Characterization of heavy metal ( cadmium and nickle ) tolerant Gram negative enteric bacteria from polluted Yamuna River , Delhi

The presence of heavy metals in aquatic environments poses a serious environmental risk. The usage of heavy metal-tolerant bacteria may serve as a cost-effective tool for bioremediation of polluted water bodies. Thus, the present study aims at the isolation and characterization of heavy metal tolerant gramnegative bacteria collected from the polluted water of the river Yamuna, Delhi, India. The water samples were collected from the downstream river and enriched separately in the nutrient broth. Appropriate dilutions were then plated in eosin methylene blue (EMB) agar supplemented with cadmium (10 to 4000 μg/ml) and nickel (10 to 3000 μg/ml). The colonies having different morphologies that were formed in the EMB agar were selected, and their metal tolerance concentration (MTC) was determined. Based on their MTC values, two isolates were identified by using morphological, biochemical, and molecular (16S rRNA gene sequencing) methods. Besides, their growth kinetics, co-metal tolerance, and antibiotic resistance were also determined. The identified isolates 2 and 8 were tolerant to cadmium (3000 μg/ml) and nickle (2000 μg/ml), respectively. The isolates were found to be closely related to Pantoea agglomerans JCM 1236 (sample 2) and Enterobacter asburiae JCM 6051 (sample 8). They were not only co-tolerant to cadmium (3000 μg/ml) and nickle (2000 μg/ml) but also showed resistance to various antibiotics. Scanning electron microscopy (SEM) showed changes in surface morphology of nickle and cadmium-treated samples 2 and 8, and the energy dispersive X-ray spectroscopy (EDX) studies revealed the presence of cadmium and nickel in both the isolates after metal treatment. The results conclude that the identified heavy metal tolerant bacteria could be useful for the bioremediation of contaminated wastewater and industrial effluents.


INTRODUCTION
The Yamuna river is one of the most polluted rivers in India due to the presence of high concentrations of toxic heavy metals (Ahmad, 2009;Misra, 2010;Sehgal et al., 2012;Kaur and Mehra, 2012).Anthropogenic and industrial activities add non-biodegradable environment pollutants like heavy metals, such as lead (Pb), chromium *Corresponding author.E-mail: nbhagat@amity.edu.Tel: +91 99871789288.Fax: +91 120 4392947.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (Cr), cadmium (Cadmium), mercury (Hg), iron (Fe), cobalt (Co), and nickel (Ni), and their derivatives into the water bodies (Islam et al., 2007).In developing countries, rapid and unorganized industrialization and urbanization have also contributed to the elevated levels of heavy metals in the water bodies (Sharma et al., 2008).In India, most of the industries are situated along the river banks for the easy disposal of the effluents that contain heavy metals (Kaushik et al., 2009;Malik et al., 2014).The industrial waste dump grounds present in the vicinity of rivers also add to the heavy load of toxic metal ions, organic ions, organic wastes, and antibiotics in the rivers (Mohiuddin et al., 2011;Haq et al., 1999).
Cadmium and nickel are among the most widespread contaminants in the aquatic environment (Kaushik et al., 2001) and they can cause serious health problems to all organism.Human consumption of such contaminated water is extremely dangerous as these heavy metals have carcinogenic and mutagenic effects.Cadmium can affect the kidney, causing renal dysfunction, especially in the proximal tubular cells as it is the main site of cadmium accumulation.It can also cause bone demineralization, either directly by damaging the bones or indirectly as a result of renal dysfunction (Blessy and Krishnamurthy, 2015;Bernard, 2008).Moreover, Ni is also known for its hematotoxic, immunotoxic, neurotoxic, genotoxic, reproductive toxic, pulmonary toxic, nephrotoxic, hepatotoxic, and carcinogenic effects (Duda-Chodak and Blaszczyk, 2008).
Stainless steel manufacturing units, electroplating factories, etc. discharge Ni into the river.Whereas, industrial processes such as electroplating of iron metals, preparation of Cadmium-Ni batteries, control rods and shields within nuclear reactors, and preparation of television phosphors discharge cadmium into the river (Singh, 2014).Therefore, it is important to remove the heavy metals from industrial effluents before they are discharged into the rivers.Chemical methods, including reverse osmosis, electrodialysis, ultrafiltration, ionexchange, phytoremediation, etc., used for heavy metal removal from the wastewater/industrial effluents are expensive and inefficient (Ahalya et al., 2003).Many authors have screened and studied different microorganisms, such as bacteria, fungi, and algae, over the past decades to identify their highly efficient metal removal biological systems (Vijayaraghavan and Yun, 2008).These microorganisms can grow in the presence of high concentrations of heavy metals and have a great potential for bioremediation of heavy metals discharged in industrial effluents (Ansari and Malik, 2007).
Previous studies have well documented the occurrence and abundance of metal-tolerant microbes in metalpolluted water bodies (Abyar et al., 2012;Jankowska et al. 2006;Wong et al. 2003).Bacterial species such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis and Staphylococcus sp.isolated from wastewater samples were found to be resistant to heavy metals and antibiotics (Filali et al., 2000).Similarly, cadmium-resistant Klebsiella and Enterobacter species and nickel resistant Actinomycetes and other bacteria have also been isolated from industrial effluents and soil samples (Alboghobeish et al., 2014;Karakagh et al., 2012).These microorganisms develop a variety of resistance mechanisms to survive in different heavy metal concentrations, but the resistance is often specific to one or few metals (Mejare and Bulow, 2001;Nies, 2003;Piddock, 2006).These heavy metal tolerant bacterial species may serve as an important and costeffective bioremediation tool for the removal of heavy metals from wastewater and industrial effluents, preventing the contamination of water bodies (Jan et al., 2014).Thus, the present study was envisaged to isolate and characterize heavy metal tolerant gram-negative bacteria from the water samples collected from the Yamuna river, Delhi, India.

