Geomicrobiology Journal, (2015) 32, 836–845 Copyright © Taylor & Francis Group, LLC ISSN: 0149-0451 print / 1521-0529 online DOI: 10.1080/01490451.2015.1010754
Isolation and Molecular Characterization of Heavy Metal-Resistant Alcaligenes faecalis from Sewage Wastewater and Synthesis of Silver Nanoparticles ALY E. ABO-AMER1,2*, ABD EL-RAHEEM R. EL-SHANSHOURY3,4, and OTHMAN M. ALZAHRANI3 1
Department of Biology, Division of Microbiology, University of Taif, Taif, Saudi Arabia Department of Botany, Division of Microbiology, Faculty of Science, Sohag University, Sohag, Egypt 3 Department of Biotechnology, Faculty of Science, University of Taif, Taif, Saudi Arabia 4 Department of Botany, Unit of Bacteriology, Faculty of Science, Tanta University, Tanta, Egypt 2
Received December 2014; Accepted January 2015
Environmental pollution with toxic heavy metals is increasing throughout the world alongside industrial development. Microorganisms and microbial products can be highly efficient bioaccumulators of soluble and particulate forms of metals, especially dilute external solutions. Microbe related technologies (Biotechnology) may provide an alternative or additive conventional method for metal removal or metal recovery. This study dealt with isolation, identification and characterization of heavy metal-resistant (Pb2C, Cd2C, Al3C, Cu2C, Ag2C and Sn2C) bacteria from sewage wastewater at Taif Province, Saudi Arabia. Nine bacterial isolates were selected by using an enrichment isolation procedure based on high level of heavy metal resistance. All the isolates showed high resistance to heavy metals with Minimum Inhibitor Concentration (MIC) ranging from 800 mg/ml to 1400 mg/ml. All nine resistant isolates showed multiple tolerances to heavy metals. On the basis of morphological, biochemical and 16S rRNA characterization, the most potent isolates (Cd1-1, Ag1-1, Ag1-3 and Sn1-1) were identified as Alcaligenes faecalis. Scanning electron microscope analysis showed that the morphology of Alcaligenes faecalis Ag1-1 was unchanged after growth in medium without and with addition of Ag2C indicative Ag2C is not toxic to the isolate under the conditions tested. The ability of Alcaligenes faecalis Ag1-1 to synthesize sliver nanoparticles was examined. The heavy metal-resistant bacteria obtained could be useful for the bioremediation of heavy metal-contaminated environment. Keywords: 16S rRNA, Alcaligenes faecalis, heavy metal resistance, scanning electron microscope, silver nanoparticles, sewage, transmission electron microscope
Introduction Pollution of soil, water and wastewater by heavy metals is a significant environmental problem (Cheng 2003). In this context, industrial and sewage sludge wastewaters have permanent toxic effects to human and the environment (Rehman et al. 2008). Lead (Pb2C) is a major pollutant that is found in soil, water and air. It is a hazardous waste and highly toxic to human, animals, plants and microbes (Low et al. 2000). Cadmium (Cd2C) is unnecessary but poisonous for humans, animals and plants (Gupta and Gupta 1998). Cadmium is one of the most toxic pollutants of the surface soil layer, released into the environment by mining and smelting activities, burning of batteries and plastics, land application of sewage sludge, atmospheric deposition from metallurgical industries, and burning of fossil fuels (Tang et al. 2006). Nickel (Ni) is the 24th-most abundant element in the Earth’s crust and has
*Address correspondence to Aly E. Abo-Amer, Department of Biology, Division of Microbiology, Faculty of Science, Taif University, Taif 888, Saudi Arabia; Email:
[email protected]
been detected in different media in all parts of the biosphere. Ni is classified as the borderline metal ion, because it has both soft and hard metal properties and can bind to sulfur, nitrogen and oxygen groups (Costa and Klein 1999). Ni has been concerned as an embryo toxin and teratogen (Chen and Lin 1998). Mercury is one of the most toxic metals in the environment. It has been released into environment in extensive quantities through natural events and anthropogenic activities (Kiyono and Hou 2006). Wastewater contains significant concentration of heavy metals that are not degraded by the conventional process of wastewater treatment. Presence of high concentrations of toxic heavy metals in wastewater directly leads to both contamination of receiving water bodies and a harmful impact on aquatic life. Using such polluted water can bring severe problems to human health. At higher concentrations, heavy metals result in toxic complex compounds in the cell that are too dangerous for any biological systems. Removal of excesses of heavy metal ions from wastewaters is essential due to their extreme toxicity towards aquatic life and humans. The uses of conventional technologies, such as ion exchange, chemical precipitation, reverse osmosis and
