soil)             associated oxidative enzymes to degrade PAHs. One
                                                                             hypothesis to account for this could be that the extra-
                    Phenantrhene Fluoranthene Pyrene        Chrysene
                                                                             cellular system secretes other compounds participating in
Control, zero       46.772.5     48.672.2      37.971.6     41.071.7         the degradative action of the enzymes. Among the different
time                                                                         possibilities, lipid peroxidation may be the catalytic
Control, 30 days    25.6711      38.076.0      38.376.7     38.376.7         mechanism for the oxidation of PAHs, as described for
Phanerochaete       23.072.1     35.174.4      33.772.8     33.874.5
                                                                             PHEN (Moen and Hammel, 1994).
chrysosporium
Phanerochaete       16.874.1     29.672.7      30.571.3     30.872.7
sordida                                                                      3.3. Effect of saline conditions and PAH concentration on
Polyporus           20.372.4     28.772.1      28.171.0     32.972.3         fungal ligninolytic activity
ciliatus
Stereum             24.070.6     32.471.1      31.672.5     45.271.6
                                                                                The evaluation of the possible inhibitory effect of salinity
hirsutum
Lentinus tigrinus   13.970.7     23.170.7      22.871.0     21.870.9         is essential for the selection of the most suitable strains for
Bjerkandera         13.371.0     17.770.7      19.771.2     20.173.7         the remediation of shorelines and coastal areas polluted
adusta                                                                       with oil spills. The concentrations of salts in the medium
Irpex lacteus       13.072.4     15.170.8      14.171.6     16.770.1         and the PAH concentrations being major factors that could
Pleurotus eryngii   13.272.4     28.470.7      32.071.5     34.970.3
                                                                             affect degradative capacity, the objective of our experi-
Phlebia radiata     16.671.0     36.270.6      37.270.8     29.971.7
                                                                             ments was to determine the levels of salinity and PAH
                                                                        ARTICLE IN PRESS
                                              ´
                                     L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21                              19


concentration which affect the degradative capability                               tion between time and species, F3, 538 ¼ 99.77, Po0:001).
of the three selected fungi, B. adusta, I. lacteus and L.                           The response of different fungi to salinity was also variable
tigrinus.                                                                           (ANCOVA, three-way interaction between time, salinity
   As mentioned in Section 2, the effect of concentration of                        and species, F3, 538 ¼ 24.31, Po0:001). Overall, the
the salts was determined in a first experiment where assays                          presence of sea water (which has average salinity values
were performed with different proportions of sea water in                           of around three-fold higher than in marsh water) had no
the culture medium. It can be seen from the dye                                     detrimental effect on the rate of decolourization for either
decolourization assay (Fig. 1), used to assess the extra-                           I. lacteus or L. tigrinus, but for B. adusta decolourization
cellular ligninolytic enzyme activity because fungal bior-                          was inhibited by 100% sea water (Fig. 1). Therefore,
emediation potential is often attributed to these enzymes,                          although the salinity experiments indicate that these fungi
that decolourization started after an initial lag of approx. 5                      can perform the degradation in a soil from a marine
days and continued throughout the 22 days of the                                    environment, the employment of this strain of B. adusta
experiment. The statistical analysis (ANCOVA) corrobo-                              would require control of the salt content to prevent
rated that time had an effect on the decolourization (F1,                           inhibition. Having in mind that this can be an ex situ
538 ¼ 1093.02, Po0:001). On the other hand, the decolour-                           technique, preconditioning of the soil to remove
ization rate varied with sea water content (ANCOVA,                                 salts would not be necessary, although it would not be
interaction between time and salinity, F1, 538 ¼ 3.87,                              advisable to use sea water for inoculum and culture media
Po0:05) and between fungal species (ANCOVA, interac-                                preparation.


