Isolation, characterization of heavy metal ... - Wiley Online Library

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Journal of Basic Microbiology 2008, 48, 168 – 176

Research Paper Isolation, characterization of heavy metal resistant strain of Pseudomonas aeruginosa isolated from polluted sites in Assiut city, Egypt S. H. A. Hassan, R. N. N. Abskharon, S. M. F. Gad El-Rab and A. A. M. Shoreit University of Assiut, Faculty of Science, Botany Department, Assiut, Egypt

Sixty six isolates of Pseudomonas spp. were isolated from wastewater of El-Malah canal located in Assiut, Egypt and were checked for their heavy metal tolerance. One isolate has tested for its multiple metal resistances and found to be plasmid mediated with molecular weight 27 Kb for nickel and lead. It was identified as Pseudomonas aeruginosa ASU 6a. Its minimal inhibitory concentration (MIC) for Cu2+, Co2+, Ni2+, Zn2+, Cr3+, Cd2+and Pb2+ were 6.3, 5.9, 6.8, 9.2, 5.8, 4.4, and 3.1 mM, respectively. Growth kinetics and the maximum adsorption capacities were determined under Ni2+ and Pb2+ stress. The latter heavy metals induced potassium efflux and were used as indicator for plasma membrane permeabilization. Keywords: Heavy metal resistance / Pseudomonas aeruginosa / K+ efflux / Lipid peroxidation Received: October 25, 2007; accepted: December 25, 2007 DOI 10.1002/jobm.200700338

*

Introduction

Among the pollutants of serious concern, toxic metals are important since they accumulate through the food chain and cause environmental hazards [1]. Application of sewage sludge on agricultural land for enhancing crop productivity has been a common practice in Egypt for many years. Nowadays serious attention is being paid to the heavy metal content of sewage sludge before its land application because of the tendency of uptake of toxic metals like Cr3+, Cu2+, Hg2+, Pb2+, Mn2+, Zn2+, and Ni2+ by food crops and plants [2]. These metals not only cause serious health hazards but also disturb the ecological status of biota [3, 4]. Bacteria require nickel as a trace element for enzymes [5]. However, it is toxic to microorganisms at higher concentrations. It frequently inhibits enzymatic activity, DNA replication, translation and transcription by binding proteins and nucleic acid [6]. Several nickel resistant bacteria have been isolated from ecosystem polluted by heavy metals [7]. Correspondence: A. A. M. Shoreit, University of Assiut, Faculty of Science, Botany Department, 71516 Assiut, Egypt E-mail: [email protected] Phone: 0020882411433 Fax: 0020882342708 © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Lead is one of non essential heavy metals; its hazardous to ecosystem, human health, has accumulated on microorganisms and is also reported to inhibit microbial growth and inactivating the cell enzymes [8]. The main sources of nickel and lead were stainless steel, batteries, gasoline paint, pigment and mineral processing [9]. Resistance to heavy metals, antibiotics, disinfectants, detergents and other toxic substances were observed in a wide variety of bacteria, especially in the genus Pseudomonas. It has been considered as one of the indicators bacteria for measuring pollution [10, 11]. Microbial survival in polluted environments depends on intrinsic biochemical and structural properties, physiological, and/or genetic adaptation [12, 13]. Microbes apply various types of resistance mechanisms in response to heavy metals encoded by chromosomal genes or plasmids [13 – 15]. Cupriavidus metallidurans CH34 (previously Ralstonia metallidurans CH34 contains two megaplasmids (pMOL28 and pMOL30), which confer resistance to a broad range of heavy metals (Cd2+, Co2+, Zn2+, Tl+ , Cu2+, Pb2+, Ni2+, Hg2+ and CrO42–). [15]. Howlett and Avery [16] reported that the toxicity of transition metals caused lipid peroxidation and cellular K+ efflux in Saccharomyces cerevisiae resulting in loss of function and membrane integridity. www.jbm-journal.com


The objective of this study is to determine heavy metals and antibiotic resistance of Pseudomonas aeruginosa, checking for its plasmid-encoded metal-tolerance, MIC, growth parameters, maximum adsorption capacities using Langmiur equation, leakage of potassium and lipid peroxidation under nickel and lead stress.

