Chemosphere 50 (2003) 137–143 www.elsevier.com/locate/chemosphere
Tolerance and uptake of heavy metals by Trichoderma atroviride isolated from sludge E. L opez Errasquın, C. V azquez
Departamento de Microbiologıa III, Facultad de Biologıa, Universidad Complutense de Madrid, Avenida Complutense s/n, 28040 Madrid, Spain Received 19 July 2001; received in revised form 8 August 2002; accepted 12 August 2002
Abstract A strain of Trichoderma atroviride, isolated from sewage sludge obtained from a water treatment plant located in Madrid (Spain), has been studied for tolerance to heavy metals (copper, zinc and cadmium) and for its capacities to uptake these metals. It was found that this fungus is capable of surviving high metal concentrations, apparently as a result of the natural selection of resistant cells. Also, growth and metal uptake have been assayed in samples where the fungus was cultured in the presence of a single metal and in the presence of a combination of two or three cations, where additive and synergistic interactions were observed. Finally, metal uptake by this strain has been studied under diﬀerent nutritional conditions. It was found that the highest values of metals removal were achieved for autolysed mycelia while the lowest levels were observed in the presence of glucose. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Heavy metals; Trichoderma atroviride; Tolerance; Uptake; Sewage sludge
1. Introduction Toxic metals, especially cadmium, copper, mercury, manganese, and zinc, are increasingly being released into the environment from industrial wastewater and other human activities. Some, for example, copper, iron, manganese and zinc, are essential micro-nutrients for most, if not all, living organisms. One of the most important functions of micro-nutrients is their role in metalloenzymes. It has also been suggested that cations increase membrane stability and that they may also play speciﬁc roles in nucleic acid structure, functions and metabolism (Dedyukhina and Eroshin, 1991). However, when the concentrations of beneﬁcial metals in the environment are excessively high, or when metals with no known essential biological functions are present, for
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instance, mercury, lead, or cadmium, can become toxic (Gadd, 1986; Dedyukhina and Eroshin, 1991). Consequently, if wastewater is untreated before being discharged, there may be adverse repercussions for the environment. The impact of heavy metals on the environment and their accretion through the food chain have promoted research aimed at developing alternative, eﬃcient and low cost wastewater puriﬁcation systems (Wilhelmi and Duncan, 1995). Conventional methods for removing dissolved heavy metals include chemical precipitation and sludge separation, chemical oxidation or reduction, ion exchange, reverse osmosis, ﬁltration, adsorption using activated charcoal, electrochemical treatment and evaporative recovery (Volesky, 1994; Domenech, 1998). However, these techniques can be expensive, they may not always be feasible and their metal-binding properties are non-speciﬁc (Price et al., 2001). These are the reasons why alternative processing methods, such as those using microbial biomass, are now being considered more seriously (Volesky, 1994).