Heavy Metal-Resistant Alcaligenes faecalis evaporative recovery for this purpose are often inefficient and/or very expensive (Volesky 1990). Therefore, there is a need for innovative treatment technologies for the removal of heavy metal ions from wastewater. Recent studies reported that microorganisms were employed to remove pollutants such as toxic organic and inorganic compounds from the environment (Abo-Amer 2007; Abo-Amer 2011; Abo-Amer 2012; Ahemad and Malik 2012; Francois et al. 2012; Mohamed and Abo-Amer 2012; Singh and Gadi 2012; Volesky 1990). Different microbes have been proposed to be efficient and economical alternatives in removing heavy metals from water (Abo-Amer et al. 2013; Ahemad and Malik 2012; Francois et al. 2012; Singh and Gadi 2012; Waisberg et al. 2003). The heavy metal-resistant microorganisms have significant role in wastewater treatment system. The detoxifying ability of these resistant microorganisms can be manipulated for bioremediation of heavy metals in wastewater. Effluents having heavy metals can be treated with these microorganisms by processes such as biosorption, bioaccumulation and bioprecipitation (Abo-Amer et al. 2013; Ahemad and Malik 2012; Francois et al. 2012; Singh and Gadi 2012). The capability of microbes to synthesize nanoparticles as an extension of the metal accumulation process has turned the attention of researchers towards microorganisms to synthesize extracellular or intracellular nanoparticles (Bhattacharya and Rajinder 2005; Catauro et al. 2004; ElShanshoury et al. 2011; Hussain et al. 2003; Kuber and D’Souza 2006; Sharma et al. 2000). The microbial synthesis of nanoparticles has emerged as a promising field of research as nanobiotechnology interconnecting biotechnology and nanotechnology (Narayanan and Sakthivel 2010). The dried cells of Corynebacterium sp. SH09 were found to produce silver nanoparticles on the cell wall, in the size range of 10– 15 nm with diamine silver complex [Ag(NH3)2]C (Zhang et al. 2005). The bacteria, Enterobacter sp. and Bacillus anthracis PS2010 (avilrulant strain) have the ability to synthesize lead nanoparticles (El-Shanshoury et al. 2012). Heavy metals are transported from wastewater and contaminate water sources and the environment. Contamination of soil, groundwater, sediment and surface water with toxic heavy metals are serious problems, which have been faced by the world recently. Therefore, this project was aimed to isolate and characterize heavy metal-resistant (Pb2C, Cd2C, Al3C, Cu2C, Ag2C and Sn2C) bacteria from sewage wastewater at Taif Province. Also, this work aimed to study the ability of heavy metal-resistant bacteria to synthesize nanoparticles. The isolated bacteria might be used to clean heavy metals from a contaminated environment.
Materials and Methods Sample Collection Samples were collected from sewage wastewater at Taif Province, Saudi Arabia. The samples were taken in sterile plastic bottles and transported to the laboratory in an ice box for bacteriological analysis.
837 Isolation of Heavy Metal-Resistant Bacteria For the selective isolation of heavy metals-resistant bacteria, heavy metal-supplemented media were used. Isolation of cultures from the water samples was carried out using a enrichment isolation procedure. Basal media; nutrient broth (NB) supplemented with heavy metals like CdC2, AgC2, CuC2, PbC2, AlC3 and SnC2 were prepared separately. Heavy metals CdC2, AgC2, CuC2, PbC2, AlC3 and SnC2 were used as CdCl2, CuSO4.5H2O, Pb (CH3COO) 2.3H2O, AgNO3, Al2(SO4)3.18H2O and SnCl2.2H2O, respectively. The concentration of each heavy metal was maintained at 100 mg/ml of the medium (heavy metal stock was 100 mg/ml). One ml of wastewater samples was added to these media and incubated at 37 C for 24–48 h. The previous cultures were directly streaked onto Nutrient Agar (NA) supplemented with 50 mg/ml heavy metal, and the inoculated plates were incubated at 37 C for 24–48 h. At the end of the incubation period, the plates were observed for any microbial growth on the media. Distinct colonies that appeared on these selective media were subcultured repeatedly on the same media for purification. Colonies differing in morphological characteristics were selected and used for further studies.
Determination of Minimum Inhibitory Concentrations (MICs) Minimum inhibitory concentrations (MICs) of the heavy metal-resistant bacterial isolates grown on heavy metals supplemented-media, against respective heavy metal were determined by gradually increasing the concentration of the heavy metal on NA plates until the strains failed to grow on the plates. The culture growing on the first concentration was transferred to the higher concentration (200, 400, 800, 1000, 1200, 1400, 1600 and 2000 mg/ml) by streaking on the plates. MIC was noted when the isolates failed to grow on plates, even after 48 h of incubation.
