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Tolerance and biosorption of copper and zinc by Pseudomonas putida CZ1 isolated from metalpolluted soil XinCai Chen, JiYan Shi, YingXu Chen, XiangHua Xu, ShengYou Xu, and YuanPeng Wang
Abstract: A strain of Pseudomonas sp. CZ1, which was isolated from the rhizosphere of Elsholtzia splendens obtained from the heavy-metal-contaminated soil in the north-central region of the Zhejiang province of China, has been studied for tolerance to copper (Cu) and zinc (Zn) and its capacities for biosorption of these metals. Based on 16S ribosomal DNA sequencing, the microorganism was closely related to Pseudomonas putida. It exhibited high minimal inhibitory concentration values (about 3 mmol Cu·L–1 and 5 mmol Zn·L–1) for metals and antibiotic resistance to ampicillin but not to kanamycin. Based on the results of heavy metal toxicity screening, inhibitory concentrations in solid media were lower than those in liquid media. Moreover, it was found that the toxicity of Cu was higher than that of Zn. Pseudomonas putida CZ1 was capable of removing about 87.2% of Cu and 99.8% of Zn during the active growth cycle, with specific biosorption capacities of 24.2 and 26.0 mg·L–1, respectively. Although at low concentrations, Cu and Zn slightly damage the surface of some cells, P. putida demonstrated high capacities for biosorption of Cu and Zn. Since P. putida CZ1 could grow in the presence of significant concentrations of metals and because of its high metal uptake capacity in aerobic conditions, this bacterium may be potentially applicable in bioreactors or in situ bioremediation of heavy-metal-contaminated aqueous or soil systems. Key words: Pseudomonas putida, copper, zinc, tolerance, biosorption. Résumé : Une souche de Pseudomonas sp. fut isolée de la rhizosphère de Elsholtzia splendens obtenue d’un sol contaminé aux métaux lourds du centre nord de la province de Zhejiang en Chine et la tolérance au cuivre (Cu) et au zinc (Zn) ainsi que les capacités de biosorption de ces métaux furent étudiées. Selon le séquençage de l’ADN ribosomal 16S, le micro-organisme était fortement apparenté à Pseudomonas putida. Il a démontré des valeurs de concentration inhibitrice minimale élevées (environ 3 mmol Cu·L–1 et 5 mmol Zn·L–1) pour les métaux et une résistance à l’ampicilline mais non à la kanamycine. À la lumière des résultats sur le criblage de la toxicité aux métaux, la concentration inhibitrice sur des milieux solides était inférieure à ceux en milieu liquide. De plus, nous avons constaté que la toxicité du cuivre était supérieure à celui du zinc. Pseudomonas putida CZ1 fut capable d’enlever environ 87,2 % du Cu et 99,8 % du Zn pendant le cycle de croissance active avec une capacité de biosorption spécifique de respectivement 24,2 et 27,0 mg·L–1. Bien que le cuivre et le zinc aient légèrement endommagé la surface de certaines cellules à de basses concentrations, les capacités de biosorption supérieures ont tout de même été démontrées pour le cuivre et de zinc. Puisque P. putida CZ1 pouvait croître en présence de concentrations significatives de métaux et à cause de sa capacité élevée d’absorption des métaux en conditions aérobies, cette bactérie pourrait être potentiellement applicable à des bioréacteurs ou à de la bioremédiation in situ de systèmes aqueux ou de sols contaminés aux métaux lourds. Mots clés : Pseudomonas putida, cuivre, zinc, tolérance, biosorption. [Traduit par la Rédaction]
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Introduction Human activities, such as mining operations and the discharge of industrial wastes, have resulted in the accumulation of metals in the environment (Babich and Stotzky 1980; Received 12 October 2005. Revision received 5 December 2005. Accepted 12 December 2005. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 4 April 2006. X. Chen, J. Shi, Y. Chen,1 X. Xu, S. Xu, and Y. Wang. Department of Environmental Engineering, Zhejiang University, HangZhou, 310029, China. 1
Corresponding author (e-mail:
[email protected]).
