Isolation and characterization of heavymetal resistant

Journal of Basic Microbiology 2012, 52, 53 – 65

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Research Paper Isolation and characterization of heavy-metal resistant microbes from roadside soil and phylloplane Rehab M. Mohamed and Aly E. Abo-Amer Division of Microbiology, Department of Botany, Faculty of Science, Sohag University, Sohag, Egypt

Contamination by heavy metals is one of the major environmental problems in many countries and these contaminants reach from various sources such as traffic cars and other activities. Soil and phylloplane samples were collected from eight traffic and two non-traffic sites in Sohag city, Egypt. Heavy metal contents of Cd2+, Zn2+ and Pb2+ of soil and phylloplane samples were determined and revealed high levels of Zn2+ and Pb2+ in traffic samples. A total of 112 bacterial and 62 fungal isolates were obtained from soil and phylloplane. Bacterial isolates were characterized on the basis of morphological, physicochemical and biochemical characteristics; and 16S rRNA gene sequences. Fungal isolates were identified according to morphological characterization. Minimal inhibitory concentrations (MICs) of Cd2+, Zn2+ and Pb2+ for each isolate were detected. All bacterial and fungal isolates demonstrated resistance to lead with MICs > 0.528 mM and > 0.211, respectively. Moreover, the maximum MICs of cadmium and zinc for bacteria were 0.821 mM and 1.471 mM, respectively, where as, MICs for fungi were 0.328 mM and 0.588 mM, respectively. The most resistant bacterial and fungal isolates were Pseudomonas aeruginosa RA65 and Penicillium corylophyllum, respectively. Therefore, P. aeruginosa RA65 was selected for further investigations. Growth curve study showed that 0.264 mM lead had no efficiently effect on the growth of P. aeruginosa RA65. Plasmid isolation evidenced by transformation studies indicated that P. aeruginosa RA65 harbored a single plasmid (~9.5 kb) which mediated heavy meal resistance. Consequently, these microbial isolates could be potentially used in bioremediation of heavy metal-contaminated environment. Keywords: Pseudomonas aeruginosa / Penicillium corylophyllum / Roadside / Soil / Phylloplane / Heavy metals / MICs / Plasmid / Transformation / Bioremediation Received: March 21, 2011; accepted: June 26, 2011 DOI 10.1002/jobm.201100133

Introduction* The pollution of soils and phylloplanes by heavy metals is a major environmental issue in many countries and these contaminants usually come from automobile sources, commercial and different industry activities like leather, agricultural, textile industries, etc. [1]. Therefore, Environmental pollution of heavy metals has achieved much attention in the recent past. Heavy metals such as lead, cadmium and zinc are the major metal pollutants of the roadside environments and are released from petrol fuel burning, leakage of oils, wear Correspondence: Aly E. Abo-Amer, Division of Microbiology, Department of Biology, Faculty of Science, Taif University, Taif (888), Saudi Arabia (Current address) E-mail: [email protected] © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

out of tyres, and corrosion of batteries and metallic parts such as radiators of motor vehicles [2]. These pollutants were known as primary sources of metallic problem in atmosphere. A variety of motor vehicles introduce a number of toxic metals into the environment adjacent to roadways [3]. Soil pollution with heavy metals is considered one of the most severe human health and environmental hazards. High levels of heavy metals not only threaten human health during the food chain, but also decrease crop production and soil microbial activity [4, 5]. Heavy metal contamination can have major effects on indigenous microbial inhabitants. For example, heavy metals may restrict microbial reproduction and decrease species formation [6]. Microorganisms like bacteria, fungi, algae and yeast are known to tolerate and accumulate heavy metals [7]. www.jbm-journal.com

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R. M. Mohamed and Aly E. Abo-Amer

Fungi and yeasts accumulate micronutrients such as Cu, Zn, Mn and non-nutrient metals, like U, Ni, Cd, Sn and Hg in amounts higher than the nutritional requirements. The potential of fungal biomass as biosorbent has been recognized for removal of heavy metals and radionucliedes from polluted waters [8]. Metalresistant fungi isolated from wastewater-treated soil belonged to genera Aspergillus, Penicillium, Alternaria, Geotrichum, Fusarium, Rhizopus, Monilia and Trichoderma [9]. Adaptation of bacteria to heavy metals is attributed to a variety of chromosomal, transposon, and plasmidmediated resistance systems [10]. The incidence of plasmid-bearing strains is more in polluted places than in the unpolluted area [11]. Pseudomonas aeruginosa AA301 isolated from Sohag soil exhibited 60-kb plasmid mediated resistance to a wide range of heavy metals such as Mn2+, Hg2+, Mg2+, Cd2+, Zn2+, and Ni2+ [12]. Heavy metal contamination in soils is one of the serious environmental problems in the world, causing significant risks to ecosystems and public health. Accordingly, for environmental conservation and human health, the development of a remediation strategy for metal-contaminated soils is urgent [13]. Phytoremediation provides more significant and benefits than traditional tools such as chemical and physical remediation technologies to accumulate heavy metals from the soil because of its safer and less expensive for humans and the environment [14 – 17]. But slow growth and low biomass of plants in heavy metalcontaminated soil may reduce the efficiency of phytoremediation [18, 19]. Therefore, the application of heavy metal-resistant and heavy metal-solubilizing microorganisms is a promising approach for increasing heavy metal bioavailability in heavy metal amended soils. Soil bacteria-assisted phytoremediation has been reported [20 – 22] however there is little information on the potential of plant-associated bacteria isolated from plants grown in heavy metal-contaminated soils on the phytoremediation of heavy metal-contaminated soils. As rapid increasing in motor vehicle numbers on Sohag city roads recently and as a consequences of a raise in industrial and commercial activities, significant amounts of some heavy metals are expected to be emitted regularly as long as these pollutants sources are active. Therefore, the objectives of this study were determination the contents of heavy metals such as lead, cadmium and zinc in roadside soil and phylloplane at different sites in relation to their natural background levels and also to isolate and characterize heavy metalresistant microorganisms from soil and phylloplane of roadsides to select strains which might be useful in heavy metal-bioremediation of the environment. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