Materials and methods Collection of samples and determination of their physicochemical properties Different polluted water samples were collected in sterile glass bottles from different sites of El-Malah canal, Assiut city (Egypt); it is about 3 km far from the main River Nile, in the west site of Assiut city which exposed to domestic sewage disposal and industrial effluent. Temperature, pH, Electrical conductivity and Heavy metals were determined by water checker model Horiba U-10 and according to Elith and Garwood [17]. Isolation of Pseudomonas spp. Pseudomonas spp. were enumerated from wastewater samples by Most Probable Number (MPN) techniques using selective acetamide broth medium [18]. Positive presumptive tubes were determined by fluorescence under long-wave ultraviolet light after incubation at 37 °C for 48 h. The positive tubes had confirmed on Glycerol Mannitol Acetamide Cetramide agar (GMAC) [19]. Sixty six isolates were selected randomly from GMAC, purified and were preserved on nutrient agar for further studies. Screening for heavy metals tolerance In order to minimize the complexation of heavy metals, the isolates were grown in tris minimal medium [20]. Tolerance to heavy metals was determined by an agar dilution method [21]. The plates containing 20 ml of above medium at different concentration (1 – 14) mM of heavy metals studied CuSO4 ⋅ 5 H2O, Co(NO3)2 ⋅ 6 H2O, Ni(NO3)2 ⋅ 6 H2O, Cr(NO3)2 ⋅ 9 H2O, (CHCOO)2Zn ⋅ 2 H2O, Cd(NO3)2 ⋅ 2 H2O and Pb(NO3)2. The plates inoculated with cultures and incubated at 37 °C for 2 days. In the case of lead toxicity test, the amount of sodium β-glycerophosphat was reduced to 0.02 g/l, omitting tris buffer and adjusting pH to 6.0 to limit metal precipitations. Identification of the isolate Pseudomonas sp. ASU 6a Among the isolates, isolate 6a showed high resistance to heavy metals. Isolate have primarily been identified by phenotypic characterization according to Brenner [22]. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Characterization of heavy metal resistant strain of P. aeruginosa

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Determination of the Minimal Inhibitory Concentration (MIC) Minimal Inhibitory Concentration (MIC) for different heavy metals were registered on agar plates of Tris minimal medium using agar dilution methods and were confirmed in broth medium, for broth medium a 5 ml of Tris minimal medium containing different concentration of heavy metals salts CuSO4 ⋅ 5 H2O, Ni(NO3)2 ⋅ 6 H2O, Cr(NO3)2 ⋅ 9 H2O, Co(NO3)2 ⋅ 6 H2O, (CHCOO)2Zn ⋅ 2 H2O, Cd(NO3)2 ⋅ 2 H2O and Pb(NO3)2 inoculated with 200 µl of an 18 h old culture of the studied bacterial P. aeruginosa ASU 6a (resistant) and E. coli DH5α (sensitive) at 37 °C for 2 d. The lowest concentration of heavy metals that completely preventing growth known as MIC [23]. Antibiotics resistance The susceptibility of P. aeruginosa ASU 6a to various antibiotics was tested by the disk diffusion method [24]. The following antibiotics discs were used: (N) Neomycin (5 mg/l), (AK) Amikacin (300 µg/l), (OFX) Ofloxacin (5 mg/l), (CIP) Ciprofloxacin (5 mg/l), (CN) Gentamicin (30 mg/l), (CAZ) Cefatazidime (30 mg/l), (NOR) Norfloxacin (10 mg/l) and (VA) Vancomycin (30 mg/l). Plasmid isolation The plasmids were isolated from the P. aeruginosa ASU 6a by the modified miniprep method [25]. The isolated plasmids were characterized by agarose gel electrophoresis according to the standard procedure of Sambrook et al. [26]. Plasmid transformation To confirm the plasmid-encoded tolerance to the metals studied, competent cells of E. coli DH5α, sensitive to heavy metals, were transformed with respective plasmids using the standard chemical method Sambrook et al. [26]. The suspensions (100 µl) of transformed E. coli DH5α were plated on tris minimal media supplemented with 3.4 or 1.44 mM of nickel or lead. Plasmid DNA of transformant and non transformant one of E. coli DH5α were compared with a resistant one by using agrose gel electrophoresis. Effect of lead and nickel on the growth kinetics of P. aeruginosa ASU 6a The growth rate and growth parameters of resistant strain P. aeruginosa ASU 6a to lead and nickel were determined according to [27, 28]. Determination of bacterial proteins P. aeruginosa ASU 6a was cultivated in tris minimal medium containing different concentrations (0, 0.85, 1.7, www.jbm-journal.com

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S. H. A. Hassan et al.