Determination of Co-resistance to Other Heavy Metals Various bacterial isolates resistant to one heavy metal were tested for their resistance to the rest of the heavy metals chosen for this study. The concentration of heavy metals in this test was 400 mg/ml. Identification and Characterization of Heavy Metal-Resistant Bacteria The pure cultures were identified on the basis of their morphology, physiology and biochemical characters as well as molecular techniques. The shapes and colors of the colonies were examined under the microscope after Gram staining. Isolates were biochemically analyzed for the activities of oxidase, catalase, V-P test, MR-VP test, starch hydrolysis and gelatin hydrolysis, motility, indole production and citrate utilization. The tests were used to identify the isolates according to Bergey’s Manual of Systematic Bacteriology (Claus and Berkeley 1986).
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16S rRNA Gene Amplification
Preparation of Bacterial Cultures for Electron Microscope
Genomic DNA was isolated and analyzed from bacterial isolates by the method of Chen and Kuo (1993). Bacterial 16S rRNA gene was amplified by using the universal bacterial 16S rRNA gene primers, F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and R (50 - GGT GTT TGA TTG TTA CGA CTT-30 ). PCR was performed with a 50-ml reaction mixture containing 1-ml (10 ng) of DNA extract as a template, each primer at a concentration of 5 mM, 25 mM MgCl2 and dNTPs at a concentration of 2 mM, as well as 1.5 U of Taq polymerase and buffer used, as recommended by the manufacturer. After the initial denaturation for 5 min at 94 C, the samples were incorporated into 35 cycles consisting of denaturation at 94 C for 1 min, annealing at 55 C for 1 min, extension at 72 C for 1 min and final extension at 72 C for 5 min. PCR products were analyzed by 1.5% (w/v) agarose gel electrophoresis in 1£ TAE buffer with ethidium bromide (0.5 mg/ml).
One liter of nutrient broth medium free of metals was prepared for each isolate and sterilized by autoclaving. Media containing heavy metals such as CdC2 (1000 mg/ml), AgC2 (1200 mg/ml), CuC2 (1200 mg/ml), PbC2 (2500 mg/ml), AlC3 (2000 mg/ml) and SnC2 (1400 mg/ml) were inoculated with overnight cultures previously prepared for isolates. The same isolates were also grown in nutrient broth without any heavy metal acted as control. Then the cultures were grown to the end of exponential phase, after constructing the growth curve. After the incubation, cultures were centrifuged at 5000 g for 20 min to obtain the cells and the pellets were washed three times with sterilized distilled water. The pellets that contain cells treated and untreated with the used heavy metal were examined by electron microscope.
Phylogeny PCR products of 16S rRNA genes of isolates were purified by using QIAgen Gel Extraction Kit and then sequenced. Sequences were matched with previously published bacterial 16S rRNA sequences in the NCBI databases using BLAST. Based on the scoring index the most similar sequences were aligned with the sequences of other representative bacterial 16S rRNA regions using Clustal X software (version 1.83). Further phylogenetic tree information and similarity index were generated and compared with known sequences. The 16S rRNA sequences of bacterial isolates were deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers: AB967975 (Alcaligenes faecalis Cd1-1), AB967977 (Alcaligenes faecalis Ag1-1), AB967978 (Alcaligenes faecalis Ag1-3) and AB967983 (Alcaligenes faecalis Sn1-1).
Electron Microscope Examination The cells of the isolates before and after the treatment with heavy metal were examined by scanning electron microscope (SEM) to detect if there is any change in the morphology of the cells as a result of metal treatment. The cells of control and treated cultures (as described before) were separated, washed twice and fixed in 2.5% buffered glutaraldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4 for 24 h at 4 C, washed three times with PBS for 10 min in each time and then centrifuged. The previous steps were followed by post fixation in 1% osmic acid for 30 min and dehydrated in a series of ethyl-alcohol (30–100%), infiltrated with acetone each concentration for 30 min. Scanning Electron Microscopy (SEM) Samples were dried in critical point drying machine using liquid CO2, The cells were mounted on stubs and coated with gold in a coater apparatus, and graphs were taken with a scanning electron microscope.
Isolation of Plasmids Plasmids of bacterial isolates were investigated with a QIAprep spin miniprep kit. The DNA samples were subjected to electrophoresis along with a standard supercoiled DNA ladder.
Growth Curve Growth of bacterial isolates were studied in 250-ml flasks containing 50 ml NB medium supplemented with different heavy metals such as CdC2 (800 mg/ml), AgC2 (1000 mg/ml), CuC2 (1000 mg/ml), PbC2 (2000 mg/ml), AlC3 (1600 mg/ml) and SnC2 (1200 mg/ml). Flasks were inoculated with 0.5 ml of overnight culture and agitated on a rotary shaker (150 rpm) at 30 C. Growth was monitored as a function of biomass by measuring the absorbance at 600 nm using spectrophotometer (Hitachi, Japan).