Materials and methods Collection of samples Soil samples were collected from different places at roadsides in Sohag city, Egypt which were H3 and H4 sites at Hommiat Street, O5 and O6 sites at Opera square, G7 and G8 sites at Gomhouria Street, GM9 and GM10 sites at Gamaa Street (Fig. 1). These locations were selected based on their vehicular densities. Other samples were collected from BG1 and BG2 sites at botanical garden (without vehicular densities) acting as a control, which are far away from traffic routes and represent natural background soil. Healthy plant leaves were also removed from trees of 1.5 – 2.0 m height above the ground located at the same places that soil samples were collected. The samples were kept in plastic bags, placed in icebox and then transported immediately to the laboratory, and stored at 4 °C. All following experiments were carried out in triplicates. Determination of heavy metal contents in soil and phylloplane To determine heavy metal contents in soil of Cd2+, Zn2+ and Pb2+, soil samples were passed through a sieve (1.7 mm mesh) to remove large pieces of debris and vegetation and then air dried at 90 °C for 24 h. Soil sam-

Figure 1. Map of Sohag city showing the locations of samples. The sites are: BG1 and BG2 at Botanical garden, H3 and H4 at Hommiat Street, O5 and O6 at Opera Square, G7 and G8 at Gomhouria Street, GM9 and GM10 at Gamaa Street. www.jbm-journal.com


ples were digested and prepared for heavy metal analysis according to Ho and Tai [23]. In order to detect heavy metal contents of Cd2+, Zn2+ and Pb2+ of phylloplane, deposits adhering to the leaves surfaces were removed by washing off leaves with 50 ml of distilled water [24]. The washing solutions were filtrated using preweighed 0.22 μm pore size filter papers. After filtration, filter papers were air-dried at ambient temperature in air for 24 h, and then re-weighed to calculate the deposit amounts. The dried filter papers were digested according to Keane et al. [25]. Metal concentrations were determined using an atomic absorption spectrophotometer (A Buck Scientific atomic absorption spectrophotometer Model 210 VGP). Isolation and characterization of microorganisms from soil and phylloplane Bacteria and fungi were originally isolated by plating serial dilutions of soils and leave deposits in saline solution (0.9% NaCl) on nutrient agar (NA) and Czapecks (CZ), respectively. Nutrient agar plates were incubated at 37 °C for 48 h while CZ plates were incubated at 28 °C for 7 d. The developed colonies were counted in plates and the average number of colonies per three plates was determined. The total number of bacteria and fungi was determined as CFU/g dry weight for soil and leaf deposits. Individual colonies of bacteria and fungi showing and having different morphological appearance on agar were selected and purified on the same media by streaking 3 – 4 times in the fresh media. The bacterial and fungal isolates were kept on slants at 4 °C and recultured every 4 weeks. The bacterial isolates were tentatively identified on the basis of classification schemes published in Bergey’s Manual of Systematic Bacteriology [26]. Also, identification of fungal isolates was performed according to Raper and Thom [27], Gilman [28] and Domsch et al. [29]. Molecular characterization (16S rRNA) of bacterial isolates For 16S rRNA gene amplification, genomic DNA was extracted from bacterial isolates as described previously by Sambrook et al. [30]. The genomic DNA was resuspended in 50 ml of TE Buffer (10 mM Tris, 1 mM EDTA), pH 8.0 and stored at – 20 °C until used in PCR amplification. The 16S rRNA gene about 1.5 kb long was PCR amplified using the universal primers: forward primer, 5′AGAGTTTGATCCTGGTCAGAACGCT-3′ and reverse primer, 5′-TACGGCTACCTTGTTACGACTTCACCCC-3′ [31]. The PCR reactions were carried out according to the © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Heavy metal resistant microorganisms

