heavy metal tolerance of filamentous fungi isolated from polluted
TRANSCRIPT
African Journal of Microbiology Research Vol. 3 (2) pp. 035-048 February, 2009 Available online http://www.academicjournals.org/ajmr ISSN 1996-0808 ©2009 Academic Journals Full Length Research Paper
Heavy metal tolerance of filamentous fungi isolated from polluted sites in Tangier, Morocco
L. Ezzouhri1, E. Castro2, M. Moya2, F. Espinola2 and K. Lairini1*
1Department of Biology, Faculty of Sciences and Techniques - Tangier, University Abdelmalek Essaadi. B. P. 416, Tangier, Morocco.
2Department of Chemical, Environmental and Materials Engineering, University of Jaén, Campus Las Lagunillas, 23071 Jaén, Spain.
Accepted 29 January, 2009
Thirty-six micro-organisms, represented by fungi and yeasts strains, were isolated from heavy metal-contaminated sites in Tangier, Morocco. Filamentous fungi isolated belonged to the genera Aspergillus, Penicillium, Fusarium, Alternaria and Geotrichum. They were screened for their resistance to heavy metals. The results revealed that the majority of the isolates were resistant to Pb, Cr, Cu and Zn, whereas to Cd, only the fungus Penicillium sp. was able to grow. The level of resistance depended on the isolate tested, as well as the site of its isolation. Minimum inhibitory concentrations (MICs) for Pb2+, Cr6+, Cu2+ and Zn2+ were also determined. Aspergillus and Penicillium isolates were the most tolerant to the heavy metals and exhibited strong growth, often exceeding the control (isolates grown in agar medium without heavy metals). Their MIC ranged from 20 - 25 mM for Pb, followed by 15 - 20 mM both for Cu and Zn and 10 - 15 mM for Cr. These fungi have shown a high level of resistance to all metals tested, which makes them attractive potential candidates for further investigations regarding their ability to remove metals from contaminated wastewaters. Key words: Metal tolerance, resistance, heavy metal, soil and water fungi, biosorption.
INTRODUCTION Contamination of sediments and natural aquatic recap-tors with heavy metals is a major environmental problem all over the world (Baldrian and Gabriel, 2002; Gavrile-sca, 2004; Malik, 2004; Srivastava and Thakur, 2006). These inorganic micropolluants are released by effluents generated from various industries such as electroplating and metal finishing industries, metallurgy, tannery, and battery manufacturing.
The introduction of heavy metal compounds into the environment generally induces morphological and physio-logical changes in the microbial communities (Vadker-tiova and Slavikova, 2006), hence exerting a selective pressure on the microbiota (Verma et al., 2001). Gene-rally, the contaminated sites are the sources of metal-resistant micro-organisms (Gadd, 1993). Therefore, it is important to explore autochthonous micro-organisms from such contaminated niches for the bioremediation of *Corresponding author. E-mail: [email protected]. Tel.:+ 212-39-39-39-54/55. Fax: + 212-39-39-39-53.
heavy metals since conventional processes such as chemical precipitation; ion exchange and reverse osmo-sis are uneconomical and inefficient for treating effluents of dilute metal concentrations (Kapoor and Viraraghavan, 1995; Gupta et al., 2000; Pagnanelli et al., 2000; Gavrilesca, 2004; Malik, 2004).
In naturally polluted environments, the microbe’s res-ponse to heavy metals toxicity depends on the concen-tration and the availability of metals and on the action of factors such as the type of metal, the nature of medium and microbial species (Hassen et al., 1998). Fungi and yeast biomasses are known to tolerate heavy metals (Gavrilesca, 2004; Baldrian, 2003). They are a versatile group, as they can adapt and grow under various ex-treme conditions of pH, temperature and nutrient availa-bility, as well as high metal concentrations (Anand et al., 2006). They offer the advantage of having cell wall mate-rial which shows excellent metal-binding properties (Gupta et al., 2000). Generally, microbial biomasses have evolved various measures to respond to heavy metals stress via processes such as transport across the cell
036 Afr. J. microbiol. Res.
Sea
Sites of sampling
Industries
Beach
Principal road 38
Moghogha River
Legend
Figure 1. Localisation of sampling sites in Moghogha River.
