Heavy metal resistance Bacterium isolated from KrishnaGodavari ...

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 7, 2011 © Copyright 2010 All rights reserved Integrated Publishing Association Research article

ISSN 0976 – 4402

Heavy metal resistance Bacterium isolated from KrishnaGodavari basin, Bay of Bengal

Gunaseelan C 1 , Ruban. P 2 1 Dr. Mahalingam Center for Research and Development, N G M College, Pollachi642 001, Coimbatore. Tamil Nadu, India. 2 School of Marine Sciences, Department of Oceanography and Costal Area Studies, Alagappa University, Thondi Campus, Thondi623 409, Tamil Nadu, India. [email protected] ABSTRACT This is one of the series of works explaining about the pollution in the marine environment due to which there arises a population of bacteria resistant to metals. The sediment samples were collected from KrishnaGodavari basin, (lat 13 0 07’ N and 19 0 20’ N and long 73 0 22’ E) Bay of Bengal. Totally 53 different bacterial organisms were obtained from the sediment samples on the half and full strength nutrient agar media which were morphologically and phenotypically characterized by employing staining methods and all these were subjected to metal response tests with heavy metals in different concentrations. Of these isolates, 79.24% were found to be resistant to 350ppm of Mercury (11.53%), 250ppm of Cadmium (3.77%), 700ppm of Chromate (50.94%) and 250ppm of Zinc (13.20%). The resistance could have been due to the selective pressure exerted on the organisms by pollution of the marine atmosphere with heavy metals. Keywords: Marine Environment, Heavy Metals, Resistance, Morphological, Phenotypic. 1. Introduction Microbial communities in buried sediments may represent up to onethird of the earth’s biomass (Whitman et al., 1998). The release of heavy metals into our environment is still large and causes an environmental pollution problem because of their unique characteristics (Soltan et al., 2008). Contamination of the aquatic environment by toxic metal ions is a serious pollution problems, heavy metals may reach watercourses either naturally through a variety of geochemical processes or by direct discharge of municipal, agricultural and industrial wastewater (Semerjian, 2010; SrinivasaRao et al., 2010), to a lesser extent, from natural weathering (Higham et al., 1985). Mercury is the most toxic of the heavy metals (Gerlach, 1981) and occupies the sixth position in the list of hazardous compounds (Nascimento and ChartoneSouza, 2003). At elevated concentrations, soluble metal compounds can be deleterious to human health as well as to aquatic and marine environments (Semerjian, 2010; SrinivasaRao et al., 2010). Cadmium, arsenic, mercury, lead and chromium have been known to be extremely toxic at lower concentrations (ElSersy and ElSharouny, 2007), although, they have no significant biological function so far reported. Cadmium causes reduced growth rate, long lag phase, and lower cell density and may even cause death of bacteria at levels below 1 ppm (Shapiro and Keasling, 1996; Sinha and Mukherjee, 2009). Nygaerd et al., 2001 explained that low level of this metal even in higher organisms could be due to excretion of mercury through the growth of feathers or due to dilution during body growth. Highly soluble, hexavalent chromium (chromate, CrO4 2) is very toxic. As an analogue of sulfate, chromate can enter bacterial and

Received on April 2011 Published on August 2011

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Heavy Metal Resistance Bacterium Isolated from KrishnaGodavari basin, Bay of Bengal