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methods described by Chouari et al. [32]. The PCR products of 16S rRNA gene were purified using a QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions. DNA sequencing and phylogenetic tree The ~1.5 kb-PCR products of 16S rRNA genes were used for DNA sequencing. Sequence analysis of the DNA fragments was performed by the Taq dye-deoxy terminator method and an automated 373A DNA sequencer (Applied Biosystems). Selected sequences of other microorganisms with greatest similarity to the 16S rRNA sequences of bacterial isolates were extracted from the nucleotide sequence databases and aligned using CLUSTAL W (1.81) Multiple Sequence Alignment generating phylogenetic tree. The 16S rRNA gene sequences of the bacterial isolates reported in this paper were deposited in the DDBJ/ EMBL/GenBank nucleotide sequence databases with the accession numbers: AB634825 (Bacillus cereus RA81), AB634826 (Pseudomonas aeruginosa RA65), AB634827 (Bacillus subtilis RA43), AB634828 (Bacillus megaterium RA70), AB634829 (Micrococcus luteus strain RA100), AB634830 (Staphyloccus aureus RA20), AB634831 (Streptomyces sp. RA35) and AB634832 (Kocuria rosea RA16). Minimal inhibitory concentrations of heavy metals of bacterial isolates Minimal inhibitory concentrations (MICs) of Cd2+, Zn2+ and Pb2+ for microbial isolates were determined by the plate dilution method as described previously by Aleem et al. [33]. Heavy metals Cd2+, Zn2+ and Pb2+ were used as cadmium chloride, zinc chloride and lead acetate, respectively. The MICs were expressed as the lowest concentration of heavy metals that completely prevented the growth of bacteria. Isolation of plasmid DNA Plasmid miniprep method was used for isolation of plasmid DNA from bacteria as previously described [34]. The isolated plasmid DNA was analyzed by 1% agarose gel electrophoresis according to the standard procedure of Sambrook et al. [30]. Transformation of plasmid DNA To investigate whether the plasmid-mediated resistance to heavy metals under study, competent cells of E. coli DH5α, sensitive to heavy metals, were transformed with respective plasmid using the standard method described previously by Sambrook et al. [30]. One hundred micro-liter of transformed E. coli DH5α suspensions were plated on NA media supplemented with 0.273 mM www.jbm-journal.com

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cadmium chloride, 0.368 mM zinc chloride or 0.132 mM lead acetate and incubated at 37 °C for 24 h. Plasmid characterization of transformant and non transformant one of E. coli DH5α were compared with a resistant strain by 1% agarose gel electrophoresis. Effect of heavy metals on growth of P. aeruginosa RA65 Pseudomonas aeruginosa RA65 was grown in tris-minimal broth [61] amended with different concentrations of mentioned heavy metals. Cadmium chloride was added at concentrations of 0.273 mM and 0.546 mM, zinc chloride was added at concentrations of 0.368 mM and 0.735 mM, and lead acetate was added at concentrations of 0.132 mM and 0.264 mM. Another culture without heavy metals was used as a control. All cultures were incubated at 37 °C for 12 h and at 150 rpm. Cultural samples were taken at regular intervals for measuring optical density at λ = 600 nm (OD600nm) of each culture using a Spekol 11 spectrophotometer. Statistical analysis The data were evaluated by one-way analysis of variance (ANOVA) and assessed by post hoc comparison of means using lowest significant differences (LSD) using SPSS 11.0 software. Differences were considered to be significant among means ±SD standard deviation (n = 3) at a probability of (p < 0.05).

Results Heavy metal contents of soil Heavy metal contents of Cd2+, Zn2+ and Pb2+ of soil at different locations in Sohag city are presented in Table 1. Cadmium content in botanical garden soils at BG1 and BG2 sites was 0.546 μM. However, cadmium content was ranged from 1.1 μM to 1.6 μM in soils collected from sites H4 and G7, respectively. Zinc concentration was ranged around 138.2 – 183.8 μM in BG2 and BG1 sites, respectively while it was 239.1 – 900.7 μM in O6 and GM10 sites, respectively. However, lead content was around 6.069 – 10.290 μM in BG2 and BG1 sites, respectively, it was around 36.9 – 442.1 μM in H4 and GM10 sites, respectively. These results indicated that the sites H3, H4, O5 and O6 are moderately contaminated with zinc and lead. However, the locations G7, G8, GM9 and GM 10 were highly contaminated with these heavy metals in comparison to the natural background soil (Botanical garden). However, these sites are not highly contaminated with cadmium as compared to the control. There was significant variation (p < 0.05) of © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


Table 1. Heavy metal contents of soil and phylloplane at different sites. Sites

* BG1 BG2 H3 H4 O5 O6 G7 G8 GM9 GM10

Heavy metal contents in soil (μM)

Heavy metal contents of phyllpplane (μM)

Cd2+

Zn2+

Pb2+

Cd2+

Zn2+

Pb2+

0.5 0.5 1.6 1.1 1.6 1.1 1.6 1.1 1.6 1.1

183.8 138.2 275.7 294.1 275.7 239.1 477.9 465.4 735.1 900.7

10.1 6.1 39.6 36.9 211.1 224.3 428.8 436.7 389.2 442.1

0.05 0.04 0.07 0.06 0.04 0.19 0.33 0.04 0.06 0.08

7.1 4.8 5.6 3.7 31.6 47.1 72.1 62.5 116.9 136.8

2.1 2.6 7.9 10.6 21.1 29.3 37.7 52.1 94.7 62.3

* BG1 and BG2 at Botanical garden (act as control), H3 and H4 at Hommiat street, O5 and O6 at Opera square, G7 and G8 at Gomhouria street, GM9 and GM10 at Gamaa street.