3.4, 5.1 mM; 0, 0.25, 0.5, 1, 1.5, 2 mM) of Ni2+ and Pb2+, respectively incubated at 37 °C with agitation at 150 rpm for 12 h, then the bacterial cell was centrifuged at 10,000 rpm and the pellet was dried at 50 °C over night. The dried cells (0.05 g) were boiled for 1 h in 10 ml 0.1 N NaOH. After cooling, the total proteins content of bacterial cells in the supernatant were determined according to the method adopted by Lowery et al. [29]. Determination of potassium efflux Asset of Erlenmeyer flask containing 100 ml of tris minimal medium at different concentrations (0, 0.85, 1.7, 3.4, 5.1 mM; 0, 0.25, 0.5, 1, 1.5, 2 mM) of Ni2+ and Pb2+, respectively, were inoculated with 200 µl of preculture of isolate. Erlenmeyer flask was incubated at 37 °C with agitation at 150 rpm for 12 h. 5 ml aliquots were removed and centrifuged for 10 min and then the supernatant was diluted with 4 volumes of distilled deionized water. Extra-cellular K+ was measured by Jenway PFP7 flame photometer [16]. Determination of lipid peroxidation The level of lipid peroxidation was measured by determination of malondialdehyde (MDA) a breakdown product of lipid peroxidation. MDA content was determined with thio-barbituric acid (TBA) reaction. Briefly 0.25 g of fresh cells was homogenized in liquid nitrogen in 5 ml 0.1% Trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000 rpm for 5 min. To 1 ml aliquot of the supernatant 4 ml of 20% TCA containing 0.5% TBA were added. The mixture was heated at 95 °C for 15 min and cooled immediately. The developed colour was extracted with 2 ml n-butanol and the absorbance was measured at 532 nm. The level of lipid peroxidation was expressed as nmol of MDA using an extinction coefficient of 155 mM cm–1 [30]. Gel analysis The LabImage 1D [2006] program was used in molecular weight determination of plasmid DNA. Statistical analysis: Data were analyzed using oneway analysis of variance (ANOVA) with differences determined using Duncan’s test by SPSS 10.0 software. Differences were considered to be significant among means ±SE standard error (n = 3) at a probability of (P < 0.05).

Results Physicochemical properties of samples The temperature, pH and Electrical conductivity of samples ranged from 23.7 – 27 °C, 7.9 to 8.12, and © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


0.34 – 0.35 ms/cm, respectively. The concentrations of Cd2+, Cu2+, Cr3+, Pb2+ and Ni2+, expressed in mg/l ranged around 0.0117 – 0.045, 1.93 – 7.39, 0.38 – 0.88, 0.23 – 0.825 and 1.5 – 7.057 mg/l, respectively. It was noted that the obtained results exceeded the safe limit of WHO [31] it is 0.003, 0.02, 0.05, 0.001 and 0.07 mg/l of Cd2+, Cu2+, Cr3+, Pb2+ and Ni2+, respectively. Isolation and enumeration of Pseudomonas MPN of this study indicated that Pseudomonas spp. were present in very substantial numbers in the samples of wastewater investigated. It was ranged from 220 – 400 colonies/100 ml at different sites of El-Malah canal, Assiut city (Egypt). Sixty six isolates were selected randomly on (GMAC), purified and were preserved on nutrient agar for further studies. Screening for heavy metal tolerance The purified isolates of Pseudomonas spp. were tested for tolerance against essential heavy metals CuSO4 ⋅ 5 H2O, Co(NO3)2 ⋅ 6 H2O, Ni(NO3)2 ⋅ 6 H2O, Cr(NO3)2 ⋅ 9 H2O and (CHCOO)2Zn ⋅ 2 H2O and non essential ones Cd(NO3)2 ⋅ 2 H2O and Pb(NO3)2 at concentrations 1–14 mM. The numbers and percentage of the tolerated isolates to various concentrations of heavy metals ion are shown in Table (1). The results indicated that most of them grew well at low concentrations of heavy metals and their number gradually decreased as the concentration increased. For essential heavy metals studied all the isolates able to grow at concentration 2 mM, except in the case of zinc reached 7 mM. On contrast all isolates tolerate up to 2 mM of cadmium, and drastically decreased by 50% at 3.5 mM, reached null above 4.4 mM. In other wise lead is consider the most toxic heavy metals since 89% of the isolates were inhibited by 3.5 mM and since there no isolate can grow above this concentration. Among the isolates, Pseudomonas sp. ASU 6a showed high tolerance to the above mentioned metals it tolerance 3.5, 4.5, 6, 7 and 3 mM for Pb2+, Cd2+, Cu2+, Ni2+ and Zn2+ and 5 mM for Cr3+ and Co2+, respectively. The order of toxicity of heavy metals towards the isolates of Pseudomonas spp are Pb2+ > Cd2+ > Cr3+ > Co2+ > Cu2+ > Ni2+ > Zn2+; so the lead, cadmium and chromium are more toxic than Ni2+and Zn2+. Identification of isolate Pseudomonas sp. ASU 6a The isolate ASU 6a was identified according to Brenner [22], it was characterized by: single rod-shaped motile, gram-negative, non-spore forming. It gave positive reactions with the following tests: Cytochrome-C oxidase, catalase test, denitrification, gelatin hydrolysis, lipid hydrolysis, growth at 41 °C, urease activity and citrate www.jbm-journal.com