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Wong 1993). Some metals (e.g., Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, and Zn) are essential micronutrients for most, if not all, living organisms. One of the most important functions of micronutrients 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 some beneficial metals in the environment are excessively high, e.g., copper (Cu) or zinc (Zn), they can become toxic to these microorganisms and humans (Dedyukhina and Eroshin 1991; Gadd 1986). In recent years, the interest in the interactions of heavy metals with microorganisms has increased with the impact of
doi:10.1139/W05-157
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heavy metals on the environment and their accretion through the food chain (Wilhelmi and Duncan 1995). The influence of microbiological processes on the contamination of the environment by toxic metals and radionuclides is of economic and environmental significance (Gadd 1993, 2001). The study of these interactions has focused in particular on the selection of metal-resistant microorganisms from polluted environments (Hassen et al. 1998a; Hiroki 1992; Kunito et al. 1997) and on the possibility of using these bacteria to detoxify polluted environments (Rohit and Sheela 1994; Taniguchi et al. 2000; Wang et al. 1997). Microorganisms have been shown to adapt to these environments by acquiring specific resistance systems (Gadd 1990; Rosen 1996; Silver and Walderhaug 1992; Silver and Phung 1996; Trevors et al. 1986). Microorganisms have evolved resistance mechanisms that lead to the selection of resistant variants that can tolerate metal toxicity (Rosen 1996; Silver and Phung 1996; Roane and Kellogg 1996). Microorganisms resistant to antibiotics and tolerant to metals appear to be the result of exposure to metal-contaminated environments that cause coincidental selection for resistance factors for heavy metals and antibiotics (Foster 1983; Ramteke 1997). Cellular heavy metal accumulation processes are grouped together under the general term of biosorption. The mechanisms of biosorption may involve intracellular uptake and storage via active cationic transport systems, surface binding, or some undefined mechanisms (Gadd 1990). The biological and chemical characteristics of these uptake processes are important not only to improve the understanding of the role of metallic ions in basic cellular function but also for the potential use of cells with these characteristics in the detoxification of industrial effluents and the bioremediation of metalcontaminated soils (Yilmaz 2003). Various microbial species, such as Pseudomonas, have been shown to be relatively efficient in the bioaccumulation of different heavy metals from polluted effluents (Hussein 1999; Hussein et al. 2001). Accordingly, the aim of this work was to study the growth response of a recently isolated Pseudomonas sp., the CZ1 strain, obtained from heavy-metalcontaminated soil, and to evaluate the heavy metal biosorption capacity of this bacterium in the presence of a single metal and a combination of two metals during the active growth cycle. The biosorption of Cu and Zn was examined in the present study because of their widespread occurrence in soil polluted by a copper mining plant (Gümgüm et al. 1994). Furthermore, the impact of Cu and Zn ions on the cell surface after copper and zinc biosorption was evaluated using a transmission electron microscope (TEM).
Materials and methods Isolation and characterization of bacterium The soil samples were collected in sterile plastic bags from the rhizosphere of Elsholtzia splendens near an old copper mine located at the north-central of Zhejiang province of China and transported on ice to the laboratory. These soils were particularly high in Cu and Zn, up to 12 751.5 and 16 040 mg·kg–1, respectively. All plastic and glassware used in these studies were washed in 2 mmol·L–1 HNO3 and rinsed with double-deionized water before use to avoid metal contamination. The bacteria were isolated on mineral salts
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agar plates, with 5.0 g·L–1 glucose as the carbon source, and supplemented with different concentrations of heavy metal salts by the standard pour-plate technique (Koch 1981). Plates were incubated at 30 °C for ~1–3 days. The microorganisms that could tolerate 5.0 mmol·L–1 Cu or 5.0 mmol·L–1 Zn were selected and identified based on morphological features and biochemical properties using traditional methods. For further identification, genomic DNA was isolated, and the 16S rRNA gene was amplified by PCR using two general bacterial 16S rRNA primers (BSF8/20; 5′-AGAGTTTGA TCCTGGCTC AG-3′, BSR1541/20; 5′-AAGGAGGTGATC CAGCCGCA-3′). The purifying of amplified 16S rRNA gene and sequencing were carried out by Shanghai Huanuo Biotechnology Co., Ltd. The determined sequences were compared with 16S rRNA gene sequences obtained from GenBank and ribosomal RNA databases. Molecular phylogenetic studies showed that strain CZ1 was a member of the P. putida subgroup. The nucleotide