heavy metal contents in roadsides soil and botanical garden soil because of interaction of vehicular density and heavy metals content. The locations BG1 and BG2 without vehicular density had lower heavy metal contents than high vehicular densities at locations G7, G8, GM9 and GM 10. Heavy metal contents of phylloplane The contents of heavy metals Cd2+, Zn2+ and Pb2+ of phylloplanes at different sites are shown in Table 1. No much difference was detected in cadmium concentrations of phylloplanes collected from botanical garden sites (0.04 – 0.05 μM) and from all vehicular sites (0.04– 0.08 μM). Also, zinc concentrations of botanical garden samples (4.8 – 7.1 μM) were similar to those of other sites H4 and H3 (3.7 – 5.6 μM). However, zinc contents were ranged around 31.6 – 136.8 μM at other vehicular sites. Lead content was detected at higher concentrations in samples collected from vehicular areas (7.9 – 94.7 μM) than those in samples collected from nonvehicular area (2.1 – 2.6 μM). Bacterial population of soil and phylloplane Total viable counts of bacteria were ranged from ~ 6 × 103 to 8.4 × 103 CFU/g of soil samples collected from botanical garden BG1 and BG2 sites, respectively (Fig. 2). However, the bacterial counts ranged around 1.2 × 103– 2.8 × 103 CFU/g in soil samples collected from high vehicular density sites H3 and O6, respectively. Moreover, the bacterial population of phylloplane was ranged around 1.4 × 103 – 1.6 × 103 CFU/g in sites BG2 and BG1, respectively. While, the bacterial counts of phylloplane were recorded ~ 0.2 × 103 and 0.7 × 103 CFU/g at sites GM9 and H3, respectively. These results revealed that www.jbm-journal.com



Figure 2. Bacterial population (CFU/g) of soil (䊏) and phyllolane (䊐) at vehicular density and non-vehicular density sites. These sites are: BG1 and BG2 (non-vehicular density) act as control, H3 and H4 at Hommiat Street, O5 and O6 at Opera Square, G7 and G8 at Gomhouria Street, GM9 and GM10 at Gamaa Street (vehicular density).

bacterial population was lower in the soil and phylloplane samples collected from traffic roadsides with high vehicular densities than comparable samples taken

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from sites (botanical garden) far away from the roadsides. Analysis of results showed that both concentration of heavy metals and in turn vehicular density significantly affected the bacterial population of soil and phylloplane (p < 0.05), i.e, total count of bacteria reduced with increase in heavy metal concentration and in turn the counts increased as far away from the traffic roads. A total of 112 bacterial isolates were obtained from soil and phylloplane. Of these, 8 bacterial isolates were selected out of 112 isolated based on morphological, physicochemical and physiological characterization (Table 2) and identified as Bacillus cereus RA81, Bacillus subtilis RA43, Bacillus megaterium RA70, Kocuria rosea RA16, Micrococcus luteus RA100, Staphylococcus aureus RA20, Streptomyces sp. RA35 and Pseudomonas aeruginosa RA65. The percentage of these bacterial isolates from soil and phylloplane are shown in Table 3. These bacterial isolates were further identified using 16S rRNA gene sequences. The ~ 1500 bp of 16S rRNA gene sequences of the 8 bacterial isolates were amplified using the universal primers for the 16S rRNA gene and sequenced. Phylogenetic tree was constructed by aligning

Table 2. Morphological, physicochemical and biochemical characteristics of bacterial isolates. Characteristics

M. luteus RA100

Kocuria St. aureus B. cereus B. subtilis rosea RA16 RA20 RA81 RA43

B. megaterium RA70

P. aeruginosa RA65

Gram stain Shape

+ve* cocci (tetrads) –ve yellow –ve –ve +ve +ve +ve –ve –ve O* –ve –ve +ve –ve +ve ND –ve

+ve cocci (tetrads) –ve Red or rose +ve +ve +ve +ve –ve –ve –ve O +ve –ve –ve –ve ND ND –ve

+ve cocci (tetrads) –ve orange –ve +ve –ve +ve –ve +ve –ve F* –ve –ve –ve –ve –ve ND –ve

+ve bacilli

+ve bacilli

+ve bacilli

–ve* +ve bacilli (short) filamentous

+ve –ve +ve +ve +ve +ve –ve +ve –ve ND +ve +ve +ve –ve +ve –ve –ve

+ve –ve –ve +ve +ve +ve –ve +ve –ve ND +ve +ve ND +ve +ve –ve –ve

+ve –ve +ve –ve +ve +ve –ve –ve –ve ND +ve +ve ND +ve ND –ve –ve

+ve Green/blue –ve +ve ND* +ve +ve –ve +ve –ve +ve –ve –ve +ve ND ND ND

–ve red +ve –ve –ve +ve +ve –ve +ve ND +ve +ve +ve +ve –ve ND –ve

ND ND ND ND ND ND ND –ve

ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND +ve

–ve ND ND ND ND ND ND +ve

+ve ND ND ND ND ND ND +ve

–ve ND ND ND ND ND ND +ve

ND +ve +ve +ve –ve –ve +ve +ve

ND ND ND ND ND ND +ve ND

Motility Major pigment Acid from glucose Nitrate reduction Growth on 7% NaCl Catalase test Oxidase test V-P test M-R test O-F glucose Gelatin hydrolysis Starch hydrolysis Urease test Citrate test Casein hydrolysis Growth at 65 °C Acid and gas from glucose Growth at 50 °C Growth on King A Growth on King B Growth at 41 °C Growth at 4 °C PHB accumulation Arginine hydrolysis Lipase production