membrane, biosorption to cell walls, entrapment in extra-cellular capsules, as well as precipitation and transfor-mation of metals (Malik, 2004). Recent studies showed that strains isolated from contaminated sites have an excellent ability of removing significant quantities of me-tals both from aqueous solutions and electroplating effluents (Malik, 2004). El-Morsy (2004) studied 32 fungal species isolated from polluted water in Egypt for their resistance to metals and found that Cunninghamela echi-nulata biomass could be employed as a biosorbent of metal ions in wastewater. Vadkertiova and Slavikova (2006) have studied metal tolerance of yeasts isolated from polluted environments and found that there is an interspecific and intraspecific variation in the metal tolerance among tested strains. In the same way, Zafar et al. (2007) reported promising biosorption for Cd and Cr by two filamentous fungi, Aspergillus sp. and Rhizopus sp., isolated from metal-contaminated agricultural soil. The present work reports the characterization of metal-resistant micro-organisms isolated from polluted environ-
ments. The heavy metal MICs (minimal inhibitory concen-trations) for each micro-organism were determined and selection of the most resistant strains, which can be used in bioremediation of heavy metals, was done. MATERIALS AND METHODS Isolation of micro-organisms from polluted sites Samples of water and sediment from five contaminated sites in the Moghogha river (Tangier, Morocco) were collected in sterilized glass bottles, transported on ice and analyzed within 8 h (Figure 1). The studied river represents the most important emissary of Tangier for the evacuation of lixivia, municipal wastewaters and untreated industrial effluents into the marine ecosystem of Tangier bay. The impact of these inputs on the Moghogha river quality is particularly pronounced in the summer season where the river is only supplied by urban and industrial effluents. Their drainage toward the bay, without previous treatment, represents, therefore, the major source of contamination of the marine ecosystem of Tangier bay. The water samples were enumerated for micro-organisms employing a serial dilution technique. Sediment samples (1 g) were first suspen-
ded in 100 ml of sterilized water; the mixture was agitated for 20 min at room temperature and then diluted (10- to 10 000-fold). Aliquots of 100 µl of different dilutions were plated both onto Potato Dextrose Agar (PDA) and on 2% Malt Extract Agar (MEA) plates (three replicates) to ensure the growth of micro-organisms present in samples. After at least 3 days of incubation at 25°C, developed colonies were randomly picked and isolated. The results obtained both on PDA and MEA medium were similar. Therefore, only PDA medium was used in further experiments. Purified isolates were obtained by streaking repeatedly colonies in PDA medium and observation under light microscopy.
Pure cultures of isolated micro-organisms were identified using the keys of Pitt (1979) and Domsch et al. (1980). The cultures were characterized to the genus level on the basis of macroscopic characteristics (colonial morphology, colour and appearance of colony, shape) and microscopic characteristics (septation of myce-lium, shape, diameter and texture of conidia). The identification was also carried out to the species level for some isolates and micro-graph images were obtained with scanning electron microscopy (SEM). The SEM was carried out on Jeol/JSM 5800 instrument operated at a voltage of 15 kV. Screening and selection of heavy metal-resistant micro-organisms Purified isolates were screened on the basis of their tolerance to Cr6+, Pb2+, Zn2+, Cd2+ and Cu2+. A disk of mycelium was inoculated aseptically on PDA plates supplemented individually with 1 mM of heavy metal. The metal salts used were potassium dichromate, lead carbonate, zinc sulfate, cadmium sulfate and cupric sulfate. The inoculated plates were incubated at 25°C for at least 7 days. The effect of the heavy metal on the growth of the isolates tested was estimated by measuring the radius of the colony extension (mm) against the control (medium without metal) and the determination of the index of tolerance. The index is defined as the ratio of the extension radius of the treated colony to that of the untreated colony. Isolates showing resistance to Cr6+, Pb2+, Zn2+ and Cu2+ were selected for the following experiments. Determination of minimum inhibitory concentrations (MICs) The resistance of the selected isolates to Cr6+, Pb2+, Zn2+ and Cu2+ was determined by the dilution method. Metal ions were added separately to PDA medium at concentrations of 0.01 to 25 mM. The plates were inoculated with 8 mm agar plugs from young fungal colonies, pre-grown on PDA. Three replicates of each concentration and controls without metal were used. The inoculated plates were incubated at 25°C for at least 7 days. The minimum inhibitory concentration (MIC) is defined as the lowest concentration of metal that inhibit visible growth of the isolate. Heavy metal analysis in water and sediment The water and sediment samples were also analyzed for their total content of heavy metals. The sediment samples were dried at 105°C manually ground and sieved through a sieve of (500 µm pore size). A sample of 1 g was treated with 10 ml of aqua regia (25% HNO3; 75% HCl). Digestion was carried out on a hot plate until dense fumes evolved and a clear solution was obtained. The clear solution was filtered through a Millipore filter (0.45 µm) and diluted to 50 ml with distilled water. A control was included by treating 10 ml of aqua regia in the same way as the sediment samples. Simi-larly, 50 ml of water samples were dried at 100°C overnight and digested with an acids mixture (HNO3, HCl and HClO4) at 350°C until there was an abundance of white fumes. As done for sediment samples, the digested solution was filtered and made up to 50 ml
Ezzouhri et al. 037 by adding distilled water. Controls were set up by treating distilled water as described for the water samples.