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Table 1. Number of tolerated isolates of Pseudomonas spp at different concentrations of heavy metals. Metal ion 2+

Cu Co2+ Ni2+ Cr3+ Zn2+ Cd2+ Pb2+

Heavy metals concentration (mM) 1

2

3.5

4.5

5

6

7

10

13

14

66* (100) 66 (100) 66 (100) 66 (100) 66 (100) 66 (100) 66 (100)

66* (100) 66 (100) 66 (100) 66 (100) 66 (100) 66 (100) 48 (72)

59* (89) 44 (66) 48 (72) 40 (60) 66 (100) 33 (50) 7 (11)

14* (21) 12 (18) 17 (25) 4 (6) 66 (100) 20 (30) 0 (0)

9* (14) 5 (8) 17 (25) 3 (5) 66 (100) 0 (0) 0 (0)

3* (5) 0 (0) 8 (12) 0 (0) 66 (100) 0 (0) 0 (0)

0* (0) 0 (0) 4 (6) 0 (0) 66 (100) 0 (0) 0 (0)

0* (0) 0 (0) 0 (0) 0 (0) 45 (78) 0 (0) 0 (0)

0* (0) 0 (0) 0 (0) 0 (0) 12 (18) 0 (0) 0 (0)

0* (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

* Values indicated the number of tolerant isolates. Values in parentheses indicated % of tolerant isolates.

test, while it gave negative reactions with the growth at 4 °C and with starch hydrolysis. The pigmentation on King’s A gives the pyocyanin (blue green) and pyrobin (pink-red) pigments, whereas, King’s B favor the production of fluorescent (yellow green). In nutrient agar isolate 6a give pyocyanin after 10 – 12 h and give pyrobin after 24 h. The obtained results show that the latter isolate belongs to the genus Pseudomonas and identified as P. aeruginosa ASU 6a based on phenotypic methods.

Determination of MIC for P. aeruginosa ASU 6a and E. coli DH5α The minimal inhibitory concentration of the resistant strain P. aeruginosa ASU 6a and sensitive one E. coli DH5α to heavy metals were registered in tris minimal broth and on solid media are shown in Table 2. The values for resistant and sensitive one in liquid media are 6.3, 5.9, 6.8, 9.2, 5.8, 4.4, 3.1 and 1.2, 1.9, 1.7, 1.5, 1.33, 1.9, 0.96 mM, for Cu2+, Co2+, Ni2+, Zn2+, Cr3+, Cd2+and Pb2+, respectively. The MIC on solid medium for the metals studied was higher than in liquid medium and ranged from 4.8 – 11.46 mM on solid medium, while in the broth from 3.1 – 9.2 mM.

Resistance to antibiotics The resistances to antibiotic for P. aeruginosa ASU 6a are shown in Table 2. It was found that it was resistant to the following antibiotics Neomycin, Gentamicin, Cefatazidime, Norfloxacin, Vancomycin, Ofloxacin but sensitive to Amikacin and Ciprofloxacin. Plasmid profile of resistant strain and transformation The agrose gel electrophoreses pattern of the resistant strain P. aeruginosa ASU 6a shows that it has one plasmid with molecular weight of 27 kb. To determine whether the resistance markers of P. aeruginosa ASU 6a on plasmid DNA, the transformation was carried on a recipient cell of E. coli DH5α. By comparing the growth results, it was found that the transformant strain of E. coli DH5α could grow on tris minimal agar containing 3.4 or 1.44 mM of nickel and lead, respectively while the non-transformed cannot. The plasmid composition of viable transformants and original isolate were confirmed by agrose gel electrophoresis. The results showed that the transformation of the plasmids was successful. Fig. 1 shows the transformant cell lane 2, P. aeruginosa ASU 6a lane 3 and E. coli DH5α lane 4. The transformant cells showed that it had a plasmid with the same size as in the strain 6a.