Streptomyces

sp. RA35

* –ve, negative reaction; +ve, positive reaction; O, oxidative; F, fermentative; ND, not determined © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Table 3. Percentage of bacterial isolates in roadside soil and phylloplane. Bacterial isolates

Bacillus cereus RA81 Bacillus subtilis RA43 Bacillus megaterium RA70 Kocuria rosea RA16 Micrococcus luteus RA100 Staphylococcus aureus RA20 Streptomyces sp. RA35 Pseudomonas aeruginosa RA65

Percentage of isolates Soil

Phylloplane

14.81 16.7 7.4 5.56 11.11 9.26 5.56 29.6

14.45 14.45 1.93 36.36 5.5 3.6 5.5 21.81

16S rRNA sequences of different bacteria taken from NCIB and the sequences of the isolates from this study. Comparative 16S rRNA gene sequence analysis of these

isolates showed that they could be allocated in eight phylogenetically different clusters (Fig. 3). Fungal population of soil and phylloplane Total counts of fungi from soil and phylloplane are shown in Fig. 4. Fungal counts in botanical garden soil were ranged around 1.4 × 103 – 3.4 × 103 CFU/g at BG2 and BG1 sites, respectively. However, fungal populations in soil samples collected from high vehicular density sites ranged from 0.2 × 103 to 0.8 × 103 CFU/g at GM10 and G8 sites, respectively. Moreover, total counts of fungi in phylloplane collected from botanical garden sites were around 0.4 × 103 – 0.6 × 103 CFU/g at BG2 and BG1, respectively. The fungal counts were ranged around 0.2 × 103 – 0.7 × 103 CFU/g of phylloplane in vehicular density sites at GM9 and H3, respectively. These

Figure 3. Neighbour-joining phylogenetic tree of bacterial isolates based on 16S rRNA gene sequences. Bacterial isolates in each phylogenetic cluster are highlighted in bold. The GenBank accession numbers of the bacteria are shown in parentheses. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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phologically characterized (Table 4) and identified as Aspergillus niger, A. ficuum, A. flavus, A. flavus v. columnaris, A. ochracieos, A. fumigatus, A.versicolor, A. tereus, Penicillium coryllephyllum, P. purpurginum, Fusarium moniliformi, Cladosporium cladosporoides and Alternaria alternate. The percentage of these fungal isolates from soil and phylloplane are shown in Table 5.

Figure 4. Fungal population (CFU/g) of soil (䊏) and phylloplane (䊐) at vehicular density and non-vehicular density sites. These sites are: BG1 and BG2 (non-vehicular density) act as control, H3 and H4 at Hommiat Street, O5 and O6 at Opera Square, G7 and G8 at Gomhouria Street, GM9 and GM10 at Gamaa Street (vehicular density).

results indicated that both heavy metal concentrations and in turn vehicular density significantly (p < 0.05) affected on fungal counts of soil sample. A number of 62 fungal isolates from soil and phylloplane were mor-

Minimal inhibitory concentration (MIC) Minimal inhibitory concentrations of different heavy metals Cd2+, Zn2+ and Pb2+ for bacterial isolates are shown in Table 6. Minimal inhibitory concentration of Cd2+ for bacteria ranged between 0.273 mM and 0.821 mM whereas the MICs of Zn2+ fluctuated between 0.368 mM and 1.471 mM. Surprisingly, Pb2+ was detected as the highest MIC (>1.093 mM) for bacteria. However, the MIC of fungal isolates ranged between 0.109 mM and 0.328 mM in case Cd2+ while it was 0.147 mM and 0.588 mM in case of Zn2+ (Table 7). In addition, Pb2+ was the highest MIC (> 0.211 mM). According to the minimal inhibitory concentrations, heavy metals differed in their inhibitory effects on the bacte-

Table 4. a: Morphological characterization of Asperigulls isolates.

Aspergillus sp.

Colony color

Cinidophore

Condia

Condial head

Vesicle

Strigmata

A. niger

Black

Globose Hyaline rough

Radiate

Globose

Uni or bserrate

A. ficuum

Black

Globose Hyaline rough

Radiate

globose

Biserrate

A. flavus

Pale yellow green

Globose hyaline smooth

Radiate to loose columnar

Globose

Uni or biserrate

A. flavus v. columnaris

Green to brown

Globose Hyaline echinulate

Columnar

Sub-globose

Uniserrate

A. ochracieos

Golden yellow


Radiate

Globose

Biserrate

A. fumigatus

Green

Globose Hyaline echinulate

Columnar

Globose

Monserrate

A. versicolor

Green

Globose

Columnar

Globose

A. tereus

Brown

Long Hyaline smooth Straight long pigmented smooth somewhat sinuate Long Hyaline Rough Straight Long Hyaline smooth Straight Long pigmented Rough Straight Long Hyaline smooth Straight Long Hyaline smooth Straight Short Hyaline smooth Sinuate


columnar

Sub-globose

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Biserrate

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Table 4. (Continued) b: Morphological characterization of Penicillium isolates.

Penicillium sp.