Heavy metal (Pb, Cu, Zn, Cr and Cd) concentrations were deter-mined using Atomic Absorption Spectrometry (AAS 6200, SHIMADZU). Statistical analysis The experiments were set up with three replicates. Analysis of variance was performed by using statistical software (Fisher’s LSD test, Method: 95.0) to compare resistance to metal among individual isolates. RESULTS AND DISCUSSION Sediment and water analysis It is well known that a long-time exposure of water and sediment to heavy metals can produce considerable modification of their microbial populations, reducing their activity and their number (Doelman et al., 1994). In the present study, various micro-organisms were isolated from water and sediment samples collected from Mogho-gha River where heavy metals and other pollutants have been emitted in industrial effluents for several years. The heavy metals content of sediment and water samples is listed in Table 1. The concentration of Cr, Pb, Zn and Cu in the water samples was found to be above the permis-sible limits of 0.01, 0.5, 5 and 0.05 ppm, respectively (CCME, 1992). Also, higher amounts of these metals were found in sediment samples. The long time reception of untreated industrial effluents, lixivia and domestic wastewater associated with the weak hydrological cha-racteristics of Moghogha River are the main reasons for the high heavy metal content of Moghogha river sediment (Ezzouhri and Lairini, 2005). Moghogha River has a weak water flow for more than seven months of the year. These months are also characterized by a lack of rainfall, which favours the precipitation of heavy metals and their accumulation in the sediment. The present study was carried out within this period.
Fungi isolated belonged to the genera Aspergillus, Peni-cillium, Alternaria, Geotrichum and Fusarium (Figures 2, 3, 4 and 5). Species of the genus Penicillium was the most abundant in all the sites, followed by Fusarium spe-cies. The differences between the sampled sites regard-ing their richness on microbial isolates appear to be closely linked to the degree of heavy metal pollution. Generally, pollution of soil and water by heavy metals may lead to a decrease in microbial diversity. This is due to the extinction of species sensitive to the stress im-posed, and enhanced growth of other resistant species diameter of Penicillium isolates by about 2 - 37% and 3 - 63% was also observed in the presence of copper and zinc, respectively (Table 2). Similarly, Levinskaite (2002), studying the response of soil fungi to hexavalent chro-mium, reported that Trichoderma viride and Penicillium chrysogenum are the most tolerant fungi to the presence of 2 mM chromium in the medium Isolates from the genus
038 Afr. J. microbiol. Res.
Table 1. Heavy metal content and distribution of fungi at the sampled sites.
Water content Sediment content Heavy metals content (mg/l) Heavy metals content (µg/g)
Sampling sites and
description Filamentous
fungi Cr Pb Cd Cu Zn Filamentous
fungi Cr Pb Cd Cu Zn S1 (Control site)
A. alternata 0.032 1.89 0.009 0.0004 0.09 Penicillium sp. Fusarium sp. A. alternata
70.07 17.8 0.96 41.96 62.11
S2 (Industrial effluent)
Penicillium sp. Fusarium sp. A. alternata
1.06 3.75 0.21 0.002 1.1 Penicillium sp. Fusarium sp. A. alternata G. candidum
85.6 15.6 1 27 511.6
S3 (Tannery effluent)
Penicillium sp. 2.1 2.1 0.18 0.006 0.88 Penicillium sp. A. alternata G. candidum
369.0 186.2 1.60 48.3 133.3
S4 (Municipal and industrial wastewaters
Penicillium sp. 0.14 2.2 0.06 0.06 1.07 Penicillium sp. G. candidum
36.1 41.6 1.42 32.8 150.5
S5 (Industrial effluent combined with lixivia)
Penicillium sp. Fusarium sp. A. niger
0.15 1.5 0.13 0.03 1.15 Penicillium sp. Fusarium sp. A. niger
35.0 29.1 1.5 37.3 149.4
Figure 2. SEM micrograph of a Penicillium spp.