Table 2. MIC for different heavy metals and antibiotics resistance of P. aeruginosa ASU 6a and E. coli DH5α. Metal

MIC* (mM) in Tris minimal broth medium

MIC* (mM) in Tris minimal solid medium

Strain (6a)

E. coli DH5 α

Strain (6a)

E. coli DH5 α

Copper Cobalt Nickel Zinc Chromium Cadmium Lead

6.3 5.9 6.8 9.2 5.8 4.4 3.1

1.2 1.9 1.7 1.5 1.33 1.9 0.96

7.1 7.6 8.5 11.46 7.7 5.3 4.8

1.57 2.2 2.1 2 1.7 2.66 1.92

Antibiotics

Resistance

Neomycin (5 mg/l) Amikacin (300 µg/l) Ofloxacin (5 mg/l) Ciprofloxacin (5 mg/l) Gentamicin (30 mg/l) Cefatazidime (30 mg/l) Norfloxacin (10 mg/l) Vancomycin (30 mg/l)

R1 S2 R1 S2 R1 R1 R1 R1

*MIC: Minimum inhibitory concentrations; 1: resistant; 2: sensitive. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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del is 113.6 and 123 mg/g for nickel and lead, respectively. Determination of potassium efflux and lipid peroxidation Lipid peroxidation was evaluated by measuring conjugated dienes, which are early intermediates in the lipid peroxidative chain like MDA. Heavy metals affect lipid peroxidation and plasma membrane permeabilization, leading to an increase in MDA. Fig. 3A shows that the level of MDA production was increased by increasing time (3 – 8 h) also the level of MDA was higher in the toxicity of nickel than lead even in the same conc. One of the first indications of membrane damage in bacteria is an efflux of potassium ions, which is also caused by heavy metals. Fig. 3B shows that the released k+ level was markedly higher at 3.4 and 1 Mm of nickel and lead than control in the time of incubation 3, 4, 6 and 8 h.

Discussion

Figure 1. Plasmid transformation lane 1λ (DNA marker) cleaved with Hind III, lane 2 transformed strain of E. coli DH5α, lane 3 resistant strain of P. aeruginosa ASU 6a, and lane 4 is sensitive strain E. coli DH5α. 2+

2+

Effect of Ni and Pb on Growth kinetics, protein content and determination of maximum adsorption capacity (qmax) of P. aeruginosa ASU 6a The growth curve of tolerant strain P. aeruginosa ASU 6a to different concentrations of nickel and lead were shown in Fig. 2 (A, B). The heavy metals are added after 3 h of incubations. It was noted that both heavy metals cause a reduction in growth depending on metal type and toxicity as the concentration increased compared with control. Growth parameters and % of protein inhibition on P. aeruginosa ASU 6a are shown in Table 3. In general the results depict that the optical density, the maximum specific growth rate (K) and protein content decreased while the generation time (T) increased by increasing heavy metals concentration. The maximum adsorption capacity (qmax) of heat dried “non-living cells” using Langmiur adsorption mo© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In this paper we report high degree of tolerance to selected heavy metals by an isolate of P. aeruginosa ASU 6a. The limit of tolerance to the highest concentration of selected heavy metals in the media was evaluated based on the ability of the bacterium to grow on the subsequent higher concentration. In metal polluted habitat, the frequency of tolerant bacteria increases with increasing heavy metal concentration in these habituates [10]. In our study the heavy metal concentration in El-Malah canal is higher than the safe limit of WHO [31]. In comparison to previous studies on the metal tolerance of P. aeruginosa ASU 6a exhibited more resistance than other studies Hassen et al. [32] stated that three strains of P. aeruginosa (S6, S7 and S8) isolated from natural polluted environments on nutrient agar were tolerant to CuSO4, Cr2(SO4)3, CoSO4 corresponding to 1.6, 1.2 and 0.8 mM, respectively, of metal ions concentrations. In other wise Raja et al. [33] reported that P. aeruginosa showed tolerance up to concentration 3.8, 4.4, 7.6 and 11.9 mM on LB medium to Pb2+, Cd2+, Cr6+ and Ni2+, respectively. P. aeruginosa ASU 6a was able to grow at high concentration of heavy metals in liquid media which might be important for the capacity of bacterium to survive in different sources of pollution with elevated heavy metal levels. The (MIC) on solid medium was higher than in broth where the condition of diffusion, complexation and availability of metals were different from those observed on solid medium. This suggests that orgawww.jbm-journal.com



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a)

b)

Figure 2. The growth curves of resistant strain P. aeruginosa ASU 6a to nickel (A) and lead (B) at different concentration grown in tris minimal medium at 37 °C with agitation at 150 rpm for 12 h (↓) heavy metals were added after 3 h of incubations.