Colony color

Colony reverse

Conidiophre

Conidia

Matulae

Penicilli

P. coryllephyllum

Grey green

White pale yellow

Ovate Smooth hyaline

present

Asymmetrica biverticillate

P. purpurginum

Dark grey green

Red-purple

short unbranched smooth Straight short unbranched smooth Straight

Globose Hyaline smooth

present

Symmetrica biverticillate

c: Morphological characterization of other fungal isolates isolates. Other fungal genera

Colony color

Conidiophre

Conidia

Fusarium moniliformi

Chlamidospors absent

Cladosporium cladoporoides

White to light lilac, deep violet pigmentation Grey

Alternaria alternate

Grey

Macroconidia are difficult to find, microconidia are abundant, oval to clavate, zeroseptate, formed in long chans on mono phialidic conidiogenous cells. Long branched chains, zero septate, ellipsoidal or limoniform, 3 – 11 × 2 – 5 μ, pale olivacious brown, smooth. Long loosely branched chains, brown, rough

Short, smooth, pigmented –

Table 5. Percentage of fungal isolates in roadside soil and phylloplane. Fungal isolates

Aspergillus niger A. ficuum A. flavus A. flavus v. columnaris A. ochracieos A. fumigatus A.versicolor A. tereus Penicillium coryllephyllum P. purpurginum Fusarium moniliformi Cladosporium cladosporoides Alternaria alternata

Percentage of isolates (%) Soil

Phylloplane

17.8 – 3.6 – 7.1 3.6 3.6 7.1 28.6 3.6 10.7 10.7 3.6

23.5 2.9 5.9 5.9 – – 8.8 5.9 24.7 – 5.9 8.8 7.7

rial and fungal isolates. The ranking of the inhibitory effects of the three heavy metal ions on bacterial and fungal isolates declined in the order Cd > Zn > Pb. The results indicated that bacterial and fungal isolates were high resistance to lead. Among microbial isolates, P. aeruginosa RA65 exhibited high resistance to above mentioned heavy metals especially with lead so this strain was selected for further studies. Growth of P. aeruginosa RA65 in presence of heavy metals Growth curves of P. aeruginosa RA65 in presence of different concentrations of heavy metals are shown in Fig. 5. The growth of bacteria in absence of heavy metals (control) was 1.12 (OD600) after 12 h from incubation time. In presence of lead with concentrations of © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 6. Minimal inhibitory concentrations (MICs) of heavy metal of the bacterial isolates. MICs of heavy metals (mM) Cd2+ 0.273 0.546 0.821 1.093 Zn2+ 0.368 0.735 1.103 1.471 > 1.471 Pb2+ 0.132 0.264 0.396 0.528 > 0.528

Number of bacterial isolates 32 (28.5)* 79 (70.5) 1 (0.9) 0 (0) 18 (16) 89 (79) 4 (3.5) 1 (0.9) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

* Values in parentheses represent the percentage of resistant isolates.

0.132 mM and 0.264 mM, the growth was 1 and 0.96 (OD600), respectively, at the same time. These results showed that no much difference in bacterial growth in absence or presence of lead. However, when zinc was added with concentrations of 0.368 mM and 0.735 mM, the growth was 0.75 and 0.63 (OD600), respectively, at the same time. Moreover, the growth was 0.49 and 0.35 (OD600) in presence of cadmium with concentrations of 0.273 mM and 0.546 mM, respectively, at the same time. These results indicated that the lead had not affected significantly (p < 0.05) on the bacterial growth. However, the zinc or cadmium had significantly (p < 0.05) effected on the growth. www.jbm-journal.com



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Table 7. Minimal inhibitory concentrations (MICs) of heavy metal of the fungal isolates. MICs of heavy metals (mM)

Number of fungal isolates

2+

Cd 0.109 0.219 0.328 0.437 Zn2+ 0.147 0.294 0.441 0.588 > 0.588 Pb2+ 0.053 0.106 0.158 0.211 > 0.211

23 (37)* 38 (61) 1 (1.6) 0 (0) 28 (45) 30 (48) 3 (4.8) 1 (1.6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

* Values in parentheses represent the percentage of resistant isolates.

Plasmid profile of P. aeruginosa RA65 and transformation The ability of P. aeruginosa RA65 to be resistant to some heavy metals might be attributed to a genetic factor. To determine whether the resistance ability of this strain was mediated by plasmid, firstly, isolation of plasmid was carried out. Agarose gel electrophoreses of plasmid preparation confirmed the presence of one plasmid with molecular weight of ~ 9.5 kb in P. aeruginosa RA65 (Fig. 6). Secondly, E. coli DH5α was transformed with the ~ 9.5 kb plasmid and demonstrated that the transformant E. coli DH5α could grow on nutrient agar containing 0.109 mM cadmium, 0.368 mM zinc or 0.264 mM

Figure 5. Growth curve of the resistant strain P. aeruginosa RA65 grown in tris-minimal broth with different concentrations of heavy metals at 37 °C and at 150 rpm for 12 h. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 6. 1% agarose gel electrophoresis of DNA. Lane 1 represents DNA ladder, lane 2 represents plasmid preparation from P. aeruginosa RA65, lane 3 represents plasmid preparation from E. coli DH5α, and lane 4 represents plasmid preparation from transformant E. coli DH5α. The left arrows indicate the molecular weight of DNA ladder and the right arrow represents the DNA plasmid.

lead, while the non-transformed strain could not grow on these heavy metals. The presence of plasmid in the transformant strain was confirmed by plasmid preparation and agarose gel electrophoresis (Fig. 6) and indicated that the transformant strain had a plasmid with the same size (~ 9.5 kb) as in the P. aeruginosa RA65.