0045-6535/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 2 ) 0 0 4 8 5 - X
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Microbial biomass can remove and concentrate a variety of metal ions from aqueous solutions. The uptake of heavy metals by micro-organisms can be divided into two categories. One, known as biosorption, consists of a metabolism-independent binding to negatively charged free groups in several biopolymers that form the fungal cell wall. The other is an energy-dependent metal inﬂux known as bioaccumulation (Cervantes and Gutierrez-Corona, 1994; Alguacil and Merino, 1998; Gomes et al., 1998). As a consequence, micro-organisms have had to develop diﬀerent mechanisms of metal resistance that include cell membrane metal eﬄux (Kamizomo et al., 1989), intracellular chelation by metallothionein proteins (Presta and Stillman, 1997) and glutathione-derived-peptides called phytochelatins (Kneer et al., 1992) and metal compartmentalization in vacuoles (Volesky et al., 1993). Interest in processes involving heavy metal uptake by micro-organisms has increased considerably in recent years, in particular because of the biotechnological potential of micro-organisms in metal removal and/or recovery. For example, biosorption has a possible application as a process for the removal and concentration of heavy metals from wastewater. Indeed, microorganisms are potent biosorbents and may represent a base for developing suitable technology for that purpose (Nourbakhsh et al., 1994). Also, biosorbents may be used where more expensive physicochemical methods are not feasible and they are of particular value when working with large volumes of wastewater containing relatively low metal concentrations (Volesky, 1987). However, whereas conventional methods, such as synthetic ion exchangers, can now be considered as mature technologies, biosorption is still in its developmental stages, and further improvements in both performance and costs can be expected (Volesky, 1999). At present in the literature there is growing interest in the application of fungal biomass for bioremediation and a broad range of results have be reported. Traditionaly, species such as Mucorales have been described as good biosorbents (Remacle, 1990). Also, the mycelium of Rhizopus has been reported to be an excellent biosorbent for lead, cadmium, copper, zinc and uranium (Volesky, 1994), Aspergillus niger has been used to remove metals (Akthar and Mohan, 1995; Bosshard et al., 1996) and Fusarium ﬂocciferum has been used to eliminate cadmium and nickel from industrial eﬄuents (Delgado et al., 1998). The objectives of this work are the isolation of ﬁlamentous fungi indigenous to environments contaminated by heavy metals and the study of their tolerance and uptake capabilities; this work being orientated towards their application as biosorbents. Trichoderma atroviride, a fungus isolated from metal-polluted wastewater sludge, was selected for our study because it was the most abundant species present in the sludge
analysed. This fungus has been investigated for its ability to grow in the presence of three heavy metals, i.e. copper, zinc and cadmium. Copper and zinc were chosen for our study because of their high concentrations in the sludge analysed. Cadmium was also included as it is the most toxic metal found in the sludge and because it is ubiquitously distributed throughout the world. Metal uptake from culture media was also analysed. For this, individual metals and combinations of metals were added to the medium. Metal combinations were added in order to compare the response of the fungus when exposed to individual and combined metals. Finally, growth and metal removal were studied under diﬀerent nutritional conditions to try to determine the possible mechanism/s of metal uptake and tolerance.
2. Materials and methods 2.1. Micro-organism used and maintenance A strain of T. atroviride was isolated from a sludge sample taken from a wastewater treatment plant situated in the Autonomous Community of Madrid, Spain. Fungi were detected and isolated using the dilution plate method: dilutions of 101 or 103 were prepared and 1 ml of dilution was plated on Sabouraud dextrose agar containing gentamicin–chloramphenicol (Biomedics). The fungus was maintained on solid potato-dextrose agar (PDA, Scharlau) at 25 °C and, for use as stock culture, on PDA slants at 4 °C or as micro-conidial suspensions in 15% glycerol at )80 °C. 2.2. Fungal growth screen The metal tolerance of T. atroviride was determined by measuring the mycelial biomass. The fungus was grown in 100 ml Erlenmeyer ﬂasks containing 20 ml of Sabouraud liquid medium (Scharlau), which consisted of 1% peptone and 2% dextrose, pH 5.8. Peptone, rather than other nitrogen-containing organic substrates, was used because of its comparatively low metal binding (Garcıa-Toledo et al., 1985). The medium was amended with progressively increasing concentrations (in steps of 50 mg/l (ppm)) of copper, zinc or cadmium until a lethal level was achieved for each metal. The cultures were inoculated with 7 mm mycelial discs removed from the margins of 7-d-old colonies and incubated at 28 °C in an orbital shaker at 150 rpm for 72 h. The mycelia were harvested by ﬁltration through a nitrocellulose ﬁlter with a pore size of 0.45 lm (Millipore) and were dried overnight at 65 °C to determine the dry weight. Three replications of all assays were performed.