Transmission Electron Microscopy (TEM) After dehydration, samples will be embedded in Araldite 502 resin. The plastic molds will be cut at 850 nm thickness in an Ultracut ultra-microtome, and then they can be stained with 1% toluidine blue. After examination of semithin sections, ultrathin sections will be cut at thickness of 75 nm, stained with uronyl acetate for 45 min, then counterstained with lead citrate, examined and photographed using electron microscope. Statistical Analysis The experiments were carried out in triplicate. Analyses were performed according to one-way analysis of variance (ANOVA) and assessed by post hoc comparison of means using lowest significant differences (LSD) using SPSS 11.0 software. They were considered significant at the p < 0.05 level.
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Results
Table 2. MICs and effect of different concentrations of cadmium on isolates from sewage water selected on NB supplemented with 50 mg/ml AgC2
Isolation of Heavy Metal-Resistant Bacteria In the present study we identify and characterize heavy metalresistant bacteria isolated from sewage water. The enrichment isolation technique was used to isolate metal-resistant bacteria. Isolated colonies were picked up from plates according to their different form and purified by subculturing onto fresh nutrient-metal agar plates using the streak-plate technique. Fifty isolates were screened from initial level of heavy metal supplemented LB medium. Only nine bacterial isolates from sewage water samples were shown resistance to tested heavy metals of concentration 800–1200 mg/ml. Nine isolates called Cd1-1, Ag1-1, Ag1-2, Ag1-3, Ag1-4, Sn1-1, Sn1-2, Sn1-3 and Sn1-4, which were resistant to Cd, Ag and Sn were recovered from sewage water. MIC of Heavy Metals and Antibiotics The Minimum Inhibitory Concentration (MIC) of wastewater isolates against the heavy metals was determined in solid media and ranged from 800 to 1400 mg/ml. Bacterial isolates Cd1-1 showed a very high degree of resistance to Cd, MIC values were 1000 mg/ml. However, isolates Cd1-2 showed MIC of 800 mg/ml to CdC2 (Table 1). Isolates Ag1-1, Ag1-3 and Ag1-4 showed MIC of 1200 mg/ml, whereas isolated Ag1-2 demonstrated MIC of 1000 mg/ml to AgC2 (Table 2). MICs of bacterial isolates Sn1-1, Sn1-2, Sn1-3 and Sn1-4 were 1400 mg/ml to SnC2 (Table 3).
Bacterial isolates Cd1-1, Ag1-1, Ag1-3 and Sn1-1 showed coresistance to Cd2C, Pb2C (400 mg/ml each) whereas isolates Cd1-1, Ag1-1, Ag1-3, Sn1-1 demonstrated co-resistance to Cd2C, Pb2C, Ag2C, Al3C (400 mg/ml each). All the isolates showed no co-resistance to heavy metals Cd2C, Pb2C, Ag2C, Al3C, Cu2C, Sn2C (400 mg/ml each). Finally 4 isolates Cd1-1, Ag1-1, Ag1-3 and Sn1-1 were selected based on a high degree of heavy metal resistance were used for further studies. Identification and Characterization of the Most Potent Bacterial Isolates The most potent isolates (Cd1-1, Ag1-1, Ag1-3 and Sn1-1) were identified and characterized (Table 4). All isolates were Table 1. MICs and effect of different concentrations of cadmium on isolates from sewage water selected on NB supplemented with 50 mg/ml CdC2 CdC2 concentrations (mg/ml)
Cd1-1 Cd1-2
Ag1-1 Ag1-2 Ag1-3 Ag1-4
100
200
400
800
1000
1200
MIC(mg/ml)
C C C C
C C C C
C C C C
C C C C
C ¡ C C
¡ ¡ ¡ ¡
1200 1000 1200 1200
C, growth; ¡, no growth.
Gram negative, motile and rod cells. Growth of all isolates in Nutrient Broth was observed between 25 C and 37 C, but not at or above 42 C. All isolates reacted positively to oxidase, catalase, lipase and nitrate reductase tests, and negatively to methyl red, Voges–Proskauer, starch, gelatin, urease, indole, hydrogen sulphide and litmus milk tests. All isolates grew well in Palleroni’s basal medium using as a sole source of carbon of the following compounds (0.1% final concentration): acetate, fumarate, pyruvate, malate, succinate and tryptophan. All isolates did not grow in Palleroni’s basal medium with glucose, fructose, sucrose, lactose, mannose and galactose. Morphological and biochemical characteristic results showed that the bacterial isolates were related to the members of Alcaligenes faecalis.