Discussion Heavy metal contents of Cd2+, Zn2+ and Pb2+ of soil and phylloplane were determined at different sites of roadsides and compared with their natural background levels in botanical garden soil. Locations of botanical garden (without vehicular density) had lower heavy metal contents in soil than other locations of roadsides with high vehicular density. Lead content of soil was around 6.1 – 10.3 μM in non-traffic sites, where as, in traffic areas it was around 36.9 – 442.1 μM. However, zinc content of soil was 138.2 – 183.8 μM in nonvehicular areas, while it was 275.7 – 900.7 μM in traffic sites. Cadmium content of soil was similar in botanical garden and vehicular sites (0.5 – 1.6 μM). Contents of Cd2+, Pb2+ and Zn2+ in roadside soil measured by other investigators in various countries worldwide were 0.89, 444 μg/g in USA, different cities [35]; 0.70, 180, 205 μg/g in Birmingham [36]; 4.2, 1354, 513 μg/g in London [37]; 6.8, 1779, 1143 μg/g in North Wales [38]; 1.3, 247, 163 μg/g in Nigeria [39]; 0.36, 293, 509 μg/g in Ecuador www.jbm-journal.com

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[40]; 1.1, 991, 633 μg/g in Hong Kong [41]; 5.2,–, 300 – 530 μg/g in Lancaster [23]; 0.4, 1650, 180 μg/g in Auckland [42] and 0.75, 188.8, 121.7 μg/g, respectively, in Amman [43]. Also, previous work reported that lead concentration was the highest in the roadside soil and ranged from 25 to 1198 μg/g and zinc concentration ranged from 56.7 to 480 μg/g. Cadmium concentration was the lowest in the soil and varied from 0.3 to 3.8 μg/g. The levels of heavy metals in roadside soils were higher as compared to their natural background contents in British soils [44]. Moreover, recent studies reported that measurement of heavy metal pollutants Pb2+ and Zn2+ was carried out in three zones along major roadside soils of Botswana and revealed that zones FN (Pb2+ = 0.03 – 0.34 μg/g, Zn2+= 0.06 – 0.29 μg/g), NM (Pb2+ = 0.06 – 1.30 μg/g, Zn2+= 0.05 – 0.11 μg/g) and MG (Pb2+ = 0.07 – 0.49 μg/g, Zn2+= 0.04 – 0.32 μg/g) are pollution impacted as compared to GK (Pb2+ = 0.03 – 0.04) (Zn2+ = 0.03 – 0.11 μg/g) and TS (Pb2+ = 0.04 – 0.08 μg/g) (Zn2+= 0.05 – 0.10 μg/g) zones [45]. The present study indicated that high contents of Zn2+ and Pb2+ compared to botanical garden were found in traffic samples. The values of heavy metals Pb2+ and Zn2+ in roadside soil suggested that automobiles were a major source of these metals in the roadside environment. Similar results were recorded that heavy metal concentrations were decreased in sites far away from traffic road which might be attributed to emitting heavy metals from vehicle exhausts in particulate forms which are forced to settle under gravity closer to the road edge [3, 46]. Moreover, results reported that lead was strongly associated with vehicular emissions and zinc was associated with various industries and metal smelting processes [47]. Another study demonstrated that lead was extremely enriched in roadside soils, mainly due to vehicular emissions [45]. Concentrations of these heavy on phylloplane at different sites were detected. Cadmium levels in phylloplanes were very low and no much difference was detected in cadmium concentrations of phylloplanes collected from botanical garden sites (0.05 – 0.04 μM) and from all traffic sites (0.04 – 0.3 μM). Previous results reported that cadmium levels in plants at roadside were below the detection limits of the flame AA [41, 43, 48]. Also, zinc concentrations of phylloplane samples collected from botanical garden (4.8 – 7.1 μM) were less than to those of traffic sites (3.7 – 136.8 μM). Lead content was around 2.1 – 2.6 μM in botanical garden sites, where as, in traffic areas it was around 7.9 – 94.7 μM. Contents of heavy metals Pb2+ and Zn2+ of roadside phylloplanes were detected by other investigators in © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