mammalian cells readily via sulfate transport systems (Ackerley et al., 2004). The subsequent reduction of Cr(VI) by glutathione, thiols and other metabolites, and coproduction of reactive oxygen species (ROS) that damage DNA and other cellular components are the cause of the carcinogenic, mutational, and teratogenic potential of chromate (Chardin et al ., 2002; Klonowska et al., 2008). The bioremediation of heavy metals using microorganisms has received a great deal of attention in recent years, The response of microorganisms towards toxic heavy metals is of importance in view of the interest in the reclamation of polluted sites. Microorganism uptake metal either actively bioaccumulation and/or passively (biosorption) (Johncy et al., 2010). Many microorganisms have developed chromosomally or extra chromosomallycontrolled detoxification mechanisms to overcome the detrimental effects of heavy metals (Ehrlich, 1997). The objective of this study was to isolate and identify metal tolerant bacteria from a KG basin to evaluate their ability to tolerate different concentrations of mercury, cadmium, zinc and chromium. 2. Materials and Method 2.1 Study Area The sediment samples were collected on KrishnaGodavari basin, Bay of Bengal (lat 13 0 07’ N and 19 0 20’ N and long 73 0 22’ E). The sediment Samples were collected from different depths ranging from 1 to 13 meter by using gravity and pressure corers. All the samples were transported to the laboratory in a cool container (10 0 C±2 0 C). 2.2 Sample Preparation and Screening of Microbes 5 grams of the sediment was suspended in 100ml of sterilized sea water and the slurry thus obtained was used for further processes. 25 µl of the slurry samples were inoculated on half strength and full strength nutrient agar media plates by the spread plate technique under sterile conditions. The plates were incubated in the room temperature for 2436 hours until the colonies clearly developed. The colonies were counted as colony forming units and subculture based on colony morphology (color, shaped, size etc,). 2.3 Metal toxicity testing The test has been carried out as outlined by Nair et al.,(1992).The working cultures were maintained in VNSS medium (Hermansson et al.,1987) containing one of the test metals (350ppm of HgCl2,250ppm of CdCl2, 250ppm of ZnCl2, and 700ppm of K2CrO4). 3. Result Totally 53 isolates were screened from different depths of sediment samples (113meter) based on colony morphology (Table. 1). Most of the isolates were gram negative. Table 1: Morphological characterization of isolates from different depths S.no

Depth (meter)

Gram Character

Microscopic morphology

1

13 (a)

+

Rods

Organisms Bacillus spp.,

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2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

13 (b) 13 (c) 12 (a) 12( b) 12 (c) 12 (d) 12 (e) 11 (a) 11 (b) 11 (a) 10 (b) 10 (c) 10 (d) 9 (a) 9 (b) 9 (c) 9 (d) 8 (a) 8 (b) 8 (c) 8 (d) 7 (a) 7 (b) 7 (c) 7 (d) 6 (a) 6 (b) 6 (c) 6 (d) 6 (e) 6 (f) 6 (g) 5 (a) 5 (b) 5 (c) 5 (d) 5 (e) 4 (a) 4 (b) 4 (c) 3 (a) 3 (b) 3 (c)

+ + + + + + +

Cocci Rods Rods Rods Rods Rods Rods Cocci Rods Rods Rods Rods Rods Rods Rods Rods Rods Rods Rods Rods Cocci Cocci Rods Cocci Rods Rods Rods Rods Rods Rods Cocci Rods Rods Rods Cocci Cocci Rods Rods Rods Rods Rods Rods Rods

Actinobacter spp., E. coli Enterobacter spp., Legionella spp., Pseudomonas spp., Klebsiella spp., Microbacterium spp., Lactobacillus spp., Bacillus spp., Clostridium spp., Rhizobium spp., Yersinia spp., Carnybacterium spp., Serratia spp., Klebsiella spp., Proteus spp., Morganella spp., Enterobacter spp., Actinobacter spp., Bacillus spp., Enterobacter spp., Carnybacterium spp., Vibrio spp., Enterobacter spp., Microbacterium spp., Pseudomonas spp., E. coli Staphylococcs spp., Vibrio spp., Moraxella spp., Actinobacter spp., Actinobacter spp., Aeromonas spp., E. coli Pseudomonas spp., Nesaria spp., Serratia spp., Klebsiella spp., E. coli Vibrio spp., Serratia spp., Pseudomonas spp., Serratia spp.,


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45 46 47 48 49 50 51 52 53

3 (d) 2 (a) 2 (b) 2 (c) 2 (d) 2 (e) 2 (f) 1 (a) 1 (b)