For further characterization, PCR amplification of the 16S rRNA gene of the most potent isolates (Cd1-1, Ag1-1, Ag1-3 and Sn1-1) produced fragments of approximately 1500 base pairs in size (Figure 1). The PCR products were then sequenced. The resulting nucleotide sequences were compared to available sequences in the databases. A phylogenetic tree illustrating the results of 16S rRNA analysis is presented in Figure 2. The results indicated greatest similarity to members of the Alcaligenes group, which matched the conclusions of the morphological and biochemical analysis. As illustrated, the 16S rRNA sequences of the five isolates were most closely related to Alcaligenes faecalis. The 16S rRNA genes of isolate Cd1-1 shared 98.8% similarity with that of Table 3. MICs and effect of different concentrations of cadmium on isolates from water selected on NB supplemented with 50 mg/ml SnC2 SnC2 concentrations (mg/ml) Isolates 100 200 400 800 1000 1200 1400 MIC(mg/ml)
100
200
400
800
1000
MIC(mg/ml)
C C
C C
C C
C ¡
¡ ¡
1000 800
C, growth; ¡, no growth.
Isolates
Molecular Characterization of the Most Potent Bacterial Isolates
Co-resistance to Other Heavy Metals
Isolates
AgC2 concentrations (mg/ml)
Sn1-1 Sn1-2 Sn1-3 Sn1-4
C C C C
C C C C
C C C C
C, growth; ¡, no growth.
C C C C
C C C C
C C C C
¡ ¡ ¡ ¡
1400 1400 1400 1400
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Table 4. Morphological and biochemical characteristics of bacterial isolates Bacterial isolates Characteristics
Cd1-1 Ag1-1 Ag1-3 Sn1-1
Morphological Gram stain ¡ve ¡ve ¡ve ¡ve Cell morphology rods rods rods rods Motility C C C C Biochemical Catalase C C C C Oxidase C C C C Gelatin ¡ ¡ ¡ ¡ Indole ¡ ¡ ¡ ¡ VP ¡ ¡ ¡ ¡ MR ¡ ¡ ¡ ¡ Lipase C C C C Nitrate reductase C C C C Starch ¡ ¡ ¡ ¡ Cellulase ¡ ¡ ¡ ¡ Urease ¡ ¡ ¡ ¡ Hydrogen sulfide ¡ ¡ ¡ ¡ Litmus milk ¡ ¡ ¡ ¡ Citrate C C C C Utilization (as a sole carbon source) of Glucose ¡ ¡ ¡ ¡ Fructose ¡ ¡ ¡ ¡ Sucrose ¡ ¡ ¡ ¡ Lactose ¡ ¡ ¡ ¡ Mannose ¡ ¡ ¡ ¡ Galactose ¡ ¡ ¡ ¡ Acetate C C C C Fumarate C C C C Pyruvate C C C C Malate C C C C Succinate C C C C Tryptophan C C C C Growth at ¡ ¡ ¡ ¡ 4 C C C C C 25 C C C C C 30 C C C C C 37 C ¡ ¡ ¡ ¡ 42 C -ve, Gram negative; C, growth; ¡, no growth.
Alcaligenes faecalis B_IV_2L49 (JF710960), respectively. Also, the isolates Ag1-1, Ag1-3 and Sn1-1 shared 99.5% similarities with that of Alcaligenes faecalis B_IV_2L49 (JF710960). These results indicated that these isolates were new isolates of the bacterium Alcaligenes faecalis.
Fig. 1. Agarose gel (1%) electrophoresis of PCR products of 16S rRNA genes of isolates: Cd1-1 (lane 1), Ag1-1 (lane 3), Ag1-3 (lane 4) and Sn1-1 (lane 9). Marker (M) is represented.
Isolation of Plasmid Plasmid minipreps of the isolates (Cd1-1, Ag1-1, Ag1-3 and Sn1-1) were achieved. The samples of plasmid preparations were run on 1% agarose gel at 80 V. The results indicated that no plasmids were detected in all isolates.
Growth Curve To study the effect of metals on bacterial growth of most potent isolate Alcaligenes faecalis Ag1-1, the metal experiments were carried out in nutrient broth that maintained a high free metal concentration in solution. Alcaligenes faecalis Ag1-1 exhibited different growth patterns in the presence of different heavy metals (Figure 3). Alcaligenes faecalis Ag1-1 exhibited a growth curve similar to the typical bacterial growth curve over the experiment time period. The experiments showed that Alcaligenes faecalis Ag1-1 increased their growth and reached to maximum growth at 28 h. A decrease in growth (measured in terms of optical density) was observed upon different heavy metals compared to the control without metal amendment. The lower optical density values revealed that the bacterial growth was affected due to the presence of metal in the growth medium. However, the reduction of the growth curve in the presence of heavy metals used in the study was evident throughout the experiment compared to the control without metal.