nic matter components on the latter forming complexes with heavy metals (Pb2+, Cd2+ and Zn2+); thereby reducing the concentration of free metal ions. This observation is in agreement with; Babich and Stotzky [6], Mergeay [20]; Hassen et al. [32]; Hussein [34]. From table 2 the MIC is higher than observed by several authors [10, 32, 35]. However lead is more toxic than other heavy metals studied, nickel and zinc are less toxic, and the difference in toxicity could be explained by the conditions of bacterial isolation and the selectivity of microbial cultures techniques in each study, par© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ticularly with respect to the nature and specificity of growth media Chen et al. [36]. Microorganisms resistant to antibiotics and tolerant to metals appear to be the result of exposure to metalcontaminated environments that cause coincidental selection for resistance factors for both [37]. Many investigators have reported that metal tolerant environmental isolates are resistant to a wide array of antibiotics and there are correlation between heavy metal resistance and antibiotic resistance [21, 32, 38 – 41]. www.jbm-journal.com

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Table 3. Growth parameters and percentage of proteins inhibition of resistant strain P. aeruginosa ASU 6a at different concentrations of nickel and lead. Conc.

Growth parameters

(mM)

Tλ (h)

Tα (h)

a

K-h

T (h)

Ni2+

0 0.85 1.7 3.4 5.1

3 3 3 3 3

12 12 12 12 12

3.68 3.61 3.55 3.47 3.27

0.197 0.19 0.183 0.174 0.154

3.51 3.64 3.79 3.98 4.6

0 7 16 25 46

Pb2+

0 0.5 1.5 2

3 3 3 3

12 12 12 12

6.06 5.88 5.48 5.29

0.2 0.185 0.14 0.12

3.5 3.75 5 5.8

0 7 30 42

Metal ion

% of protein inhibition

Tλ: Lag period, Tα: the end of the exponential phase, a: A symbiotic level = ln(OD/OD0), K: The maximum specific growth rate and T: generation time.

Metal-tolerant bacteria have evolved various resistance and detoxification mechanisms [15]. The resistance mechanisms are chromosomally encoded or, more often, plasmids of different size and showing conjugative capabilities are carriers of metal-resistance genes [10]. In this study, P. aeruginosa ASU 6a contain plasmid with molecular weight 27 kb which responsible for metal resistance as confirmed by transformation experiments. These results indicated that the plasmid can replicate in E. coli DH5α and that it possesses the genetic information necessary for the expression of resistance of nickel and lead. The results are in agreement with [10, 35]. The degree of growth in response to metal ion varied with the metal ion supplementation in the media, growth rates of the P. aeruginosa ASU 6a in the presence of lead and nickel were consistently slower than that of control, similar observation have been reported earlier [23, 27, 32, 34, 39, 42]. It was noted that the maximum

a)

b)

Figure 3. Effect of lead and nickel on lipid peroxidation (a) and potassium efflux (b), the ANOVA was carried out by using SPSS 10.0 comparisons among means ±SE standard error (n = 3), different letters show significance according to Duncan test (P < 0.05). © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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specific growth rate (K) and protein content decreased while the generation time increased by increasing heavy metals concentration. Either nickel or lead decreases the total protein content of the bacterium at high concentration. These results are in agreement with many investigators [32, 43, 44]. In our study P. aeruginosa ASU 6a show maximum adsorption capacity than other biosorbents. Hussein et al. [34] isolated strain of P. putida have a maximum adsorption capacity for nickel was 54 mg/g. In other wise Sar et al. [45] investigated that the maximum adsorption of nickel by a lyophilized biomass of P. aeruginosa was 265 mg/g. Chang et al. [46] showed that inactivated cells and resting cells of P. aeruginosa PU21 were (79.5 and 84.77 mg/g) for lead, respectively. Lipid peroxides are unstable and decompose to form a complex series of compounds including reactive carbonyl compounds. Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) upon decomposition. Measurement of malondialdehyde and 4-hydroxyalkenals has been used as an indicator of lipid peroxidation Esterbauer et al. [47]. Heavy metal-induced plasma membrane permeabilization and toxicity is markedly dependent on fatty acid composition [48]. The accumulation of lipid peroxidation products, such as lipid hydroperoxides, within the hydrophobic core of plasma membranes can result in disturbances in the arrangement of phospholipid moieties and impairment of membrane function [48] this may be manifested as K+ loss. The fact that the susceptibility of fatty acids to lipid peroxidation increases with the degree of fatty acyl chain unsaturation [49] indicates a possible link with our results. Furthermore, metals, such as lead, nickel, are known to be capable of inducing freeradical production and promoting oxidative stress through their redox-cycling activity.

Acknowledgements The Authors are highly grateful to Dr. Mohamed M.H. El-Defrawy, Prof. of Genetics in Biotechnology Lab, Faculty of Agriculture, Assiut University for his help in plasmid isolation and transformation methods.