Sample collection
Water samples were collected from the Yamuna river from three different sites: a) Sarita vihar barrage, b) Income tax office (ITO) barrage, and c) Okhala barrage.The samples were collected in sterile plastic bottles following standard sampling techniques and sent to the laboratory for further processing.Sampling bottles were treated with 1% nitric acid followed by rinsing with deionized water three times.After washing, bottles were sterilized by autoclaving and dried.At first, the collected samples were added to the nutrient broth (250 ml) and incubated overnight at 37°C.Serial dilution and spread plating method was used to isolate the colonies.Appropriate dilutions were plated in eosin methylene blue (EMB) agar (Hi Media, India) supplemented with cadmium (10 to 4000 µg/ml) and nickle (10 to 4000 µg/ml).The colonies with different morphologies formed on EMB agar plates were further studied.For long-term preservation and maintenance, the microbial cultures were stored as 15% glycerol stocks in airtight vials at −80°C.

Determination of maximum tolerance concentration (MTC) of heavy metals
After preliminary selection of cadmium and nickle tolerant isolates the MTC of Cadmium and Ni was determined.For the quantitative determination of heavy metal tolerance, the isolated strains were inoculated in EMB broth supplemented with increasing concentrations of cadmium and nickle (10 to 5000 µg/ml).The broth tubes were incubated at 37°C for 48 h in an incubator shaker (150 rpm).The highest concentration of metal ions at which bacterial growth was observed (as indicated by optical density, OD, at 600 nm) and was defined as the maximum tolerance concentration (MTC) of the respective heavy metal for the isolated strain (Schmidt and Schlegel, 1994).EMB broth without the metal ions was also inoculated with the isolated strains to serve as controls.All experiments were performed in triplicates.

Identification of the bacterial isolates
Two isolates, samples 2 and 8 were selected on the basis of their MTC values for further identification and characterization.The isolates were characterized morphologically (Gram staining).Biochemical characteristic like indole test, MR-VP, urease, catalase, citrate were also taken into consideration for characterization of these isolates (Cappucino and Sherman, 2002).