Figure 3. SEM micrograph of Aspergillus niger.
Figure 4. SEM micrograph of Alternaria alternata.
Figure 5. SEM micrograph of Geotrichum candidum.
Table 2. Tolerance index of isolated micro-organisms able to grow on PDA amended with 1 mM of metal.
Metallic cations Isolates*
Pb Cr Cu Zn Cd Penicillium sp. (S1S) 0.96 0.92 0.51 0.88 0 Penicillium sp. (S2W) 0.79 0.76 0.79 0.54 0 Penicillium sp. (S2S) 0.98 0.68 0.98 0.74 0 Penicillium sp. (S3W) 1.12 1.23 0.96 0.47 0.80 Penicillium sp. (S3S) 1.02 1.10 0.63 0.50 0.44 Penicillium sp. (S4W) 0.92 1 0.72 0.37 0.38 Penicillium sp. (S4S) 0.90 0.82 0.98 0.97 0.55 Penicillium sp. (S5W) 0.83 0.82 0.76 0.72 0.22 Penicillium sp. (S5S) 1.01 0.93 0.98 0.47 0.62 Fusarium sp. (S1S) 0.81 0.74 0.91 0.92 0 Fusarium sp. (S2W) O.71 0.64 0.72 0.80 0 Fusarium sp. (S2S) 0.94 0.87 0.99 1.03 0 Fusarium sp. (S5W) 0.72 0.78 0.60 0.76 0 Fusarium sp. (S5S) 0.94 0.98 0.79 0.97 0 Aspergillus niger (S5W) 1.08 0.94 0.97 1.04 0 Aspergillus niger (S5W) 1.03 0.95 1 0.97 0.23 Alternaria alternata (S1W) 0.72 0.53 0.90 0.93 0 Alternaria alternata (S1S) 0.89 0.53 0.93 0.88 0 Alternaria alternata (S2W) 0.94 0.85 0.98 0.72 0 Alternaria alternata (S2S) 1.04 0.93 0.86 0.73 0.13 Alternaria alternata (S3S) 0.59 1.02 1.05 1.09 0.31 Geotrichum candidum (S2S) 1 0.19 0.85 0.75 0 Geotrichum candidum (S3S) 0.79 0.82 0.82 0.70 0 Geotrichum candidum (S4S) 1.02 0.66 0.83 0.83 0
*S1-5: sampling sites; S: isolate of sediment and W: isolate of water Alternaria showed the same response pattern to the presence of metal ions in the medium (Table 2). The growth of A. alternata S2S was increased by about 4% at 1 mM lead, while it was slightly decreased in the pre-sence of the other metals. Growth of A. alternata S3S was weakly inhibited in the presence of lead, whereas it was stimulated by Zn, Cu and Cr byabout 9, 5 and 2%, respectively. The remainder of the A. alternata strains showed a developed growth in the presence of the metals tested. The growth inhibition was about 2 - 14%, 6 - 28%, 7 - 28% and 7 - 47% at 1 mM Zn, Pb, Cu and Cr, respectively.
With regard to Fusarium isolates, all Fusarium isolates were sensitive to the presence of metal ions in the growth medium, except Fusarium sp. S2S, which was stimulated by 3% at 1 mM Zn, The growth inhibition was about 6 - 29%, 2 - 36%, 11 - 40% and 3 - 24% at 1 mM concen-tration of Pb, Cr, Cu and Zn, respectively. Sanyal et al. (2005) reported that lead ions are not toxic to the fungus Fusarium oxysporium, which readily grows after exposure to the metal ions.
Similar responses to metals were shown by isolates from the Geotrichum genus. In the presence of lead, two
Ezzouhri et al. 039 of the Geotrichum strains were resistant, while copper, zinc and chromium exerted an inhibitory effect on fungal growth. The lowest growth inhibition was 15 - 30% and the highest was observed for Geotrichum candidum S2S in the presence of chromium (about 81%). Falih (1998) reported that G. candidum survived and grew at concen-trations of 400 µg/ml Cu and Pb in Czapek-Dox medium, while Price et al. (2001) reported that a Geotrichum iso-late was able to grow on plates contaminated with copper up to 5 mM.