                           1.2                                                                  1.2

                           1.0                                                                  1.0

                           0.8                                                                  0.8
                                                                                    A520/A350
               A520/A350




                           0.6                                                                  0.6

                           0.4                                                                  0.4

                           0.2                                                                  0.2

                           0.0                                                                  0.0
                                 0   5       10                15        20   25                      0        5        10      15    20   25
               (A)                           Time (days)                            (B)                                 Time (days)

                           1.2                                                                  1.2
                           1.0                                                                  1.0

                           0.8                                                                  0.8
                                                                                    A520/A350
               A520/A350




                           0.6                                                                  0.6

                           0.4                                                                  0.4

                           0.2                                                                  0.2

                           0.0                                                                  0.0
                                 0   5       10                15        20   25                      0        5        10       15   20   25
               (C)                           Time (days)                            (D)                                 Time (days)

                                                              1.2

                                                              1.0

                                                              0.8
                                                  A520/A350




                                                              0.6

                                                              0.4

                                                              0.2

                                                              0.0
                                                                    0     5    10                15       20       25
                                                  (E)                         Time (days)

Fig. 1. Decolourization rates of the dye Poly R-478 by the white-rot fungi Lentinus tigrinus ( Â ), Bjerkandera adusta (&) and Irpex lacteus (m) incubated
in culture media prepared with different proportions of sea water: (A) 100%, (B) 75%, (C) 50%, (D) 25% and (E) 0%.
                                                                        ARTICLE IN PRESS
20                                            ´
                                     L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21


                           1.2                                                                  1.2

                           1.0                                                                  1.0

                           0.8                                                                  0.8




                                                                                    A520/A350
               A520/A350



                           0.6                                                                  0.6

                           0.4                                                                  0.4

                           0.2                                                                  0.2

                           0.0                                                                  0.0
                                 0   5       10                15        20   25                      0        5        10      15    20   25
               (A)                           Time (days)                            (B)                                 Time (days)

                           1.2                                                                  1.2
                           1.0                                                                  1.0

                           0.8                                                                  0.8




                                                                                    A520/A350
               A520/A350




                           0.6                                                                  0.6

                           0.4                                                                  0.4

                           0.2                                                                  0.2

                           0.0                                                                  0.0
                                 0   5       10                15        20   25                      0        5        10       15   20   25
               (C)                           Time (days)                            (D)                                 Time (days)

                                                              1.2

                                                              1.0

                                                              0.8
                                                  A520/A350




                                                              0.6

                                                              0.4

                                                              0.2

                                                              0.0
                                                                    0     5    10                15       20       25
                                                  (E)                         Time (days)

Fig. 2. Decolourization rates of the dye Poly R-478 by the white-rot fungi Lentinus tigrinus ( Â ), Bjerkandera adusta (&) and Irpex lacteus (m) incubated
in culture media containing different PAHs concentrations (mg lÀ1): (A) 50, (B) 10, (C) 5, (D) 1 and (E) 0.



   In the second experiment evaluating the effect of PAHs                           Acknowledgements
on dye decolourization, the effect of time on the levels of
absorbance ratio (A520/A350) was significant, confirming                                This work was funded by the Spanish Commission of
decolourization with time (ANCOVA, F1,587 ¼ 1200.33,                                Science and Technology (CICYT; Project VEM2003-
Po0:001). The effect of PAHs on the fungi (Fig. 2) was to                           20089-CO2-01).
progressively slow decolourization by all three strains with
increasing concentration up to 50 mg lÀ1. (ANCOVA,                                  References
interaction between time and PAH, F1,588 ¼ 243.01,
Po0:001). Different species showed different rates of                               ASTM, 1999. American Society for Testing and Materials. Methods:
decolourization (ANCOVA, interaction between time and                                  D422-63, D2974-87, D4972-95a, D3974-81.
                                                                                    Barr, D.P., Aust, S.D., 1994. Mechanisms white-rot fungi use to degrade
species, F3,588 ¼ 62.80, Po0:001) and reacted differently to                           pollutants. Environmental Science and Technology 28, 78A–87A.
PAH concentration (ANCOVA, three-way interaction                                    Bogan, B.W., Lamar, R.T., 1996. Polycyclic aromatic hydrocarbon-
between time, PAH and species, F3,588 ¼ 16.37,                                         degrading capabilities of Phanerochaete laevis HHB-1625 and its
Po0:001). According to the time course decolourization                                 extracellular ligninolytic enzymes. Applied and Environmental Micro-
rate (Fig. 2) and the statistical results, one could say that                          biology 62, 1597–1603.
                                                                                    Brodkorb, T.S., Legge, R.L., 1992. Enhanced biodegradation of
the utilization of L. tigrinus, B. adusta and I. lacteus in                            phenanthrene in oil tar-contaminated soils supplemented with
bioremediation technologies would have to be confined to                                Phanerochaete chrysosporium. Applied and Environmental Microbiol-
PAH concentrations between 5 and 10 mg lÀ1.                                            ogy 58, 3117–3121.
                                                            ARTICLE IN PRESS
                                           ´
                                  L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21                            21


