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C. EDWARD RAJA *, G. S. SELVAM * AND KIYOSHI OMINE #. ABSTRACT. The pollution of the environment with toxic heavy metals is spreading throughout the ...
ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF HEAVY METAL RESISTANT BACTERIA FROM SEWAGE C. EDWARD RAJA *, G. S. SELVAM * AND KIYOSHI OMINE #

ABSTRACT The pollution of the environment with toxic heavy metals is spreading throughout the world along with industrial progress. Microorganisms and microbial products can be highly efficient bioaccumulators of soluble and particulate forms of metals especially dilute external solutions. Microbe related technologies may provide an alternative or addition to conventional method of metal removal or metal recovery. The present study deals with isolation, identification and characterization of heavy metal resistant bacteria was isolated from sewage water collected in and around Madurai district, South India. Initially, a total of 300 isolates were screened from sewage water. The four isolates were selected based on high level of heavy metal and antibiotic resistances. On the basis of morphological, biochemical, 16S rDNA gene sequencing and phylogeny analysis revealed that, the isolates were authentically identified as Proteus vulgaris (BC1), Pseudomonas aeruginosa (BC2), Acinetobacter radioresistens (BC3) and Pseudomonas aeruginosa (BC5). The sewage isolates showed optimum growth at 30 °C and pH 7.0. The identified isolates were resistant to cadmium (Cd), nickel (Ni), lead (Pb), arsenic (As), chromium (Cr) and mercury (Hg). The minimal inhibitory concentration (MIC) of sewage isolates against Cd, Cr, Ni, Pb, As and Hg was determined in solid media. All the sewage isolates resistant to Cd (4-7mM), Cr (0.7mM), Ni (6.75-8.5mM), Pb (6mM), As (6.5-15 mM) and Hg (0.75 mM). The multiple metal resistances of these isolates were also associated with resistance to antibiotics ampicillin, tetracycline, chloramphenicol, kanamycin, erythromycin, streptomycin and nalidixic acid. The identified heavy metal resistant bacteria could be useful for the bioremediation of heavy metal contaminated sewage and waste water. Keywords: Heavy metal resistant bacteria, Antibiotic resistance, 16S rDNA, Sewage water

INTRODUCTION Heavy metal pollution of soil and wastewater is a significant environmental problem (Cheng, 2003). Wastewaters from the industries and sewage sludge applications have permanent toxic effects to human and the environment (Rehman et al., 2008). Cadmium (Cd) is nonessential but poisonous for plants, animals, and humans (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, atmospheric deposition from metallurgical industries, incineration of plastics and batteries, land application of sewage sludge, and burning of fossil fuels (Tang et al., 2006). Lead (Pb) a major pollutant that is found in soil, water and air is a hazardous waste and is highly toxic to human, animals, plants and microbes (Low et al., 2000). Nickel (Ni) is the 24th most abundant element in the earth crust and has been detected indifferent 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 implicated *

as an embryotoxin and teratogen (Chen and Lin, 1998). Hexavalent chromium (Cr(VI)) and trivalent chromium (Cr(III)) are the most prevalent species of chromium in the natural environment (Chung et al., 2006). Major sources of chromium pollution include effluents from leather tanning, chromium electroplating, wood preservation, alloy preparation and nuclear wastes due to its use as a corrosion inhibitor in nuclear power plants (Thacker et al., 2006). Mercury is one of the most toxic metals in the environment. It has been released into environment in substantial quantities through natural events and anthropogenic activities (Kiyono and Hou, 2006). Arsenic (As), a toxic heavy metal element, is widely distributed in nature (Nriagu, 2002). The sources of arsenic arised from various natural sources like weathered volcanic, marine sedimentary rocks, fossil fuels, minerals, water, air, living organisms and anthropogenic activities including mining, agricultural chemicals, wood preservatives, medicinal products, industry activities (Mandal and Suzuki, 2002). Removal of excesses of heavy metal ions from wastewaters is essential due to their extreme toxicity towards aquatic life and humans.

Department of Biochemistry, School of Biological Sciences, Madurai Kamaraj University, Madurai, India- 625021; # Department of Civil Engineering, Kyushu University, Japan. ([email protected]) 1

EDWARD RAJA ET AL

h) conditions was noted at 600 nm to determine the growth.