various countries worldwide. Contents of Pb2+ in roadside phylloplane were 180, 134 and 7.3 μg/g in Auckland [42], Hong Kong [41] and Amman [43], respectively. Also, Zn2+ contents in roadside phylloplane were 37 – 114, 32 – 85, 30 – 180, 124 and 98.7 μg/g in Belgium [49], USA [48], Dutch Coast [50], Hong Kong Harrison) and Amman [43], respectively. In the present study, lead content was detected at higher concentrations of phylloplane samples (7.9 – 94.7 μM) collected from vehicular areas than those in botanical garden (2.1–2.6 μM) suggesting that automobiles are a major source of this metal in the roadside environment. These results were in agreement with those reported by Celik et al. [51] that contents of heavy metals was high in plants located at industrial and highway roadsides because of anthropogenic activities and elevated traffic density. Heavy metal contents were higher in soils than in phylloplanes which is might be attributed to meteorological condition since the phylloplanes are subjected to windy condition. Heavy metal-resistant microorganisms were counted, isolated and characterized from roadside soil and phylloplane. Heterotrophic bacteria were isolated and counted from soil and phylloplane samples at roadsides. The results revealed that the higher heavy metal concentrations the lower bacterial counts which indicates the effect of heavy metals concentrations on the microbial flora of the environment. Similar results reported that high concentrations of heavy metals not only cause serious health hazards but also disturb the ecological status of biota [11, 52]. Isolation of bacteria (Tables 3 and 6) from these heavy metal-contaminated sites indicated that these bacterial isolates were resistant to heavy metals. Fungi were also isolated and counted from soil and phylloplane. The present results indicated that fungal counts were lower in soil samples near the roads than comparable samples away from the road (botanical garden). However, the fungal counts were higher in phylloplane samples in some sites near the roads than comparable samples away from the road. This result might be attributed to acidic condition (low pH) around the roads which is favorite to fungi growth. Far away from the traffic roads and heavy metal concentrations had no significant effect on fungal counts in phylloplane. Similar result reported that the pH values were lower in soil closer to road junctions which caused increase in fungal count and reduction in counts of most bacterial species [46]. Fungal strains (Tables 5 and 7) were isolated from these heavy metal-contaminated area suggested that these isolates have capability to resist heavy metals. Previous data indicated that bactewww.jbm-journal.com


ria and fungi isolated from soil at different road junctions were capable of growing in presence of toxic heavy metals such as lead [53]. Minimal inhibitory concentrations of heavy metals under study for bacterial isolates were determined. A very high resistance to lead was noticeable among bacterial and fungal isolates. The high resistance to lead may be due to high lead contamination in soil and phylloplane from which the bacteria and fungi were isolated. Konopka et al. [54] isolated bacteria from heavy metal contaminated soils in Indianapolis-India, and reported that 10 to 100% of bacterial isolates were heavy metal resistant. Recent studies reported that a bacterial strain which showed a very high degree of resistance to heavy metals, especially to lead, it grew well in sucrose-minimal salts low-phosphate (SLP) medium containing lead (2.639 mM) [52]. Moreover, other results reported that high MICs of Pb+2, Cu+2, Zn+2, Hg+2, Co+2, Cd+2, Cr+3 Te+2, As+2 and Ni+2 were determined for bacteria isolated from two different sites in Sohag province, Egypt, which were 1200 μg/ml for lead, 800 μg/ml for both cobalt and arsenate, 1200 μg/ml for nickel, 1000 μg/ml for copper and less than 600 μg/ml for other metals [55]. On the other hand, heavy metal resistant fungi such as Aspergillus, Penicillium, Alternaria, Geotrichum, Fusarium, Rhizopus, Monilia and Trichoderma were isolated from the agricultural soils polluted with industrial and municipal water [56]. Moreover, Penecillium ochrochrolon has been reported to grow in a saturated solution of cupper sulfate [57]. The present data revealed that most resistant fungal isolate was Penicillium coryllephyllum which had MICs of 0.437 mM and 0.588 mM for Cd+2 and Z+2, respectively. Also, P. aeruginosa RA65 was determined to be the most resistant strain to heavy metals with MICs of 0.821 mM, 1.471 mM and > 0.528 mM for Cd2+, Zn2+ and Pb2+, respectively. Growth of P. aeruginosa RA65 in presence of different concentrations of heavy metals was determined. The growth was not significantly affected by lead however it was significantly affected by zinc or cadmium. Other results reported that growth of Acidocella facilis was decreased in presence of ZnSO4, CdSO4, CuSO4 or NiSO4 [58]. Moreover, recent studies reported the inhibitory effect of Cd, Pb, Zn or Cu on the growth of Agrobactrium tumefaciens CCNWRS33-2 isolated from root nodules of wild legumes growing in gold mine tailings in northwest of China [6]. Resistance capability of P. aeruginosa RA65 to heavy metals might be as a result of a genetic factor. The present study revealed that heavy metal resistance of P. aeruginosa RM65 is mediated by 9.5 kb plasmid. Our © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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findings were similar to the results reported that 100% of resistant P. aeruginosa strains isolated from heavy metal-contaminated regions were harboring plasmids [59]. Also, 60 kb plasmid mediated resistance to a wide range of heavy metals such as Mn2+, Hg2+, Mg2+, Cd2+, Zn2+, and Ni2+ was detected in P. aeruginosa AA301 isolated from Sohag soil, Egypt [12]. The incidence of plasmid-carrying bacteria is higher in metal-polluted sites than in the unpolluted regions [11]. Transfer of resistance genes can occur between different genera and species of bacteria by horizontal conjugation [60], therefore, the isolate P. aeruginosa RA65 could act as reservoir of resistance genes. In addition, the high incidence of heavy metal resistance detected in this work indicates the potential of these microorganisms as bioremediation agents.

Acknowledgement The authors gratefully thank Prof. Simon C. Andrews, Department of Microbiology, Reading University, England; for his assistance.

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