16S rRNA gene amplification
After biochemical characterization, isolates were identified on the molecular basis using 16S rDNA sequencing for the accurate identification of bacterial isolates.First, the genomic DNA was extracted from the bacterial culture by Triton prep Method.The partial 16S rDNA was amplified by using the universal primers, 27 F (5'-AGA GTT TGA TCC TGG CTC AG -3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3').Polymerase chain reaction (PCR) was performed using thermal cycler (Applied Biosystem, USA ) with 50 µl reaction mixture containing 1 µl (10 ng) of DNA extract as a template, each primer with a concentration of 10 picomole/µl, 25 mM MgCl2, and 2 mM dNTPs along with 1.5U of Taq polymerase and buffer as per the manufacturer's recommendations (GCC Biotech, Kolkata, India).The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 1 min, extension at 72°C for 1 min and 30 s, and final extension at 72°C for 10 min.The PCR products were analyzed by 1.5% w/v agarose gel electrophoresis (1× TAE) buffer with ethidium bromide (0.5 µg/ml).

Molecular characterization
The PCR product was sequenced by capillary sequencing method using ABi 3500 Genetic Analyzer machine as per the manufacturer's instructions.The 16S rRNA gene sequences were compared with the known bacterial 16S rRNA gene sequences using NCBI ADVANCED BLAST so as to identify the most similar sequence alignment (Altschul et al., 1997).Based on the scoring index, the most similar sequences were aligned with the sequences of other representative bacterial 16S rDNA regions by using multiple alignment software program, ClustalW (Thompson et al., 1994).A phylogenetic tree and a similarity index were generated and compared with the known sequences.The phylogenetic tree was constructed using the UPGMA method, and the tree reliability was tested by Bootstrap method using MEGA version 4 (Tamura et al., 2007).

Determination of co-metal tolerance
The bacterial isolate tolerant to one heavy metal was tested for its tolerance to other heavy metals included in the study.The cadmium-tolerant bacterial isolate (sample 2) was inoculated in EMB broth supplemented with nickle (10 to 4000 µg/ml).The tubes were incubated at 37°C for 48 h.Similarly, the nickle-tolerant strain (sample 8) was grown on Cadmium supplemented broth (10 to 4000 µg/ml).The cometal tolerance was confirmed by agar plate grid method.Series of EMB agar plates supplemented with various concentration of cadmium (10 to 4000 µg/ml) and nickle (10 to 4000 µg/ml) were made.A grid of 5 × 5 was made on each plate and each box was numbered.Selected isolates were streaked in same numbered box on each plate.Plates were incubated at 37°C for 48 h.The isolates that grew on both cadmium and nickle supplemented plated were selected and the maximum metal concentration of each metal on which they were also noted.

Effect of heavy metals on bacterial growth
The bacterial isolates were grown in 20 ml nutrient broth (HiMedia) Bhagat et al. 129 and incubated overnight at 37°C in an incubator shaker (150 rpm) for preparing the inoculum.One ml of this culture was further inoculated into 50 ml nutrient broth containing 0 (control) to 3000 µg/ml of cadmium and 0 (control) to 2000 µg/ml of nickle.The flasks were then incubated at 37°C in an incubator shaker.The bacterial growth was monitored at specific time intervals by recording absorbance (OD) at 600 nm.All the experiments were performed in triplicates, and the results are represented as average values.Analysis of the variance was performed using the statistical software GraphPad Prism (version 4.00 for Windows, GraphPad Software, Inc., San Diego, CA) to compare the growth curves at different metal concentrations.

Effect of metal (Ni and Cadmium) on Morphology of bacteria:
In order to determine the effect of metals on the morphology, the metal-treated and untreated bacterial cells were analyzed by scanning electron microscope (SEM).The samples 2 and 8 were grown in nutrient broth containing cadmium (3000 µg/ml) and nickle (2000 µg/ml) in separate flasks for 24 h at 37°C.The bacterial cultures grown without the metal in a separate flask were taken as normal control.Post growth, cells from treated and control were harvested at a density of 10 8 to 10 9 cells ml.An aliquot of each culture was centrifuged at 5000 rpm for 5 min.The supernatant was discarded, and the pellet was washed with phosphate buffer saline.The samples were fixed with 2.5% glutaraldehyde for 1 h at 37°C and pelleted again and then washed thrice with de-ionized water.
The samples were then dehydrated by suspending in a series of acetone and alcohol (30 to 100%) solution for 5 min each and were kept in 100% alcohol each for 1 h.Then, each sample was centrifuged and dried and spread on a small glass slide followed by its coating with a very thin layer of gold alloy.The morphology of prepared specimen was analyzed by using SEM, and the metal concentration was determined by energy dispersive spectrometer (EDS) (Zeiss, operating voltage 15 kV).