The uses of conventional technologies, such as ion exchange, chemical precipitation, reverse osmosis and evaporative recovery for this purpose is often inefficient and /or very expensive (Volesky, 1990). There is a need for innovative treatment technologies for the removal of heavy metal ions from wastewater. Different microbes have been proposed to be efficient and economical alternative in removal of heavy metals from water (Waisberg et al. 2003). The objective of this study is to determine heavy metals and antibiotic resistance of bacteria, MIC, growth studies, and biochemical and molecular characteristics were used to exploit these isolates for clean-up of industrial wastewater and sewage.

Determination of MIC Maximum resistance of the selected isolates against increasing concentrations of Cd on LB agar plates was evaluated until the strains unable to grow colonies on the agar plates. The initial metal concentration used 0.1 mM was prepared from 1 M stock solution. The stock solutions of CdCl2, K2Cr2O7, NiCl2, Pb (NO3)2, Na2As2O3 and HgCl2 were prepared in sterile deionized water and sterilized by autoclaving at 121oC for 15 min. The culture grow at a given concentration were subsequently transferred to the next concentration. Based on the evaluation, minimum inhibitory concentration (MIC) was determined at 30oC for 5 days.

MATERIAL AND METHODS Isolation of bacteria

Determination of antibiotic resistance

The sewage water samples were collected in and around Madurai districts, South India. The samples were collected in sterile plastic container and transport to laboratory for bacteriological analysis. The bacterial isolates were screened on Luria Bertani (LB) agar plates supplemented with 5 mg/l concentration of each metal one time by the standard pour plate method. Plates were incubated at 30oC for 5 days and colonies differing in morphological characteristics were selected and used for further studies.

Resistance to antibiotics was determined on Mueller Hinton agar plates (Hi Media, India). Inhibition zone was noted after 48 h incubation, resistance was recorded as positive. Strains were considered susceptible when the inhibition zone was 12 mm or more in diameter. Tests were performed in triplicate. The following antibiotics were tested, ampicillin (100 μg/ml), tetracycline (20 μg/ml), chloramphenicol (30 μg/ml), Kanamycin (30 μg/ml), neomycin (30 μg/ml), erythromycin (50 μg/ml), streptomycin (30 μg/ml), nalidixicacid (10 μg/ml).

Identification and characterization of the sewage bacteria

Growth studies

Selected sewage isolates were grown on MacConkey agar media (HiMedia, India). The shape and colours 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).

Growth studies of sewage bacterial isolates was studied in 250 ml flasks containing 50 ml LB medium supplemented with 0.1mM concentration of Cd, Cr, Ni, Pb, As and Hg. Flasks were inoculated with 0.5 ml of overnight culture and agitated on a rotary shaker (150 rev/min) at 30oC. Growth was monitored as a function of biomass by measuring the absorbance at 600 nm using spectrophotometer (Hitachi, Japan). 16S rDNA gene amplification

Determination of optimal growth conditions

Genomic DNA was isolated and analyzed from sewage bacteria by the method of Chen and Kuo (1993). Bacterial 16S rDNA was amplified by using the universal bacterial 16S rDNA primers, F (5 ’- AGA GTT TGA TCC TGG CTC AG – 3’) and R (5’- GGT GTT TGA TTG TTA CGA CTT - 3’). PCR was performed

The optimal growth conditions with reference to pH and temperature were determined. The isolates were grown in LB medium with different pH values (5, 6, 7, 8, and 9) and incubation was carried out at temperature 25 o C, 30 o C, 37 o C and 40 o C. The optical density of the log phase growing cultures (8-10

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EDWARD RAJA ET AL with a 50-μl reaction mixture containing 1-μl(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(Fermentas, Hanover, Germany). After the initial denaturation for 5 min at 94oC, there was 35 cycles consisting of denaturation at 94oC for 1 min, annealing at 55oC for 1 min, extension at 72oC for 1 min and final extension at 72oC for 5 min. PCR was carried out in a gene AMP PCR system 2700 (Applied Biosystems, California, USA). PCR products were analyzed by 1.5% (w/v) agarose gel electrophoresis in 1x TAE buffer with ethidium bromide (0.5 μg/ml).

RESULTS Isolation of heavy metal resistant bacteria In the present study we identify and characterize heavy metal resistant bacteria isolated from sewage water. Three hundred colonies were screened from initial level of heavy metal supplemented LB medium. 20 isolates were selected in the secondary screening from sewage water. Finally four strains were selected based on high degree of heavy metals and antibiotic resistances were used for further studies. The strains BC1, BC2, BC3 and BC5 was Gram-negative, rod shaped motile bacteria. The sewage isolates showed optimum growth at 30 °C and pH 7.0. The biochemical characteristics of sewage bacteria were shown in table 1.

