Uptake of zinc and copper by halophilic bacteria ... - Springer Link

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Uptake of Zinc and Copper by Halophilic Bacteria Isolated from the Dead Sea Shore, Jordan FOUAD A. AL-MOMANI,*,1 ADNAN M. MASSADEH,2 AND YAZAN A. HADAD1 1

Department of Biotechnology, Faculty of Science and Art, and 2Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, P. O. Box 3030, Irbid 22110 Jordan Received April 18, 2006; Revised June 22, 2006; Accepted November 10, 2006

ABSTRACT Ten Gram-positive and gram-negative bacterial cultures were recovered from nine water, mud, and soil samples collected from the Dead Sea shore at Suwaymah. All bacterial cultures were able to grow at 10% NaCl and at 45°C. They were able to grow in nutrient media supplemented with 1250 ppm of Zn. Most of them, except cultures 2 and 8, were able to grow in nutrient medium supplemented with 1000 ppm of Cu. After 2 wk of incubation of these 10 cultures at different concentrations (5, 25, 100, and 500 ppm), stock solutions of both Zn and Cu elements, the maximum absorption using atomic absorption spectrometry for Zn was achieved by culture 7 at 11.2%, 1.0%, 38.4%, and 84.54%, respectively, from the previous stock solutions, whereas the maximum absorption of the same concentration of Cu was achieved by culture 3 at 6.2%, 55.56%, 85.66%, and 90.82%, respectively, of the different concentrations. After 3 wk of incubation, the estimated absorption for Zn was achieved by cultures 2, 9, and 10 at 19.2%, 16.68%, 42.92%, and 76.5%, 18.2%, 21.56%, 32.22%, and 77.43%, and 20.8%, 23.52%, 32.22%, and 82.84% of the previous stocks. The maximum absorption of the same concentration of Cu was achieved by culture 3 at 32.6%, 49.88%, 90.44%, and 91.86%, respectively. The accumulation of the absorbed metals was found to be maximum in the protoplast of all cultures. The accumulation at the cell wall was maximum for cultures 2 and 6 for Zn and Cu, respectively, and between the cell wall and the plasma membrane, it was maximum for cultures 2 and 8 for Zn and Cu, respectively. Index Entries: Copper; zinc; halophilic; atomic absorption spectrometry (AAS); Dead Sea; Jordan. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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INTRODUCTION Removal of toxic metals from the environment is of worldwide interest. There have been numerous efforts to develop harmless biological approaches ranging from microbes to viable plants. Viable plants require the metals to be in the aqueous phase so that the absorption takes place in the roots and is susceptible to the strong binding or precipitation of metals (1). In addition, dead or dry plant leaves involve some disadvantages concerning the disposal of the outcome product. Whereas yeast requires a complex nutrient for growth, bacteria are well recognized for their low growth requirements and ability to adapt in new environments (1). More precisely, halotolerant bacteria are the best candidates to be directly inoculated into toxic saline environments along with accumulating metal ions on their surface, cell wall, and intracellular (2). These halophilic bacteria are characterized by their ability to adapt in a halodeficient environment as well as in the presence of high salt concentrations, with an optimum at 0.05 M to 2.5 M salts (3). Homeostasis is maintained by transport ATPases both in and out of the cell (4). However, in these bacteria, the removal of metal is enhanced in the presence of sodium or potassium ions (5). Studies showed that Cu2+ interacts mainly with phosphate, amine, and bidentate carboxyl ligands (6). Zinc ions, however, appear to travel in Zn2+–Cl– complexes and might behave in relation to the salinity of outer surroundings (7). Halophilic bacteria were applied in methanogenesis for waste treatment (8). However, recently, Massadeh et al. reported the ability of the bacteria in the removal of Cd and Pb from their solutions (2). In earlier studies, the susceptibility of bacteria to heavy metals was traditionally tested by the plate count method (7,9–11) rather than in solution. In this study, the removal efficiency of Zn and Cu of 10 different halophilic bacterial isolates obtained from the Dead Sea shore (a hypersaline lake, Jordan side) is presented.