Screening, isolation, and MTC of bacteria
The main goal of this study was to isolate and characterize cadmium and nickel tolerant gram negative bacteria from different sites of polluted Yamuna river.For this purpose, 50 isolates were selected on the basis of color, size and morphology from EMB plates.Further screening was done on the basis of maximum metal  1).

Molecular characterization
The partially amplified and sequenced 16S mRNA gene was analyzed for sequence homology by using BLAST.
The partial nucleotide sequence of sample 2 revealed 99% similarity with Pantoea agglomerans JCM1 and of sample 8 with Enterobacter asburiae JCM 6051.Phylogenetic analysis characterized them within the gram-negative bacteria.The phylogenetic trees derived from 16S rRNA sequence data of the two samples with other related species are represented in Figures 1 and 2, respectively.The partial sequences of samples 2 and 8 were submitted to GenBank NCBI database under the accession numbers KP410394 and KP233878, respectively (Table 2).

Co-metal tolerance
The cadmium-tolerant bacterial isolate sample 2 demonstrated bacterial growths in nutrient broth supplemented with Ni, with an MTC of 2000 µg/ml.Similarly, the nickle-tolerant sample 8 showed MTC for cadmium at 3000 µg/ml.Co metal tolerance of the two metals was verified by grid method on EMB plate supplemented with various concentration of cadmium and nickel (10 to 5000 µg/ml).

Growth studies of isolated bacteria
The growth curve of samples 2 and 8 in the presence of increasing concentrations of cadmium and nickle (0 to 3000 µg/ml) are depicted in Figures 3 and 4, respectively.The growth curves of both samples exhibited similar phases in the presence of heavy metals.However, the growth rate was retarded in the presence of heavy metal ions relative to control (ANOVA, p < 0.05).

Antibiotic resistance
The resistance of cadmium and nickle tolerant bacteria towards popular antibiotics was estimated to investigate the relationship between metal resistant and antibiotic resistance.Antibiotic susceptibility of these for carbapenem group of antibiotics was also screened to ascertain the emergence of resistant varieties to these antibiotics.Both the isolates showed resistance to antibiotics, viz.vancomycin, augmentin, cefpodoxime, and rifampicin.Moreover, sample 8 also demonstrated resistance to aztreonam.The results of antibiotic susceptibility testing of the two isolates are presented in Table 3.

Effect of metals (Cadmium and Nickle) on morphology of bacteria
The effect of metals on cell morphology was demonstrated through the SEM analysis.Distinct changes were observed in the bacterial cell shape and surface features.There were some sticky appearances evident from the overlapping of bacterial cells with each other.Some cells even showed rough surface structure and blister-like protrusions.SEM photomicrographs of samples 2 and 8 cultured with and without heavy metals (at respective      MTC values) are presented in Figure 5(a to f).It was revealed that both the bacterial isolates were loosely bound rod shaped with an average length of 1.878 ± 0.02 and 2.779 ± 0.18 µm, respectively under standard conditions.After growing in nickle (2000 µg/ml)supplemented media, the sample 2 cells showed rods of 1.7 ± 0.288 µm length with a rough surface, and sample 8 showed rods of 2 ± 0.33 µm dimension.The nickletreated cells also appeared in aggregates due to some extra polysaccharide secretions (EPS).Cadmiumexposed (3000 µg/ml) cells of sample 2 showed morphological changes, that is, small rods of dimension 1.517 ± 0.227 µm.Cadmium-treated (3000 µg/ml) cells of sample 8 also became smaller, that is, the rods were of 1.82 ± 0.35 µg/ml dimension, showing secretion of EPS around them.EDS analysis detected 4 ± 0.05% cadmium and 9.5 ± 1.01% Ni in metal treated sample 2. While cadmium and nickle-treated sample 8 showed a presence of 5.08 ± 0.88% cadmium and 7.18 ± 0.16% Ni, respectively (Table 4).