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2.3. Growth and metal uptake from multiple-metals media
2.5. Metal determination
In further studies to access possible interactions between the copper, zinc and cadmium, combinations of two or three of these metals were added to the culture medium. For this the T. atroviride was cultured in 250 ml Erlenmeyer ﬂasks containing 50 ml of broth (1% peptone and 2% dextrose) amended with a non-inhibitory concentration of each metal (50 mg/l) in order to maintain metal stress. Cultures were incubated for 96 h under the same conditions as described before. After this incubation time, the mycelial masses were harvested in order to determine their dry weights and culture broths were retained in order to measure the metal concentration in the ﬁltrates. This experiment was performed twice.
Metal concentration in the ﬁltrates was measured by inductively coupled plasma atomic emission spectrometry. Metal uptake was estimated as the amount of metal (mg) per unit of mycelium dry weight (g) (Delgado et al., 1998; Vieira and Volesky, 2000):
2.4. Metal removal under diﬀerent nutritional conditions As the composition of the medium may have a direct eﬀect on metal uptake, metal uptake was analysed under diﬀerent nutritional conditions. Mycelia produced after 72 h (160 mg) in Sabouraud liquid medium was utilised as a preinocule and was transferred to one of three diﬀerent conditions: Sabouraud liquid medium (1% peptone, 2% dextrose), dextrose-free liquid Sabouraud medium (1% peptone) and saline solution. All these broths contained a non-inhibitory concentration of one metal, e.g. 200 mg of copper/l, 125 mg of zinc/l, or 1.25 mg of cadmium/l, and they were incubated in an orbital shaker at 28 °C for 96 h at 150 rpm. The dry weights of the biomass and the metal concentrations in the ﬁltrates were determined and the assay was analysed in duplicate.
q ¼ ½ðCi Cf Þ=mV where q ¼ metal uptake (mg metal/mg biomass), Ci ¼ initial metal concentration (mg/l), Cf ¼ final metal concentration (mg/l), m ¼ quantity of dry biomass (mg), V ¼ suspension volume (ml). 3. Results and discussion T. atroviride was isolated from a sludge sample polluted with heavy metals, which included copper, zinc and cadmium. The concentration of each metal in the sample of sludge analysed was 215 ppm of copper, 1281 ppm of zinc and 2.25 ppm of cadmium (these data correspond to the mean value for four measurements). In vitro assays with T. atroviride conﬁrmed a high tolerance to copper, zinc and cadmium (Fig. 1). In the case of copper, we observe that T. atroviride survived at concentrations between 0 and 300 mg/l with almost constant levels of biomass. However, the growth dramatically decreased at 350 mg/l, where an 80% reduction was observed, while no growth was detected at 400 mg/l. Furthermore, this micro-organism was capable of surviving at higher concentrations of zinc, i.e. up to 750 mg/l, with a more gradual reduction in growth, e.g. a 50% reduction at 200 mg/l. Finally, the most toxic metal was cadmium, as could be expected, with a 50% reduction in
Fig. 1. Zinc, copper and cadmium toxicity in T. atroviride. The dry weight of 72-h-old mycelia is expressed as percentage growth.
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Fig. 2. Biomass weight for individual metals and for binary or ternary metal combinations.