References [1] Rani, D.B.R. and Mahadevan, A., 1993. Patterns of heavy metal resistance in marine Pseudomonas MRI. Indian J. Exp. Biol., 31, 682 – 686. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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[2] Bhowal, S.K., Chakraburty, A.K. and Dhur, B., 1987. Heavy metal contamination in the sewage sludge of Calcutta metropolitan area. Indian J. Environ. Health, 1, 66 – 71. [3] Malik, A. and Jaiswal, R., 2000. Metal resistance in Pseudomonas strains isolated from soil treated with industrial wastewater. World J. Microbiol. Biotechnol., 16, 177 – 182. [4] Moore, J.W. and Ramamoorthy, A., 1984. Heavy Metals in Natural Waters. Applied Monitoring and Impact Assessment, Springer Verlag, New York. [5] Hausinger R.P., 1987. Nickel utilization by microorganism. Microbiol. Rev., 51, 22 – 42. [6] Babich, H. and Stotzky, G. 1983. Toxicity of Nickel to microbes: environmental aspects. Adv. Appl. Microbiol., 29, 195 – 265. [7] Stoppel, R.D. and Schlegel., H.G., 1995. Nickel resistant bacteria from anthropogenically nickel-polluted and naturally nickel-percolated ecosystems. Appl. Environ. Microbiol., 61, 2276 – 2285. [8] Nies, D.H., 1999. Microbial heavy-metal resistance. Appl. Microbiol. Biotechnol., 51, 730 – 750. [9] Padmavathy, V., Vasudevan, P. and Dhingra, S.C., 2003. Thermal and spectroscopic studies on sorption of nickel(II) ion on protonated baker’s yeast. Chemosphere, 52, 1807 – 17. [10] Piotrowska-Seget, Z., Cycon, M. and Kozdroj, J., 2005. Metal-tolerant bacteria occurring in heavily polluted soil and mine spoil. Appl. Soil Ecol., 28, 237 – 246. [11] Malik, A., 2004. Metal bioremediation through growing cells. Environ. Int., 30, 261 – 278. [12] Ehrlich, H.L., 1997. Microbes and metals. Appl. Microbiol. Biotechnol., 48, 687 – 692. [13] Wuertz, S. and Mergeay, M., 1997. Modern Soil Microbiology. Marcel Dekker, New York. [14] Nies D.H., 2003. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol. Rev., 27, 313 – 339. [15] Ledrich M. L, Stemmler S, Gilly P. L., Foucaud L. and Falla J., 2005. Precipitation of silver-thiosulfate complex and immobilization of silver by Cupriavidus metallidurans CH34 BioMetals, 18, 643 – 650. [16] Howlett, N.G. and Avery, S.V., 1997. Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation. Appl. Environ. Microbiol., 63, 2971 – 2976. [17] Elith, M. and Garwood, S., 2001. Investigation into the levels of heavy metals within the Manly Dam Catchment. In: Freshwater Ecology Report. Department of Environmental Sciences. University of Technology, Sydney. [18] Hedberg, M., 1969. Acetamide agar medium selective for Pseudomonas aeruginosa. Appl. Microbiol., 17, 481. [19] Mossel, D.A. and Indacochea, L., 1971. A new cetrimide medium for the detection of Pseudomonas aeruginosa. J. Med. Microbiol., 4, 380 – 382. [20] Mergeay, M., 1995. Heavy metal resistance in microbial ecosystems. Mol. Microb. Ecol. Manual, 6, 7 – 17. [21] Sabry, S.A., Ghozlan, H.A. and Abou-zeid, D.M., 1997. Metal tolerance and antibiotic resistance patterns of bacterial population isolated from sea water. J. Appl. Microbiol., 82, 245 – 252. www.jbm-journal.com