Electron Microscope Analysis Scanning electron microscope revealed that the morphology of the most potent isolate Alcaligenes faecalis Ag1-1 (Figure 4) was unchanged after growth in medium without and with addition of Ag. This suggests that Ag is not toxic to the isolate under the conditions tested. On other hand, the other isolates (Alcaligenes faecalis Ag1-3, Alcaligenes faecalis Cd1-1, Alcaligenes faecalis Sn1-1 and Alcaligenes faecalis Sn4-1) showed differences in cell dimensions after treated with heavy metals (Figures 5 and 6). Transmission electron microscope indicated that Alcaligenes faecalis Ag1-3, after treatment with 1200 mg/ml AgNO3, could synthesize nanostructure particles from AgNO3. TEM analysis proved that the spherical shape and average size of the intracellular particles was 27 nm (Figure 7).
Discussion Investigations focused on the isolation and characterization of the aerobic bacterial strains with metal tolerance to identify potential candidates for heavy metal bioremediation. In the present study we identified and characterized heavy metal-resistant bacteria isolated from sewage water. Fifty isolates were screened from initial level of heavy metal supplemented LB medium. Only nine bacterial isolates from sewage water samples showed resistance to heavy metals of concentration 800–1200 mg/ml that were tested. The characteristic
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Fig. 2. Phylogenetic tree of bacterial isolates (Cd1-1, Ag1-1, Ag1-3 and Sn1-1) based on the nucleotide sequences of the 16S rRNA genes. The tree was constructed by neighbor-joining method, and genetics distances were computed by the Jukes–Cantor model. The number presented next to each node shows the percentage bootstrap value of 1000 replicates. The GenBank accession numbers of the bacteria are presented in parentheses. The sequence from Pseudomonas lutea OK2 was treated as the out-group. The scale bar indicates the genetic distance.
results showed that the bacterial isolates were closed to the members of Alcaligenes faecalis. Previous results reported that 10 heavy metal-resistant bacteria were isolated from oxidation ditch of wastewater treatment plant of Bagmati Area Sewerage Project. These include chromium resistant Staphylococcus spp, Escherichia coli, Klebsiella spp; cadmium resistant Acinetobacter spp,
Fig. 3. Growth curve of Alcaligenes faecalis Ag1-1 in the presence of heavy metals.
Flavobacterium spp, Citrobacter spp; nickel-resistant Staphylococcus spp, Bacillus spp; copper-resistant Pseudomonas spp; and cobalt-resistant Methylobacterium spp. (Rajbanshi 2008). All these isolates showed high resistance to heavy metals with Minimum Inhibitor Concentrations (MICs) for heavy metals ranging from 150 mg/ml to 500 mg/ml (Rajbanshi 2008). However, our results reported that The Minimum Inhibitory Concentrations (MICs) of isolates against the heavy metals ranged from 100 to 2500 mg/ml. Also, Filali et al. (2000) reported a wastewater bacterium isolates Psuedomonas aeroginosa, Klebsiella pneumoniae, Proteus mirabilis and Staphylococcus resistant to heavy metals. Bacteria subjected to high levels of heavy metals in their environment have adapted to this stress by developing various resistance mechanisms. These mechanisms could be utilized for detoxification and removal of heavy metals from polluted environment (Ahmed et al. 2005). The ability of bacteria to grow in the presence of heavy metals might be helpful in the waste water treatment where microorganisms are directly involved in the decomposition of organic matter in biological processes for wastewater treatment (Filali et al. 2000). In our study nine resistant isolates showed multiple tolerances to heavy metals. Multiple tolerances happen only to toxic compounds that have similar mechanisms causing their toxicity. Subsequently heavy metals are all similar in their toxic mechanisms, multiple tolerances are common phenomena among heavy metal-resistant bacteria.
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Fig. 4. Scanning electron micrographs of cells of Alcaligenes faecalis Ag1-1 (A and B) and Alcaligenes faecalis Ag1-3 (C and D) before (A and C) and after (B and D) Ag –treated.
Pollution by chemicals including heavy metals is a problem that may have a negative impact on the environment. The most abundant pollutants in the wastewater and in sewage are heavy metals. Human activities such as mining operations and the discharge of industrial wastes have resulted in the accumulation of metals in the environment and eventually
are accumulated through the food chain, leading to serious ecological and health problems. Therefore, the heavy metalresistant bacteria isolated in this study could be a potential agent for bioremediation of heavy metals pollution. Recent study reported that Providencia rettgeri MAM-4 isolated from wastewater had ability to remove Al3C, Co2C,
Fig. 5. Scanning electron micrographs of cells of Alcaligenes faecalis Cd1-1 before (A) and after (B) Cd–treated.