Cadmium (Cd) at a concentration of 1 mM showed the strongest inhibition towards isolates from the genera Aspergillus, Fusarium, Alternaria and Geotrichum. Only Penicillium isolates were able to grow. The level of resis-tance differed among Penicillium isolates. Cd-resistance of isolates was found to be independent of the pollution level at the site of isolation (Table 2). Similar results were reported by Baldrian and Gabriel (2002) who found that various strains of Piptoporus betulinus, originating from metal-contaminated sites, did not have the same level of tolerance to Cd. Roane and Pepper (2000) reported that the differences in resistance levels were probably due to the potential variation in the mechanism of resistance. Perfus-Barbeoch et al. (2002) have reported that Cd exerted severe inhibition of physiological processes in micro-organisms, such as growth and photosynthesis at concentration less than 2 ppm, and often in the ppb range. Similarly, Lilly et al. (1992) have found that the addition of only 0.1-0.2 mM Cd led to severe inhibition of a Schizophyllum commune strain. Massaccesi et al. (2002) reported that various filamentous fungi isolated from the sediments of industrially polluted streams removed 63 to 70% Cd during a 13-day growth period.
The screening test revealed heterogeneity in the heavy metal tolerance of our isolates (Figures 6, 7, 8, 9 and 10). Similar results were reported by other researchers (Verma et al., 2001; Zafar et al., 2007. The resistance against individual metals was much more dependent on the isolate than on the sites of its isolation. Brown and Wilkins (1985) suggested no relation between zinc tolerance of Amanita muscaria hooker and Paxillus involutus, and its concentration in the medium. Jones and Hutchinson (1988) demonstrated also that whatever the concentration in the medium, comparable tolerance rates were observed for isolates originating from metal-contaminated and uncontaminated sites.
Isolates of the same genus could present a marked difference in the levels of metal resistance. Major differ-rences in Cu, Zn and Cd tolerance have been found among our isolates. The variation in the metal tolerance may be due to the presence of different types of tole-rance processes or resistance mechanisms exhibited by different isolates. Sun and Shao (2007) demonstrated that both intracellular bioaccumulation and extracellular biosorption contributed to the high resistance of Peni-cillium sp. Psf-2 to lead. Sintuprapa et al. (2000) sugges-ted that ion exchange and intracellular accumulation in
040 Afr. J. microbiol. Res.
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Figure 7. Chromium index of tolerance among isolates. Method: 95.0 LSD percentages.
Ezzouhri et al. 041
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Figure 8. Copper index of tolerance among isolates. Method: 95.0 LSD percentages.
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Figure 9. Zinc index of tolerance among isolates. Method: 95.0 LSD percentages.
042 Afr. J. microbiol. Res.
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Figure 10. Cadmium index of tolerance among isolates. Method: 95.0 LSD percentages.
the form of polyphosphate precipitation are the mecha-nism of Zn2+ uptake by living cells of Penicillium sp. The resistance of the bacterium Ralstonia mettalidurans CH34 to lead is mediated by a P-type ATPase, which can transport lead out of the cell (Mergeay et al., 2003). Cop-per resistance in Aspergillus niger is due to an active process involving copper metallothionein synthesis (Kermasha et al., 1993). The detoxification of chromium by A. niger may be mediated by an enzymatic antioxidant system such as peroxidase, catalase and ascorbate peroxide (Srivastava and Thakur, 2006).From this preli-minary test, heavy metal-resistant filamentous fungi were selected and the minimal inhibitory concentration (MIC) to Cr, Pb, Cu and Zn was determined. No determinations were made for cadmium since the majority of the tested fungi were unable to grow in the presence of this metal. Minimum inhibitory concentrations (MICs) The MICs of the four metal ions against the studied fun-gal isolates are shown in Table 3. The growth rate of the fungi exhibited a lag, retarded, similar and enhanced rate of growth in the presence of heavy metal relative to the control. The growth pattern appears to suggest tolerance development or adaptation of the fungi to the presence of heavy metals. At lower metal ions concentrations, the tested fungal isolates were very resistant and exhibited strong growth, usually exceeding the control. Higher me-tal ions concentration caused a reduction in growth and
increased the length of the lag phase compared to the control. If the growth of fungi in a metal-free medium was observed after a day, metal prolonged the lag-phase, depending on the metal used and its concentration. In some cases, the fungus grew relatively quickly, even after a long lag phase. A reduction in the growth rate is a typical response of fungi to toxicants (Gadd, 1993), whereas the lengthening of the lag phase is not always present. Jones and Hutchinson (1988) reported an in-crease in lag time among different ectomycorrizal Basi-diomycetes cultivated on Zn- and Cd-amended media. Darlington and Rauser (1988) did not find any depen-dence of lag time on Cd concentration in P. involutus.