Nucleotide sequencing and Phylogeny

Analysis of sequencing results

A DNA fragment was eluted by using QIAgen Gel Extraction Kit. PCR product was sequenced by 3730x1DNA synthesizer (Applied Biosystems, California, USA). Sequences were matched with previously published bacterial 16S rDNA sequences in the NCBI databases using ADVANCED BLAST (Altchul 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 ClutalX software version 1.83(Jeanmougin et al., 1998). Further phylogenetic tree, similarity index was generated and compared with known sequences.16S rDNA sequences of sewage bacteria have been deposited in GenBank.

Comparative analysis of the sequences with already available database showed that the strains were closed to the members of genus Proteus, Acinetobacter and Pseudomonas species. The highest sequence similarities of sewage bacteria as follows: BC1, Proteus vulgaris (98% similarity to Proteus vulgaris strain ATCC 29905, accession number DQ 885257); BC2, Pseudomonas aeruginosa (98% similarity to Pseudomonas sp. L25, accession number DQ 300308); BC3, Acinetobacter radioresistens (97% similarity to Acinetobacter sp. strain SW3, accession number AY 568500); BC5, Pseudomonas aeruginosa (98% similarity to Pseudomonas aeruginosa PD100, accession number AY 025034). Phylogeny based on ClustalX clearly indicates that BC2, BC1, BC3, BC5, are a strain of Pseudomonas aeruginosa, Acinetobacter radioresistens, Proteus vulgaris, and Pseudomonas aeruginosa. The 16s rDNA sequences were submitted in the Gen Bank under the accession numbers EF 683085-EF 683088.

Atomic force microscopy analysis Sewage bacteria cells were cultured in LB medium and incubated at 37oC with agitation for 24 h. Cells were harvested by centrifugation at 4000 rev/min for 15 min and washed twice with milli Q water. Samples for AFM analysis were mounted on the cover glass and mounted directly on the specimen metal disc using a double adhesive tape. Samples were scanned at different areas using AFM (Shimadzu SPM 9500-2j). For high resolution, contact mode micro cantilever was used for the analysis. Digital images were stored in the computer and processed.

Growth studies of sewage bacteria Growth studies of BC1, BC2, BC3 and BC5 were carried out in LB medium supplemented with Cd, Cr, Ni, Pb, As (200 mg/l) and Hg (100 mg/l) were prepared from stock solutions. The measurements from the cultures incubated for 24 h were in good agreement according to bacterial resistance for each heavy metal. The order of resistance of metals to strains BC1, BC3, BC5 and BC2 was found to be As >Ni = Pb > Cd > Cr > Hg, As >Ni = Pb > Cd > Cr > Hg, As =Pb = Cd > Ni > Cr > Hg, As = Pb= Cd >Ni > Cr > Hg,

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EDWARD RAJA ET AL As >Ni = Pb > Cd > Cr > Hg on the LB agar plates. When the sewage bacteria were grown in LB medium, BC2, BC3 and BC5 showed the high peak values with Pb when compared to other metals. But BC1 showed high peak values in the presence of Ni. There is no growth values observed for sewage bacteria in the presence of Cr and Hg. In general the growth rate of the sewage bacteria in the presence of heavy metal was consistently slower than the control (Figure 1a, b, c, d).

Table1- Biochemical characteristics of sewage bacteria Sewage bacteria

BC2

BC3

BC5

White Negative rod +

Light Brown Negative rod +

Yellow Negative rod +

Light Brown Negative rod +

+ + + + + +

+ + + + + + +

+ + + +

+ + + + + + +

-

+

-

+

Utilization of Glucose Fructose Sucrose Lactose Mannose Galactose

+ + -

+ + -

+ + -

+ + -

Growth at 4 ƕC 25 ƕ C 30 ƕ C 37 ƕ C 42 ƕ C

+ + -

+ + + -

+ + -

+ + + -

Morphological Colony colour Gram nature Cell morphology Motility Biochemical Catalase Oxidase Casein Gelatin Arginine Indole VP MRVP Starch (H) Citrate MacConkey agar Pseudomonas Isolation agar