biomass at 125 mg/l and no detectable growth at 300 mg/l. However, although cadmium was the least tolerated metal, T. atroviride survived at concentrations which result toxic to other micro-organisms (Baldrian et al., 1996). Other authors have reported tolerance levels that are similar to those reported here. For example, GarcıaToledo et al. (1985) observed that Rhizopus stolonifer and Cunninghamella blakesleeana ceased to grow with 450 and 500 mg/l of copper, respectively in the culture medium. However, other fungi are less resistant to copper, for example, Aspergillus ﬂavipes tolerates 200 mg/l (Babich and Stotzky, 1983) at maximum. Also, Balsalobre (2000) found the resistance levels of yeast to zinc to be between 65 and 2288 mg/l. In the case of cadmium, Baldrian et al. (1996) observed that the basidiomycetes Phanerochaete chrysosporium, Pycnoporus cinnabarinus and Pleurotus ostreatus stopped growing when a concentration of 11.2 mg/l of cadmium was added to their culture medium. It is therefore apparent that these fungi are much more sensitive to cadmium that the strain isolated for this work. This high tolerance to copper, zinc and cadmium observed in T. atroviride could be attributed to the fact that the fungus was isolated from a sludge sample containing high levels of metals. It is known that microorganisms isolated from natural environments contaminated with heavy metals often exhibit tolerance to multiple pollutants because they have adapted to such environments (Ashida, 1965). Indeed, several authors have demonstrated in vitro adaptation of fungi to heavy metals (Garcıa-Toledo et al., 1985). Industrial eﬄuents are usually composed of several diﬀerent metal ions. As a consequence, the release of such eﬄuents into the environment expose native biota to multiple pollutants. The response of micro-organisms to exposure to multiple metals may diﬀer from their response to individual metals, and additive, synergistic or antagonic interactions may occur between metals (Babich and Stotzky, 1983). Therefore, the biomass
obtained and the metal uptake recorded when T. atroviride was cultured in the presence of a single metal were compared with the levels recorded when two or three metals were present in the culture medium. In Fig. 2, we observe that copper, zinc and cadmium, when assayed as single metals (50 mg/l), only moderately inhibit the growth of T. atroviride compared to the biomass obtained under control conditions (without metal). This was also compared to the cases where a combination of copper plus zinc or copper plus cadmium (at a concentration of 50 mg/l per metal) were assayed. However, Fig. 2 reveals a substantial decline in biomass when T. atroviride was cultured with zinc plus cadmium or with a combination of the three metals. Thus, the toxicity of the combination of zinc and cadmium for the growth of T. atroviride was greater than the sum of the toxicity of individual metals. This ﬁnding was independent of the presence or absence of copper. Therefore, there is a synergistic interaction between zinc and cadmium. Combinations of several ions also aﬀected metal uptake, resulting in an important reduction in the q values for most cases (Fig. 3). This decrease varied from 75% (uptake of copper and zinc in a combination of these) to 15% (uptake of copper in the presence of cadmium). Indeed, these results are in agreement with those obtained by several authors (Townsley et al., 1986; Castro et al., 1992; Chang and Huang, 1998). These authors concluded that the selective accumulation of heavy metals by micro-organisms is determined by interionic competition in which metal cations compete for the binding sites on the cell wall. However, we have observed a contrary eﬀect, i.e., a stimulation of uptake when other cations were present in the medium. The most important result was that of copper in a threemetal combination, where a doubling of uptake was observed compared to copper alone. In addition, when the removal values obtained by several authors for other fungi are compared with those for T. atroviride the removal rates for T. atroviride are higher. For example, A. pendulus can remove 0.09 mg copper/g fungal dry weight
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Fig. 3. Metal uptake by T. atroviride when cultured with individual metals and binary or ternary metal combinations.
and A. niger can remove 0.52 mg copper/g dry weight (Price et al., 2001). Some of the highest copper (1.59) and cadmium (2.68) removal amounts, expressed in mg of metal retained per 100 mg biosorbent, were obtained with dried mycelium of Rhizopus arrhizus (Fourest and Roux, 1992). Copper, zinc and cadmium removal by the T. atroviride biomass was studied under various diﬀerent nutritional conditions that might inﬂuence metal uptake, namely, Sabouraud liquid medium, dextrose-free Sabouraud liquid medium and saline solution. Fig. 4 shows the values of the estimated metal uptake, and that the same tendency was observed with the three cations assayed. The results depicted in Fig. 4 indicate that the best nutritional conditions for metal uptake were furnished by the saline solution, followed by the dextrosefree Sabouraud liquid medium, in which the biomass was greatly reduced and the mycelia were partially au-
Fig. 4. Metal uptake under diﬀerent nutritional conditions: Sabouraud medium, glucose-free Sabouraud medium and saline solution.