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[22] Brenner, D.J., Krieg, N.R., Staley, J.T. and Garrity, G.M., 2005. Bergey’s Manual of Systematic Bacteriology. Springer Verlag New York. [23] Yilmaz, E.I., 2003. Metal tolerance and biosorption capacity of Bacillus circulans strain EB1. Res. Microbiol., 154, 409 – 415. [24] Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disc diffusion method. Am. J. Clin. Pathol., 45, 493 – 496. [25] Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic. Acids Res., 7, 1513 – 1523. [26] Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. [27] Filali, B.K., Taoufik, J., Zeroual, Y., Dzairi, F.Z., Talbi, M. and Blaghen, M., 2000. Wastewater bacterial isolates resistant to heavy metals and antibiotics. Curr. Microb., 41, 151 – 156. [28] Ghosh, S., Mahapatra, N.R. and Banerjee, P.C., 1997. Metal resistance in Acidocella strains and plasmid-mediated transfer of this characteristic to Acidiphilium multivorum and Escherichia coli. Appl. Environ. Microbiol., 63, 4523 – 4527. [29] Lowry, O.H., Rosebrough, N.H., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 291 – 297. [30] Zaho, S.J., Xu, C.C. and Zou, Q., 1994. Improvements of the method for measurement of malondialdehde in plant tissue. Plant Phsiol. Commun., 30, 207 – 210. [31] WHO (World Health Organization) 2005. Guidelines for Drinking-water Quality, Vol. 1. WHO, Geneva. [32] Hassen, A., Saidi, N., Cherif, M. and Boudabous, A., 1998. Resistance of environmental bacteria to heavy metals. Bioresour. Technol., 64, 7 – 15. [33] Raja, C.E., Anbazhagan, K. and Selvam, G.S., 2006. Isolation and Characterization of a metal-resistant Pseudomonas aeruginosa Strain. World J. Microbial. Biotechnol., 22, 577 – 585. [34] Hussein, H., Farag, S., Kandil, K. and Moawad, H., 2005. Tolerance and uptake of heavy metals by Pseudomonads. Process Biochem., 40, 955 – 961. [35] Ünaldi, M.N., Korkmaz, H., Arikan, B. and Coral, G., 2003. Plasmid-encoded heavy metal resistance in Pseudomonas sp. Bull. Environ. Contam. Toxicol., 71, 1145 – 1150.

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[36] Chen, X.C., Wang, Y.P., Lin, Q., Shi, J.Y., Wu, W.X. and Chen, Y.X., 2005. Biosorption of copper (II) and zinc(II) from aqueous solution by Pseudomonas putida CZ1 Colloids and Surfaces B: Biointerfaces, 46, 101 – 107. [37] Spain, A., 2003. Implications of microbial heavy metal tolerance in the environment. Rev. Undergr. Res., 2, 1 – 6. [38] Bezverbnaya, I.P., Buzoleva, L.S. and Khristoforova, N.S., 2005. Metal-resistant heterotrophic bacteria in coastal waters of Primorye. Russ. J. Mar. Biol., 31, 73 – 77. [39] Pal, A., Choudhuri, P., Dutta, S., Mukherjee, P.K. and Paul, A.K., 2004. Isolation and characterization of nickelresistant microflora from serpentine soils of Andaman World J. Microbiol. Biotechnol., 20, 881 – 886. [40] Soltan, E.M., 2001 Isolation and characterization of antibiotic and heavy metal resistant Pseudomonas aeruginosa from different polluted waters in Sohag Distrit, Egypt. J. Microbiol. Biotechnol., 11, 50 – 553. [41] Ramteke, P.W., 1997. Plasmid mediated co-transfer of antibiotic resistance and heavy metal tolerance in coliforms. Indian J. Microbiol., 37, 177 – 181. [42] Sharma, R., Rensing, C., Rosen, B.P. and Mitra, B., 2000. The ATP hydrolytic activity of purified ZntA, Pb(II), Cd(II), Zn(II)-translocating ATPase from Escherichia coli. J. Biol. Chem., 275, 3873 – 3878. [43] Chen, C. and Wang, J., 2007. Response of Saccharomyces cerevisiae to lead ion stress. Appl. Microbiol. Biotechnol., 74, 683 – 687. [44] Fahmy, S.M., 2001. The behavioral responses of Rhodobacter capsulatus B10 and 37b4 to cadmium and zinc toxicity. MSc. thesis. Faculty of science. Assiut University. AssiutEgypt. [45] Sar, P., Kazy, S., Asthana, R. and Singh, S., 1999. Metal adsorption and desorption by lyophilized Pseudomonas aeruginosa. Int. Biodet. Biodeg., 44, 101 – 110. [46] Chang, J., Law, R. and Chang, C.C., 1997. Biosorption of lead, copper and cadmium by biomass of Pseudomonas aeruginosa PU21. Wat. Res., 31, 1651 – 1658. [47] Esterbauer, H., Schaur, R.J. and Zollner, H., 1991. Chemistry and Biochemistry of 4-Hydroxynonenal, Malonaldehyde and Related Aldehydes; Free Rad. Biol. Med., 11, 81 – 128. [48] Van, G.G. and Sevanian, A., 1994. Lipid peroxidationinduced membrane structural alterations. Methods Enzymol., 233, 273 – 288. [49] Stohs, S.J. and Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Rad. Biol. Med., 18, 321 – 336.

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