MATERIALS AND METHODS Sample Collection Nine different samples (3 soil, 3 mud and 3 water) were randomly collected from the Dead sea shore at Suwaymah as mentioned in the previous study (2). Each sample consists of 5 subsamples. Samples were placed in sterilized containers.

Microbial Isolation and Characterization Isolation from Soil and Mud Ten grams were suspended in a volume of 100 mL of 10% NaCl, shook at 140 rpm for 90 min; after the solution settled, 1-mL aliquot of Biological Trace Element Research

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supernatant was mixed with 9 mL of 10% NaCl for further study. From the appropriate dilution, 100 µL were spread over the nutrient medium supplemented with 250 ppm of Zn and Cu and incubated at 37°C for 5 d (12,13).

Isolation from Water An aliquot of 10 mL was suspend in 100 mL of 10% NaCl after 24 h of incubation at 37°C, an aliquot of 1 mL was added to 9 mL of 10% NaCl for further study, and from the appropriate dilution, 100 µL were spread over saline agar medium as indicated in the previous subsection on isolation.

Characterization Gram staining, phonotypical appearance, biochemical and physiological activity, acid production form simple sugars, and hydrolysis activity were done as reported in the previous study (2).

Heavy Metal Suspension Preparation Ten thousand ppm of Zn(NO3)2·6H2O and Cu(NO3)2·3H2O (Scharlau, Spain) were prepared as stock solutions in deionzed water. From these stocks, working concentrations of 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, and 2500 ppm of Zn(NO3)2·6H2O and Cu(NO3)2·3H2O were prepared with SW-10 supplemented with 5 g yeast extract (14). The pH of all media was adjusted to 7.2. These concentrations were similar to previous studies on metal tolerance of halophilic bacteria (15–17).

Estimation of Heavy Metal Uptake by Tolerant Isolates To 25 mL of SW-10 broth medium supplemented with 5, 25, 100, and 500 ppm of cadmium and lead nitrate, an aliquot of 100 µL of 106 bacterial culture was added to each flask and incubated at 37°C for 2-wk and 3-wk intervals and shaken at 180 rpm (from each treatment, three flasks were used). At the end of each interval, bacterial culture were centrifuged at 500g for 10 min, and from the supernatant, 5 mL were analyzed by atomic absorption spectrometry (AAS) to determine the heavy metal concentrations. The differences from the start concentration indicated the uptake of heavy metals by the bacterial culture. SW-10 broth with the treated concentration was used as a negative control without bacterial inoculum to determine the experimental condition effect and to be used as a blank for the study.

Determination of Heavy Metal Accumulation in Bacteria Accumulation in Cell Wall Surface After centrifugation of the bacterial cultures of all the treatments, the pellets were washed three times with deionized H2O mixed with 0.1 M Biological Trace Element Research

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Al-Momani, Massadeh, and Hadad Table 1 Survival of 10 Isolates of Moderately Halophilic Bacteria at Different Zn and Cu Concentrations

Note: + = growth; – = no growth.

sodium citrate for 10 min each time with a 3-vol size of the pellet to release all of the positive charge from the cell wall. The concentration of heavy metal in the washed water was estimated using AAS.

Accumulation Between the Cell Wall and the Plasma Membrane The cell wall was hydrolyzed or opened using a 4-mL concentration of (1 mg/mL) lysozyme solution and 6 mL of its buffer (18) and incubated at 30°C for 1 h under the hypertonic condition of 10.3% of sucrose in order to protect the protoplast from rupture. After the incubation, the suspenBiological Trace Element Research

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Fig. 1. (A) Percentage of Zn uptake after 2 wk at different concentrations in aqueous solutions by the 10 isolates; (B) percentage of Zn uptake after 3 wk at different concentrations in aqueous solutions by 10 isolates.

sion was centrifuged and the concentration of heavy metals were estimated from the supernatant using AAS.