tolysed. It should be noted that T. atroviride remove almost 16 fold more zinc in saline solution (28.2 mg/l) than in Sabouraud medium (1.8 mg/l). Also, incubation of live T. atroviride in the presence of dextrose (1%) did not result in increased metal uptake. Jones and Gadd (1990) and Ross (1993) have previously indicated that glucose resulted in an increase in the uptake of metal ions. These workers reported that stimulation of metal uptake by glucose is the result of enhanced membrane transport protein synthesis and/or increased transplasmalemma proton motive forces. Avery and Tobin (1992) showed that the presence of glucose resulted in
E. Lopez Errasquın, C. Vazquez / Chemosphere 50 (2003) 137–143
stimulation of Sr2þ uptake, which was attributed to metabolism-dependent accumulation. When the mycelia were transferred to medium without dextrose, T. atroviride was capable of uptaking more metal than in the presence of dextrose. Two possible and alternative explanations for our ﬁndings can be postulated here. The ﬁrst is the existence of an active detoxiﬁcation process when there is an abundant source of carbon, thereby reducing the amount of metal in the cell; the second is the triggering of autolysis when no carbon source is available. The high levels of biosorption recorded for the saline solution were attributed to operation of the same mechanism: autolysed mycelia. These results suggest that uptake occurred as a consequence of physical binding to cell surfaces rather than of an active process, since the mycelia appeared to be partially degraded. Our results also suggest that the fungal cell wall plays an important role in metal uptake (biosorption). If biosorption takes place by physical binding to charged groups on the cell wall, it would follow that the breakdown of the fungal wall could account for the high levels of uptake. The increased biosorption due to autolysis of the fungus may be due, not only to the increased surface area of the autolysed cell wall, but also to exposure of intracellular binding sites after denaturation of the biomass (Avery and Tobin, 1992), thus providing access to additional negatively charged binding sites in addition to those present on the surface of the fungal cells. Interest in the metal uptake by micro-organisms is not new. However, recently there has been a considerable resurgence of interest in the ﬁeld, in particular in the biotechnological potential of microbial biomass as a mechanism for metal uptake. However, as certain researchers have noted, most studies on microbial uptake of heavy metals have employed laboratory stock cultures with the result that researchers have had to extrapolate their ﬁndings to potentially desirable industrially derived micro-organisms. As in several previous studies (Horikoshi et al., 1981; Nakajima et al., 1981; Townsley et al., 1986; Volesky, 1987; Castro et al., 1992; Brady and Duncan, 1994; Delgado et al., 1998; Volesky, 1999), the present experiment has, as its ultimate goal, the development of a metal removal process based on micro-organisms. However, in contrast to other studies (Townsley et al., 1986) the novel feature of our experiment is that we have investigated metal uptake by the biomass of a naturally occurring micro-organism that had been isolated from sludge polluted with heavy metal ions. With this in mind, the results of our study on metal uptake allow certain potentially interesting and useful conclusions to be drawn. Similar studies have observed that the biosorptive function, which is known to be a property of the cell wall, may be enhanced by rendering organisms non-viable either by heat or chemical sterilisation or by crushing (Galun et al., 1983; Townsley et al.,
1986; Avery and Tobin, 1992; Ben Omar et al., 1997). Thus, naturally (autolysis) or artiﬁcially (by heating or crushing) degraded mycelia should exhibit higher uptake levels, probably as a consequence of a larger available surface area and of the destruction of the cell membranes, thereby resulting in exposure of intracellular components and surface binding sites (Townsley et al., 1986; Avery and Tobin, 1992). Further studies designed to elucidate the mechanisms of metal tolerance developed by T. atroviride are needed. Acknowledgements The authors thank Dr. K.J. McCarthy for his helpful revision of the English version of this manuscript. This research was supported by the Comunidad Aut onoma de Madrid (CAM) under projects 07M/0280/1999 and 07M/0029/1999. The authors are thankful to Dr. M. Larrea and A. Miranda of the Centro de Espectrometrıa At omica, UCM, Madrid, for metal analysis. E. L opez Errasquın received a fellowship from the Comunidad Aut onoma de Madrid.
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