DISCUSSION
Heavy metal contamination of aquatic environments is a major global concern as huge amounts of heavy metals are discharged into water bodies as effluents of mining, metallurgy and electroplating industries (Mahato et al., 2014;Singh, 2014;Tiwari et al., 2015).Sehgal et al. (2012) studied heavy metal contamination in water and soil from 13 different sites of the Delhi segment of Yamuna basin.Average heavy metal concentration at different locations in the river water varied in the order of Fe > Cr > Mn > Zn > Pb > Cu > Ni > HgAs > Cd.So, heavy metal removal from industrial effluents is a crucial requirement in wastewater treatment plants.Several chemical methods used for this purpose are expensive and not environmental friendly.In contrast, bioremediation of heavy metals by microorganisms is cost-effective and compatible with the environment (Jan et al., 2014).Bacterial populations isolated from heavy metal contaminated sites may have the ability to tolerate a higher concentration of metals.Hence, in the present study, cadmium and nickle-tolerant bacteria were isolated from the polluted Yamuna river which has been contaminated with heavy metal.The enrichment of stagnant water samples collected from heavily polluted sites led to the isolation of bacteria that have significant potential to tolerate high cadmium and nickel concentrations.Isolate 2 was able to grow at 2000 µg/ml of cadmium ion concentration and isolate 8 at 3000 µg/ml in the liquid medium.The values are significantly variable than the tolerance values reported in some of the previous studies.Isolates Klebsiella pneumoniae, Pseudomonas aeruginosa, and Bacillus cereus were found to tolerate and exclude cadmium ion (Cd 2+ ) from the cell surface (Kafilzadeh et al., 2013).Earlier studies have reported cadmium-resistant bacteria community isolated from sewage sludge contaminated by cadmium ions (50 µg/ml) and the predominance of gram-negative bacteria with 5.08 ± 0.88% cadmium (Chovano et al., 2004).Similarly, isolate 8 demonstrated tolerance for nickel (2000 µg/ml).Ni-tolerant Acinetobacter isolates with MTC of 6.5 mM had been screened from Torsa  River, West Bengal, India (Bhadra et al., 2006).Other studies have reported the gram-negative Ni-tolerant isolates, exhibiting MTC range of 1 to 24 mM for nickle, isolated from the industrial effluents wastewater treatment plant of Paonta Sahib, Himachal Pradesh, India (Virender et al., 2010).Multimetal tolerant Alcaligenes xylosoxidans, with the tolerating range of 2.0 to 4.0 mM for NiCl 2 and 1 mM cadmium was characterized by Sevgi et al. (2010).
Pseudomonas fragi, Staphylococcus spp.and Bacillus spp. with MTC ranging from 150 to 500 μg/ml was isolated from industrial effluents (Patel et al., 2006;Rajbanshi, 2008).Thus, many such bacteria with a varying range of MTCs have been reported in various studies from different parts of the world.Our isolates (sample 2 and 8) showed tolerance to much higher concentration of cadmium and nickle (3000 and 2000 μg/ml).
The preliminary identification results suggested that isolates 2 and 8 are gram-negative rods representing the family Enterobacteriaceae.A combination of biochemical tests and 16S rRNA gene sequencing revealed that the two isolates share 99% similarity with Pantoea agglomerans JCM 1236 and Enterobacter asburiae JCM 6051, respectively.Both isolates exhibited co-tolerance to cadmium and nickel.The microbiota isolated from cocontaminated environments could exhibit tolerance to more than one metal ion and, thus, co-tolerance may be a common natural response (Gadd and Sayer, 2000;Malik et al., 2002).Since the heavy metals are similar in their toxic mechanisms; multiple tolerances were the common phenomena among the heavy metal tolerant bacteria (Mikolay and Nies, 2009;Ansari and Malik, 2007).The growth rate of the bacterial isolates in the presence of the heavy metals (cadmium and nickle) was consistently slower than that of the control (p < 0.05).The growth pattern suggests tolerance development or adaptation of bacteria to the presence of heavy metals in the aquatic environment.
All the isolates were tested for their resistance to common antibiotics, that is, rifampicin, augmentin, vancomycin, and cefpodoxime.A correlation exists between the two beneficial traits, that is, metal tolerance and antibiotic resistance, in bacteria.It may be because the resistance genes to both antibiotics and heavy metals may be located closely together on the same plasmid in bacteria.Thus, they are more likely to be transferred together in the event of conjugation (Filali et al., 2000;Malik and Aleem, 2011;Nies, 1992).This phenomenon would be favorable for bacteria to survive in heavy metal contaminated water bodies.The microbial tolerance to heavy metals is attributed to various detoxifying mechanisms such as complexation by exopolysaccharides, binding of metal in bacterial cell envelopes, metal reduction, metal efflux or using them as terminal electron acceptors in anaerobic respiration (Haferburg and Kothe, 2010).The SEM analysis (Figure 5) showed distinct changes in the cell size and surface features.The cellular changes and presence of exopolysaccharides around the cell can be interpreted as a possible strategy of the cell to accumulate more metals.
Several studies have reported the potential use of metal-resistant microorganisms in the treatment of heavy metal contaminated wastewater bodies (Karakagh et al., 2012;Shakibaie et al., 2010).Researchers have characterized Pseudomonas aeruginosa KUCADMIUM1 showing biological removal of cadmium at the level of 75 to 89% of total content (Sinha and Mukherjee, 2009) and Klebsiella pneumoniae CBL-1 at a concentration of 1500 mg/ml (Rehman and Shamim, 2012).Ni-tolerant Micrococcus sp. was also studied for its applicability in bioremediation of industrial waste water (Congeevarama et al., 2007).A bacterial consortium of metal-resistant bacteria including Enterobacteriaceae members, isolated from the river Yamuna, had been studied as a potential source for generation of electricity (Malik et al., 2014).Similarly, the bacteria isolated in the present study can be further explored for their ability to remove heavy metals.