Intracellular Accumulation Protoplast pellets were mixed with 10 mL nitric acid for protoplast opening. Concentrations of heavy metals were measured by AAS. The total efficiency of each bacterial culture removal was calculated by dividing the difference of both elements concentrations over the first concentration and multiply by 100%; the efficiency of each cell to remove the heavy metal is calculated by dividing the difference of heavy metals concentration over the total colony-forming unit (CFU) and multiplying by 100%.

Results and Discussion Ten halophilic isolates were recovered from nine samples (soil, water, and mud) collected from the Suwaymah region of the Dead Sea. The survival activity of the 10 isolates in different concentrations of Zn and Cu indicated that all isolates were able to survive up to 1250 ppm Biological Trace Element Research

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Fig. 2. (A) Percentage of Cu uptake after 2 wk at different concentrations in aqueous solutions by the 10 isolates; (B) percentage of Cu uptake after 3 wk at different concentrations in aqueous solutions by the 10 isolates.

of Zn and up to 1000 ppm of Cu, except isolates 2 and 8 (see Table 3). The variation of heavy metals tolerance could be the result of the variation of their genetic makeup. After 2 wk, the highest absorption or removal percentages of Zn from 5 ppm was achieved by isolate 10, with 19%; for 25 ppm, it was achieved by isolate 4, with 8.76%; for 100 ppm, it was achieved by isolates 2 and 5 with 40.92% and 41.2%, respectively; and for 500 ppm, it was achieved by isolate 7, with 84.54% (See Fig. 1A). This might be the result of the greater availability of ions or the high positive charge. After 3 wk, the highest absorption or the highest removal percentages of Zn from 5 ppm was achieved by isolate 10, with 20.8%, and for 25 ppm, it was achieved by isolate 5, with 28.32%. For 100 ppm, the highest absorption was achieved by isolate 2, with 42.92%, and for 500 ppm, it was achieved by isolate 10, with 82.84% (Fig. 1B). The reason behind the highest absorption variation with time at 25 ppm and 500 ppm might be the result of differences in adaptation to the heavy metal concentration. After 2 wk, the highest absorption or removal percentages of Cu from 5 ppm was achieved by isolate 8, with 38%, and for 25 ppm, it was achieved by the isolates 3 and 7, with 55.56% and 66.16%, respectively. For 100 ppm, it was achieved by isolate 3, with 85.66%, and for 500 ppm, it was also achieved by isolate 3, with 90.82% (See Fig. 2A). After 3 wk, the highest absorption or removal percentages of Cu from 5 Biological Trace Element Research

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Table 2 Percentage of Bioaccumulation of 25 ppm Cu and Zn in Different Cellular Parts of the Three Most Efficient Moderately Halophilic Bacteria

ppm was achieved by isolates 2, 8, and 9, with 34.4%, 38.8%, and 40.2%, respectively, and for 25 ppm, it was achieved by isolates 6, 7, and 8, with 63.48%, 77.16%, and 52.24%, respectively. For 100 ppm, it was achieved by isolates 1, 2, and 3, with 57.16%, 56.15%, and 90.44%, respectively, and for 500 ppm, it was achieved by isolate 3, with 91.86% (Fig. 2B). The efficiency of removal of heavy metals with time is the result of cellular adaptation and acclimatization that could enhance some gene expression to utilize these heavy metals in their living biomass. The accumulation of the uptake of Zn or Cu of three selected isolates was detected in three cellular parts (cell wall, plasma membrane, and cytoplasm). The analysis found that the highest Zn accumulation in the cell wall was achieved by culture 6, with 0.072%, and that accumulated in each bacterial cell wall was 0.015 × 10–4% per viable cell; the absorption between the cell wall and the plasma membrane was achieved by culture 8, with 0.100%, and accumulation in cytoplasmic fluid was 6.86%, achieved by culture 2 (Table 2). The highest accumulation of Cu in the cell wall, Biological Trace Element Research

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Table 3 Characterization of the 10 Moderately Halophilic Isolates (from the Dead Sea Shore) and Their Biochemical Tests

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between the cell wall and the plasma membrane, and in the cytoplasm fluid was by culture 2 (Table 2).

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