sequence coding for the 16S rRNA gene of P. putida CZ1 has been submitted to the GenBank database under accession No. DQ181650. Determination of minimal inhibitory concentration Analytical-grade CuSO4·5H2O and Zn(NO3)2·6H2O were used to prepare 0.1 mmol·L–1 stock solutions. The metal solutions were sterilized using 0.22 µm pore size sterile filters. Nutrient agar and mineral salts (as mentioned above) agar plates were supplemented with different concentrations (~0.1– 6.0 mmol·L–1) of heavy metals, adjusted to pH 5.0, and then inoculated. Analysis of metal resistance was performed by mid-log-phase cultures in 5 mL of liquid medium. Cells were streaked on nutrient agar and mineral salts agar plates containing different concentrations of metal salts. Growth was recorded after 1–3 days of incubation at 30 °C. The lowest concentration of metal that completely prevented growth was termed as the minimal inhibitory concentration (MIC). Antibiotic resistance To determine resistance to antibiotics, Pseudomonas CZ1 was tested against ampicillin and kanamycin. The bacterium was grown in nutrient medium at 30 °C for 24 h. A 0.1 mL fraction from the culture was plated onto nutrient agar plates containing different concentrations of antibiotics. Growth was recorded after 24 h of incubation at 30 °C. Effects of metals on bacterial growth These experiments involved growing the bacterium in liquid media that contained a range of metal concentrations (~1.0–8.0 mmol·L–1). A culture grown in the absence of metal served as the control. Nutrient medium (100 mL, adjusted to pH 5.0) with different concentrations of metal salts was inoculated with 2 mL of mid-log-phased cultures of P. putida CZ1. The cultures were incubated by shaking at 30 °C for 51 h, during which the growth was monitored as absorbance at 600 nm using a spectrophotometer. Metal accumulation Liquid cultures were grown in 100 mL nutrient medium with added metals (each at 0.5 mmol·L–1 final concentration) in 250 mL Erlenmeyer flasks by shaking at 30 °C. Culture samples (2 mL) drawn at different stages were centrifuged at © 2006 NRC Canada
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Can. J. Microbiol. Vol. 52, 2006 Table 1. Heavy metal tolerance cultured in different conditions and antibiotic sensitivity profile in Pseudomonas putida CZ1. Minimum inhibitory concn. (mmol·L–1) Metal
Isolated
Nutrient plates test
Mineral salts plates test
Antibiotic (concn.; mg·L–1)
Cu Zn
5.0 5.0
1.0 3.0
3.0 4.0
Ampicillin (100) Kanamycin ( Pb > Cu > Mn. This difference in toxicity could be explained by © 2006 NRC Canada
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Fig. 3. Transmission electron microscopy images of living Pseudomonas putida CZ1 cells (a–b) before accumulation; (c–d) after Cu accumulation; and (e–f) after Zn accumulation. Magnifications are at (a) × 12 000, (b) × 40 000, (c) and (d) × 30 000, (e) × 40 000, and (f) × 50 000. Arrows show some of the changes of the cells before and after metal accumulation.
the conditions of bacterial isolation and the selectivity of microbial culture techniques adopted in each study, particularly with respect to the nature and specificity of growth media (Hassen et al. 1998b). The inhibitory concentrations of Cu and Zn in solid media were lower than those in liquid
media (except for Cu in mineral salts medium), in contrast with the results of Hassen et al. (1998a, 1998b) and Yilmaz (2003), which showed that inhibitory concentrations in solid media were much higher than those in liquid. As pointed out by Trevors et al. (1985), there is a problem with defining © 2006 NRC Canada
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exactly what is meant by resistance to heavy metals. To determine the MICs of heavy metals, most studies have used the medium that best supports the growth of the microorganisms or a group of microorganisms. A number of problems are associated with this approach. Metal-binding capacity of the microorganisms, chelation to various components of the media, and formation of complexes can each cause a reduction in the activities of free metals. Therefore, the activity of a free metal ion, ordinarily considered to be the toxic metal species that ultimately determines the microbial response to the metal, rarely approaches the total metal concentration added to media (Angle and Chaney 1989). Of particular importance is the sorption or chelation of metals to unspecified organic compounds found in most biological media. For example, interactions of mercury with the constituents of Luria–Bertani broth (LB) have been studied by Chang et al. (1993) during the growth of P. aeruginosa PU21 (possessing a mercuric reductase). This study showed that 30%–40% of the mercury (Hg2+) formed complexes with tryptone and yeast extract of the medium within 140 h of contact. As previously stated, metals and various components of nutrient media could interact, thus complicating the interpretation of the data. The test of toxicity in solid media investigated here could be useful in the evaluation of metal toxicity in