Conclusion
The increased levels of heavy metals in the river water lead to accumulation of heavy metals in the adjoining soil and plants grown on these contaminated soils, leading to great harm to humans and animals.Such contaminated environment poses pressure on microbes to develop various mechanism to survive in high metal concentration.So these microorganism can be promising tool for removal of these metals from the contaminated river bodies.The present study reports the identification of two bacterial strains with 99% similarity to Pantoea agglomerans JCM1 and Enterobacter asburiae JCM 6051 with a tolerance to high concentrations of cadmium (3000 µg/ml) as well as nickel (2000 µg/ml).These isolates can be termed as heavy metal tolerant bacteria that may have the potential to remove heavy metals from contaminated effluents before discharging them in river waters.

Figure 1 .
Figure 1.Phylogenetic relationship of sample 2 and closely related sequences based on partial 16S rRNA gene sequence.The phylogeny for the 16S bacterial identification data was constructed using the UPGMA method, and the tree reliability was tested by Bootstrap method for the Test of Phylogeny option in MEGA 4 software.

Figure 2 .
Figure 2. Phylogenetic relationship of sample 8 and closely related sequences based on the partial 16S rRNA gene sequence.The phylogeny for the 16S bacterial identification data was constructed using the UPGMA method, and the tree reliability was tested by Bootstrap method for the Test of Phylogeny option in MEGA 4 software.

Figure 3 .
Figure 3. Growth pattern of the isolate sample 2 in the presence of a) Cadmium (1000 to 3000 µg/ml) and b) Ni (500 to 2000 µg/ml); control: Isolate grown in media without any metal supplementation.Anova was performed using GraphPad Prism (version 4).

Figure 4 .
Figure 4. Growth pattern of the isolate sample 8 in the presence of a) Cadmium (1000 to 3000 µg/ml) and b) Ni (500 to 2000 µg/ml); control: Isolate grown in media without any metal supplementation.Anova was performed using GraphPad Prism (version 4).

Table 1 .
Morphological and biochemical characterization of the two selected bacterial isolates.

Table 2 .
Molecular characterization of the bacterial isolates.

Table 3 .
Antibiotic susceptibility of the two bacterial isolates.

Table 4 .
Metal concentration as detected by EDX analysis.