Zinc is essential for all organisms, although at high con-centrations it can be toxic (Balsalobre et al., 2003). The majority of fungi tested were able to grow at a zinc con-centration of 12.5 mM or higher (Figures 11 and 12). The fungal colour and morphology were both affected by high Zn concentrations. Their mycelia became thick in comparison with the control. Moreover, the growth of Penicillium isolates on agar media at high zinc ions concentrations was accom-panied with the secretion of a yellow sub-stance, which is probably a response to the stress imposed by the presence of Zn in the medium. The zinc MICs were in the range 15 - 20 mM, 12.5 - 20 mM and 12.5 - 15 mM for isolates of the genera Aspergillus, Penicillium and Fusarium, respectively. Similar results were reported by Vadkertiova and Slavikova (2006), who found that Pichia anomala, Candida krusei and Cryptococcus laurentii tolerated high concentrations of zinc (up to 20 mM). Castro-Silva et al. (2003) reported
Ezzouhri et al. 043
Table 3. Minimal inhibitory concentrations (MICs) of fungal isolates
MIC (mM) Fungi strains
Pb2+ Cr6+ Cu2+ Zn2+ Penicillium sp. (S3S) 12.5<MIC<15 20<MIC<25 15<MIC<20 15<MIC<20 Penicillium sp. (S4W) 20<MIC<25 12.5<MIC<15 7.5<MIC<10 12.5<MIC<15 Penicillium sp. (S5S) 7.5<MIC<10 15<MIC<20 7.5<MIC<10 15<MIC<20 Fusarium sp. (S5S) 12.5<MIC<15 20<MIC<25 10<MIC<12.5 12.5<MIC<15 Aspergillus niger (S5W) 20<MIC<25 12.5<MIC<15 15<MIC<20 20<MIC<25 Aspergillus niger (S5S) 25<MIC<30 10<MIC<12.5 15<MIC<20 15<MIC<20
Figure 11. Picture showing growth of Aspergillus niger (S5W) after exposure to different concentrations of zinc ions for 10 days.
Figure 12. Picture showing growth of Penicillium sp. (S3S) after exposure to different concentrations of zinc ions for 14 days.
044 Afr. J. microbiol. Res.
Figure 13. Picture showing growth of Aspergillus niger (S5S) after exposure to different concentrations of copper ions for 10 days.
Figure 14. Picture showing growth of Fusarium sp. (S5S) after exposure to different concentrations of copper ions for 10 days.
a similar level of zinc resistance by yeast strains isolated from coal-mining environments. Levinskaite (2001) de-monstrated that the growth rate of P. atratmentosum 25SL decreased slowly as the Zn ions concentration increased up to 40 mM.
Copper is a co-factor in numerous enzymatic processes and represents the third most abundant transition metal found in living organisms (Brandolini et al., 2002). The growth of all fungi tested was decreased after addition of copper in comparison with zinc. The blue colour of the isolates’ mycelia on agar media amended with copper may be due to binding of Cu ions to the fungal cell wall
(Figures 13 and 14). A similar observation was also made by Anand et al. (2006), who found that the mycelia of T. viride turned blue on agar media at all concentrations of Cu (II). The MICs for copper was in the range of 15 – 20 mM and 7.5 - 20 mM for isolates belonging to the genera Aspergillus and Penicillium, respectively. Similarly, iso-lates belonging to the genus Fusarium tolerated copper up to 12.5 mM. Collett (1992) found that amongst several strains of Antrodia vaillantii tested, some were able to tolerate up to 40 mM of Cu, whereas others were not able to grow at 3 mM of Cu in the medium.
Hexavalent chromium (Cr6+) is the toxic form of chro-
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Figure 15. Picture showing growth of Aspergillus niger (S5W) after exposure to different concentrations of chromium ions for 10 days.