sewage sludge and contaminated soil, where conditions of diffusion, complexation, and availability of metals were different from those observed in liquid media. The speciation of metals was a very important determinant in understanding the quantitative aspects of metal toxicity. In spite of its limits, described previously, the liquid media test allowed a good evaluation of metal toxicity in polluted environments, such as industrial effluents, incinerator residues, landfill municipal refuse, and sewage sludge leachates (Hassen et al. 1998a). Since most metal–microbe interactions are initiated at the level of uptake, the uptake mechanism is likely to be closely linked to the mechanism of metal resistance in the microorganism. The heavy-metal-tolerant bacterium P. putida CZ1 was capable of removing significant concentrations of Cu and Zn during the active growth cycle. In previous studies, it had been shown that the metal biosorption capacity of microbial cells varies with their growth phase (Maceskie and Dean 1984; Volesky et al. 1993). In our studies, high amounts of Cu and Zn were accumulated during the late-logarithmic phase or stationary phase of strain CZ1. The Cu uptake profile was similar to that found in Cu biosorption by Escherichia coli during the late-logarithmic phase (Baldry and Dean 1980), while in another study, reported by Chang et al. (1997), Cu biosorption was not affected by the growth phase. They indicted that the number of Cu-adsorbing sites on the cells was independent of cell growth. It has been reported that the cell envelopes and peptidoglycan layer isolated from P. putida 5-x play important roles in the adsorption of Cu2+ (Wang et al. 2003). Thus, it is likely that the cell wall components of P. putida CZ1 play roles in the increase of Cu adsorption capacity for cells from the late-logarithmicphase or stationary-phase cultures. In view of the results of metal accumulation experiments, it was concluded that soil isolate CZ1 was not only tolerant to heavy metals, but it also bound considerable amounts of heavy metals from the growth
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medium. This suggests that this bacterium may be able to remove metals from different sources of pollution. The influence of Cu and Zn ions on P. putida CZ1 cells were evaluated using TEM images. Some forms of damage on cell surfaces were observed in the cell section images obtained with the TEM technique. Though Cu and Zn below the MICs slightly damage the surface of some living cells, CZ1 cells still demonstrated higher capacities for biosorption of Cu and Zn compared with those reported for other bacteria (Yilmaz 2003). Moreover, visible electron dense particles (which would indicate the presence of Cu precipitates) in the living cells after Cu and Zn biosorption may indicate that the mechanism of biosorption of Cu and Zn involves not only surface binding but also intracellular uptake and storage via active cationic transport systems. In general, while microbial metal resistance includes a variety of strategies to deal with toxic metal concentrations in the environment (Roane et al. 1996), these strategies are either to prevent entry of the metal into the cell or to actively pump the metal out of the cell. Such resistance can be divided into two classes: metal dependent and metal independent (Roane and Pepper 1997). Pseudomonas putida CZ1, with a metal-dependent intracellular accumulation of Cu and Zn, came from an old copper mine with 12 751.5 mg·kg–1 Cu and 16 040 mg·kg–1 Zn. Under high metal stress, it may become necessary for resistant microorganisms to use specifically directed metal-resistance mechanisms, including ATP-dependent efflux pumps and intracellular sequestration, which may be more effective at detoxifying the increased metal penetrating cell membranes under high bioavailable metal conditions. Further investigations of the components of the visible electron dense particles will greatly improve our understanding of the uptake mechanisms of Cu and Zn by P. putida CZ1 cells. The results of this study show the potential applicability of the recently isolated heavy-metal-tolerant soil strain P. putida CZ1 in the treatment of heavy-metal-containing solutions or soil. Because efficient metal removal and growth over a range of metal concentrations under aerobic conditions are advantageous, this organism may be employed for metal remediation in simple reactors or even in situ. However, many aspects of metal–microbe interactions remain unexplored, and further development and application are necessary.
Acknowledgements This work was funded by the National Key Natural Science Foundation of China (40432004), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Zhejiang Province (G50437), and the Natural Science Foundation of Zhejiang Province (Y504109). Special thanks go to Mr. Perera Anton for revising the English. The authors also thank the centre of analysis and measurement of Zhejiang University for making electron microscope photographs.
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