Bumpus, J.A., 1989. Biodegradation of polycyclic aromatic hydrocarbons        Levin, R.D., Lias, S.G., 1982. Ionization potential and appearance
   by Phanerochaete chrysosporium. Applied and Environmental Micro-              potential measurements 1971–1981. National Standards Reference
   biology 55, 154–158.                                                          Data Series 71, 95–112.
Cavalieri, E.L., Rogan, E.G., 1985. Role of radical cations in aromatic       Moen, M.A., Hammel, K.E., 1994. Lipid peroxidation by the manganese
   hydrocarbons carcinogenesis. Environmental Health Perspectives 64,            peroxidase of Phanerochaete chrysosporium is the basis for phenan-
   69–84.                                                                        threne oxidation by the intact fungus. Applied and Environmental
Field, J.A., de Jong, E., Feijoo, G., de Bont, J.A.M., 1992. Biodegradation      Microbiology 60, 1956–1961.
   of polycyclic aromatic hydrocarbons by new isolates of white-rot           Okeke, R.C., Agbo, H.U., 1996. Influence of environmental parameters
   fungi. Applied and Environmental Microbiology 58, 2219–2226.                  on pentachlorophenol biotransformation in soil by Lentinula edodes
Glenn, J.K., Gold, M.H., 1983. Decolorization of several polymeric dyes          and Phanerochaete chrysosporium. Applied Microbiology and Bio-
   by the lignin degrading basidiomycetes Phanerochaete chrysosporium.           technology 45, 263–266.
   Applied and Environmental Microbiology 45, 1741–1747.                      Pointing, S.B., 2001. Feasibility of bioremediation by white-rot fungi.
Gold, M.H., Glenn, J.K., Alic, M., 1988. Use of polymeric dyes in lignin         Applied Microbiology and Biotechnology 57, 20–33.
   biodegradation assays. In: Wood, W.A., Kellog, S.T. (Eds.), Methods        Sims, R.C., Overcash, M.R., 1983. Fate of polynuclear aromatic
   in Enzymology, vol. 161. Biomass Part B: Lignin, Pectin and Chitin.           compounds (PNAs) in soil-plant systems. Residue Reviews 88, 1–68.
   Academic Press, San Diego, pp. 74–78.                                      Tien, M., Kirk, T.K., 1988. Lignin peroxidase of Phanerochaete
Hammel, K.E., Tien, M., Kalyanaraman, B., Kirk, T.K., 1985. Mechan-              chrysosporium. In: Wood, W.A., Kellog, S.T. (Eds.), Methods in
   isms of oxidative Ca–Cb cleavage of a lignin model dimmer by                  Enzymology, vol. 161. Biomass Part B: Lignin, Pectin and Chitin.
   Phanerochaete chrysosporium ligninase. Stoichiometry and involve-             Academic Press, San Diego, pp. 238–249.
   ment of free radicals. Journal of Biological Chemistry 260, 8348–8353.     Zheng, Z., Obbard, J.P., 2002. Oxidation of polycyclic aromatic
Kirk, T.K., Farrell, R.L., 1987. Enzymatic ‘‘combustion’’: the microbial         hydrocarbons (PAH) by the white-rot fungus Phanerochaete chrysos-
   degradation of lignin. Annual Review of Microbiology 41, 465–505.             porium. Enzyme and Microbial Technology 31, 3–9.

								
To top