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Fig. 6. Scanning electron micrographs of cells of Alcaligenes faecalis Sn1-1 before (A) and after (B) Sn–treated.
and Cu2C efficiently from aqueous media. This study has revealed that P. rettgeri could be employed as an effective and economic technology for the removal such metal elements from polluted environment (Abo-Amer et al. 2013). Our most recent study reported that a total of 120 Azotobacter sp. isolated from soils irrigated with wastewater and groundwater showed resistance to Zn2C, Cd2C, Cu2C, Pb2C and Mn2C (Abo-Amer et al. 2014). There might three general mechanisms for resistances to heavy metal ions which are the specificities of plasmid-
determined metal resistances and two general resistant mechanisms, which are efflux pumping and enzymatic detoxification (generally reduction- oxidation reactions to convert more toxic to less toxic metal-ion species). However, the present results showed no plasmids in the most potent isolates indicative the heavy metal resistant genes located on the chromosome. The genes encoding resistance to heavy metals in bacteria are present either on the bacterial chromosome, on the plasmids, or on both (Nies and Brown 1997; Silver and Phung 1996).
Fig. 7. Transmission electron micrograph of Alcaligenes faecalis Ag1-1 showing cellular accumulation of Ag nanoparticles.
844 Examination of cells of the isolate by SEM indicated distinct changes in cell sizes and surfaces in metal-treated cells. Investigating the cells of isolate after treatment with 1200 mg/ml AgNO3 using TME indicated that the isolate Alcaligenes faecalis Ag1-3 was able to synthesize nanostructure particles from Ag. It was clear that these nanoparticles were synthesized intracellularly within the periplasmic space of Alcaligenes faecalis Ag1-3 cells. The present study estimates that the identified bacteria were used to remediate heavy metal contaminated wastewater and sewage. Therefore, the ability of bacterial isolates to grow in the presence of heavy metals would be helpful in the wastewater treatment where microorganisms are directly involved in the decomposition of organic matter in biological processes for wastewater treatment.
Conclusions Nine bacterial strains were isolated from sewage water by using an enrichment isolation technique based on a high level of heavy metal resistance of Pb2C, Cd2C, Al3C, Cu2C, Ag2C and Sn2C. The bacterial isolates showed high resistance to heavy metals with MICs ranging from 100 mg/ml to 2500 mg/ml. All resistant isolates showed multiple tolerances to heavy metals. The most potent isolates were characterized and identified as Alcaligenes faecalis. The cells of Alcaligenes faecalis Ag1-1 could synthesize intercellular nanoparticles of 27 nm. Therefore, the ability of bacterial isolates to grow in the presence of heavy metals would be helpful in wastewater treatment.
Funding This work was supported by the research grant from Taif University, Contract No. 1/434/2466.
References Abo-Amer AE. 2007. Involvement of chromosomally encoded genes in malathion utilization by Pseudomonas aeruginosa AA112. Acta Microbiol Immunol Hungarica 54:261–277. Abo-Amer AE. 2011. Biodegradation of diazinon by Serratia marcescens DI101 and its use in bioremediation of contaminated environment. J Microbiol Biotechnol 21(1):71–80. Abo-Amer AE. 2012. Characterization of a strain of Pseudomonas putida isolated from agricultural soil that degrades cadusafos (an organophosphorus pesticide). World J Microbiol Biotechnol 28:805–814. Abo-Amer AE, Abu-Gharbia MA, Soltan EM and Abd El- Raheem WM. 2014. Isolation and molecular characterization of heavy metal-resistant Azotobacter chroococcum from agricultural soil and their potential application in bioremediation. Geomicrobiol J 31: 551–561. Abo-Amer AE, Ramadan AB, Abo-State M, Abu-Gharbia MA and Ahmed HE. 2013. Biosorption of aluminum, cobalt, and copper ions by Providencia rettgeri isolated from wastewater. J Basic Microbiol 53: 477–488.