Figure 16. Picture showing growth of Penicillium sp. (S5S) after exposure to different concentrations of chromium ions for 14 days.
mium released during industrial processes such as leather tanning and pigment manufacture (Srivastava and Thakur, 2006). All isolates studied tolerated more than 10 mM of Cr. The most tolerant isolate belonged to the genus Fusarium with a MIC of 25 mM. Penicillium and Aspergillus isolates were also very tolerant to chromium (up to 10 and 15 mM, respectively) (Figure 15). A similar level of resistance to chromate ions (10 mM) by Entero-
bacter cloacae under both aerobic and anaerobic condi-tions was reported by Wang et al. (1989). Bader (1999) found that Monilia sp. and Penicillium sp. showed high resistance to Cr up to 10 mM (0.52 g/l). The morphology of isolates was highly affected by the presence of Cr. Their mycelia became diffuse compared with the control (Figure 16). The growth rate of fungi tested was reduced and their conidiogenesis was also slowed down. Similar
046 Afr. J. microbiol. Res.
Figure 17. Picture showing growth of Aspergillus niger (S5S) after exposure to different concentrations of lead ions for 10 days.
result was reported in the study of Levinskaite (2002), where growth and conidiogenesis of T. viride and P. chrysogenum were slowed down at 1.5 - 2 mM Cr in the medium.
Lead ions appeared less toxic in comparison with the others metals studied. All isolates studies were able to grow in plates amended with lead with MICs ranging from 20 to 25 mM, 7.5 - 25 mM and 12.5 - 15 mM for the genera Aspergillus, Penicillium and Fusarium, respect-tively. Isolates of the Aspergillus genus showed a differ-rence in their tolerance to metals. The growth of Asper-gillus isolates on agar media containing a high lead con-centration was accompanied with the removal of white colouration of PDA medium around the colony (Figure 17). This is probably due to a period of adaptation where cells of the Aspergillus isolate synthesized some enzymes essential for the uptake of lead, as reported by Pelmony (1993).
The results obtained affirmed that the response of the isolates to heavy metals depended on the metal tested, its concentration in the medium and on the isolate considered. Our results were comparable with those reported by Badar et al. (2000), Verma et al. (2001), Bai and Abraham (2003), Malik (2004), Zouboulis et al. (2004), Zafar et al. (2007) and Yoshida et al. (2006). Metals such as copper and zinc are essential to biological actions, however, all metals, whether essential and inessential will tend to show toxicity at certain levels. Their toxicity may be presented differently, depending on the isolate and its site of isolation. Some isolates were tolerant, while others reacted negatively even at low metal ion concentrations. This could be explained by the heterogeneity of pollution in the locality from which the tested isolates originated. However, although some
authors found that micro-organisms isolated from conta-minated sites were more tolerant than those from natural environments (Massaccesi et al., 2002; Malik, 2004), there are also studies that do not confirm this (Jones and Hutchinson, 1988; Howe et al., 1997; Rudawska and Leski, 1998). They reported very little differences in metal tolerance between strains from polluted and unpolluted sites. The resistance of our isolates appears not to be correlated with the sites of their isolation. In this way, isolates originating from highly metal-contaminated sites have shown a comparative metal resistance to those iso-lated from uncontaminated sites (S1). Thus, the presence of metals may not have acted as a selective pressure for metal-resistant fungi. Indeed, five of the eight most Cr-sensitive strains in the present study, that is, Penicillium sp. (S2W and S2S), Fusarium sp. (S2W and S5W) and G. candidum (S2S), were collected in areas with a high level of Cr contamination. Howe et al. (1997) isolated a strain of Sclerodema citrinum from copper-polluted soil that was unable to grow even at a 10-imes lower copper concentration than other fungal strains tested. Baldrian and Gabriel (2002) found that three of the four most Cd-sensitive strains were isolated from areas with a high level of Cd contamination.
Various genera and also isolates of the same genus did not necessarily have the same heavy metal tolerance. The variation in the metal tolerance may be due to the presence of one or more strategies of tolerance or resis-tance mechanisms exhibited by fungi. It must also be taken into account that the contamination at the polluted sites is usually not caused by a single metal and that the selec-tion is probably driven either by the most toxic element or by more different metals acting synergistically (Baldrian and Gabriel, 2002). Gadd and Sayer (2000) reported that
the microbiota isolated from co-contaminated environ-ments could exhibit resistance to more than one ion and consequently, co-tolerance may be a common natural response.
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