MIC of heavy metal and antibiotics Sewage bacteria BC1, BC2, BC3 and BC5 showed very high degree of resistance to all heavy metals, MIC values varying concentration from 0.7- 16 mM. Among the heavy metals arsenic and nickel was less toxic, where as chromium and mercury were highly toxic to all strains. The strains BC2 and BC5 showed resistance to all antibiotics, in contrast BC1 sensitive to all antibiotics except ampicillin. In other hand the strain BC3 resistance to ampicillin, chloramphenicol, naldixic acid, and kanamycin. MIC of heavy metal and antibiotics were shown in table 2. Microscopic observations of sewage bacteria Sewage bacterial cells were imaged with AFM operated in contact mode in liquid conditions (Figures 2a, b, c, d). The examination of sewage bacteria (BC1, BC2, BC3, and BC5) have demonstrated significant morphological differences that depend on the bacterial growth conditions and imaging environment. Bacteria imaged in liquid appeared to have smooth surfaces and apparently greater resolution of cell surface structures.

BC1

Note: + positive; - negative Table 2- MIC of heavy metals and antibiotics against sewage bacteria Heavy metals/antibiotics BC1 BC2 BC3 BC5

DISCUSSION

Arsenic Cadmium Chromium Lead Mercury Nickel Ampicillin Tetracycline Chloramphenicol Kanamycin Erythromycin Streptomycin Nalidixicacid

The ability of microbial stains to grow in the presence of heavy metals would be helpful in the waste water treatment where microorganisms are directly involved in the decomposition of organic matter in biological processes for waste water treatment, because often the inhibitory effect of heavy metals is a common phenomenon that occurs in the biological treatment of waste water and sewage (Filali et al., 2000). In the present study high degree of heavy metals resistance associated with multiple antibiotic resistances was detected in sewage bacteria.

16 4 6 8.5 12 (R) 22(S) 21(S) 18(S) 15(S) 19(S) 14(S)

6.5 7 0.7 6 0.75 6.75 10(R) 7 (R) 17(S) 12(R) 11(R) 12 (R) 6(R)

13 4 0.7 6 8.5 12(R) 22(R) 11(R) 12(R) 14(S) 18(S) 10(S)

6.5 7 0.7 6 0.75 6.75 NZ 5(R) 22(S) 11(R) 12(R) 10(R) NZ

Note : Heavy metal concentration in mM; R- Resistant; S- Sensitive; NZ- No Zone

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In most of the studies, metal resistance has been reported to hold an association with antibiotic resistance (Verma et al., 2001). Under conditions of metal stress, metal and antibiotic resistance in microorganisms possibly helps them to adopt faster by the spread of resistant factors than by mutation and natural selection (Silver and Misra 1988). Growth rate of the sewage isolates in the presence of heavy metal (Cd, Ni, As and Pb) were consistently slower than that of the control, similar results have been reported earlier (Pal et al., 2004; Edward Raja et al., 2006). In this study, Pseudomonas species were resistance to cadmium 7mM in TY agar plates. In contrast, only 2mM Cd resistance was observed from sewage of Morocco (Filali et al., 2000). The present results showed Proteus and Acinetobacter species were highly resistant to As (16, 13mM) and Ni (8mM). But nickel resistant isolates tolerating were screened from West Bengal, India (Bhadra et al., 2006) and from serpentine outcrops of central Italy (Mengoni et al. 2001). In this study sewage bacteria were resistant to Pb at the concentration of 6 mM in TY medium. In contrast, lead resistant isolates were resistant to 0.6 mM and 2.5 mM in minimal medium (Roane, 1999). Bacteria exposed to high levels of heavy metals in their environment have adapted to this stress by developing various resistance mechanism.

Fig1. Growth studies of Sewage bacteria

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EDWARD RAJA ET AL

BC1

BC2

BC3

BC5

Fig. 2. Atomic force micrograph of sewage bacteria. Proteus vulgaris (BC1), Pseudomonas aerugionsa (BC2), Acinetobacter radioresistens (BC3) and Pseudomonas aerugionsa (BC5).

Bacteria exposed to high levels of heavy metals in their environment have adapted to this stress by developing various resistance mechanism. These mechanisms could be utilized for detoxification and removal of heavy metals from polluted environment (Ahmed et al., 2005).

According to these results, the present study evaluates that the identified bacteria were used to remediate heavy metal contaminated waste water and sewage.

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