Abo-Amer et al. Ahemad M, Malik A. 2012. Bioaccumulation of heavy metals by zinc resistant bacteria isolated from agricultural soils irrigated with wastewater. Bacteriol J 2:12–21. Ahmed N, Nawaz A, Badar U. 2005. Screening of copper tolerant bacterial species and their potential to remove copper from the environment. Bull Environ Contam Toxicol 74: 219–226. Bhattacharya D, Rajinder G. 2005. Nanotechnology and potential of microorganisms. Crit Rev Biotechnol 25:199–204. Catauro M, Raucci MG, De Gaaetano FD, Marotta A. 2004. Antibacterial and bioactive silver-containing Na2O, CaO and 2SiO2 glass prepared by sol-gel method. J Mater Sci Mater Med 15(7): 831-–837. Chen CY, Lin TH. 1998. Nickel toxicity to human term placenta: in vitro study on lipid peroxidation. J Toxicol Environ Health A 54:37–47. Chen WP, Kuo TT. 1993. A simple and rapid method for the preparation of gram negative bacterial genomic DNA. Nucl Acids Res 21:2260. Cheng S. 2003. Heavy metal pollution in China: Origin, pattern and control. Environ Sci Pollut Res 10:192–198. Claus D, Berkeley RCW. 1986. Genus Pseudomonas. In: Sneath PHA, Mair NS, Sharpe ME, editors. Bergey’s Manual of Systematic Bacteriology, Vol 1. Baltimore, MD: Williams and Wilkins, p140–219. Costa M, Klein CB. 1999. Nickel carcinogenesis, mutation, epigenetics, or selection. Environ Health Perspect 107438–439. El-Shanshoury AR, El-Silk SE, Ebeid ME. 2011. Extracellular biosynthesis of silver nanoparticles using Escherichia coli ATCC 8739, Bacillus subtilus ATCC 6633 and Streptococcus thermophilus ESh1 and their antimicrobial activities. ISRN Nanotechnol 24:1-–7. El-Shanshoury AR, El-Silk SE, Ebeid ME. 2012. Rapid biosynthesis of cadmium sulfide (CdS) nanoparticles using culture supernatants of Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633 and Lactobacillus acidophilus DSMZ 20079T. Afri J Biotechnol 11 (31):7957–7965. Filali BK, Taoufik J, Zeroual Y, Dzairi FZ, Talbi M, Blaghen M. 2000. Waste water bacteria resistant to heavy metals and antibiotics. Curr Microbiol 41:151–156. Francois F, Lombard C, Guigner J, Soreau P, Brian-Jaisson F, Martino G, Vandervennet M, Garcia D, Molinier AL, Pignol D, Peduzzi J, Zirah S, Rebuffat S. 2012. Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury. Appl Environ Microbiol 78:1097–1106. Gupta UC, Gupta SC. 1998. Trace element toxicity relationships to crop production and livestock and human health: implications for management. Comm Soil Sci Plant Anal 29:1491–1522. Hussain I, Brust M, Papworth AJ, Cooper AI. 2003. Preparation of acrylate-stabilized gold and silver hydrosols and gold-polymer composite films. Langmuir 19(11):4831–4835. Kiyono M, Hou HP. 2006. Genetic engineering bacteria for environmental remediation of mercury. J Health Sci 52:199–204. Kuber CB, D’Souza SF. 2006. Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Coll Surf B 47 (2):160–164. Low KS, Lee CK, Liew SC. 2000. Sorption of cadmium and lead from aqueous solution by spent grain. Proc Biochem 36:59–64. Mohamed RM, Abo-Amer AE. 2012. Isolation and characterization of heavy-metal resistant microbes from roadside soil and phylloplane. J Basic Microbiol 52(1):53–65. Narayanan KB, Sakthivel N. 2010. Biological synthesis of metal 270 nanoparticles by microbes. Adv Coll Interf Sci 156:1–13. Nies DH, Brown NL. 1997. Two-component systems in regulation of heavy metal resistance. In: Silver S, Walden W, editors. Metal Ions in Gene Regulation. New York: Chapman and Hall, p77–103. Rajbanshi A. 2008. Study on heavy metal resistant bacteria in Guheswori sewage treatment plant. Our Nature 6:52–57. Rehman A, Zahoor A, Muneer B, Hasnain S. 2008. Chromium tolerance and reduction potential of a Bacillus sp. ev3 isolated from
Heavy Metal-Resistant Alcaligenes faecalis metal contaminated wastewater. Bull Environ Contam Toxicol 81:25–29. Sharma PK, Balkwill DL, Frenkel A, Vairavamurthy MA. 2000. A new Klebsiella planticola strain (Cd-1) grows anaerobically at high cadmium concentrations and precipitates cadmium sulfide. Appl Environ Microb 66(7):3083–3030. Silver S, Phung LT. 1996. Bacterial heavy metal resistance: new surprises. Annu Rev Microbiol 50:753–789. Singh N, Gadi R. 2012. Studies on biosorption of Pb (ii) by the nonliving biomasses of Pseudomonas oleovorans and Brevundimonas vesicularis and its removal from wastewater samples. Eur J Sci Res 69:279–289.
845 Tang XY, Zhu YG, Cui YS, Duan J, Tang L. 2006. The effect of ageing on the bioaccessibility and fractionation of cadmium in some typical soils of China. Environ Int 32:682–689. Volesky B. 1990. Biosorption of Heavy Metals. Boca Raton, FL: CRC Press, p408. Waisberg M, Joseph P, Hale B, Beyersmann D. 2003. Molecular mechanism of cadmium carcinogenesis. Toxicol 192:95–117. Zhang HR, Li QB, Lu YH, Sun DH, Ling XP, He N, Zheng SZ. 2005. Biosorption and bioreduction of diamine silver complex by Corynebacterium. J Chem Technol Biotechnol 80(3): 285–290.