+ + +

E. coli Enterococcus spp., Moraxella spp., Serratia spp., Klebsiella spp., Clostridium spp., Proteus spp., Enterococcus spp., Micrococcus spp.,

Rods Cocci Rods Rods Rods Rods Rods Cocci Cocci

+ Positive; Negative 3.1 Metal toxicity testing Of the 53 cultures, totally 79.24% of bacterium was found to be resistant to mercury, cadmium, zinc and chromium. In this 11.53% bacterium were resistant to 350ppm of Mercury, 3.77% bacterium resistance to 250ppm of Cadmium, 13.20% bacterium were resistance to 250 ppm of Zinc and 50.94% bacterium were resistant to 700ppm of Chromate, (Table. 2). Table 2: Metal response tests with isolates from different depths. Depth (meter)

Metals K2CrO4

HgCl2

CdCl2

ZnCl2

350ppm

250ppm

700ppm

250ppm

Organisms Bacillus spp., Actinobacter spp., E. coli Enterobacter spp., Legionella spp., Pseudomonas spp., Klebsiella spp., Microbacterium spp., Lactobacillus spp., Bacillus spp., Clostridium spp.,

13 (a) 13 (b) 13 (c) 12 (a) 12 (b) 12 (c) 12 (d) 12 (e) 11 (a) 11 (b) 11 (a)

+ + + + + +

+

Rhizobium spp., Yersinia spp.,

10 (b) 10 (c)

+

Carnybacterium spp., Serratia spp.,

10 (d) 9 (a)

+

+

+

Klebsiella spp.,

9 (b)

+

Proteus spp.,

9 (c)

+

Morganella spp.,

9 (d)

+

Enterobacter spp.,

8 (a)

+

+

+

Actinobacter spp.,

8 (b)

+

+

Bacillus spp.,

8 (c)

+

+


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Enterobacter spp., Carnybacterium spp., Vibrio spp., Enterobacter spp., Microbacterium spp., Pseudomonas spp., E. coli Staphylococcs spp.,

8 (d) 7 (a) 7 (b) 7 (c) 7 (d) 6 (a) 6 (b) 6 (c)

+ +

+

+

Vibrio spp., Moraxella spp., Actinobacter spp., Bacillus spp., Aeromonas spp., E. coli Pseudomonas spp., Nesaria spp., Serratia spp., Klebsiella spp., E. coli Vibrio spp., Serratia spp., Pseudomonas spp., Serratia spp., E. coli Enterococcus spp., Moraxella spp., Serratia spp., Klebsiella spp., Clostridium sp Proteus spp., Enterococcus spp., Micrococcus spp.,

6 (d) 6 (e) 6 (f) 6 (g) 5 (a) 5 (b) 5 (c) 5 (d) 5 (e) 4 (a) 4 (b) 4 (c) 3 (a) 3 (b) 3 (c) 3 (d) 2 (a) 2 (b) 2 (c) 2 (d) 2 (e) 2 (f) 1 (a) 1 (b)

+

+ +

+ + + + + + + + + + + +

+ +

Sensitive; + Resistance 4. Discussion Presence of metal tolerant bacterium in a given environment may be an indication that such area is affected by heavy metals. Such an area may foster adaptation and selection for heavy metal resistant organisms (Clausen, 2000). Isolation of bacteria from metal polluted environment would represent an appropriate practice to select metal resistant strains that could be used for heavy metal removal and bioremediation purposes (Malik, 2004). The results from this study of resistance of bacterial isolates to metals from the marine sediments of various depths reflects the extent and character of pollution, on one hand, and the level of adaptation of natural bacteria to their surrounding, on the other. In this study it was found that all the isolates showed varied population along the sediment columns. Microorganisms undergo selection pressures in the presence of toxic compounds and develop resistance (Hideomi et al., 1977). Enumeration of these resistant bacteria needs reservation as it not only depends on the nutritional status of the organisms but also on the organic content of the Gunaseelan C, Ruban P International Journal of Environmental Sciences Volume 1 No.7, 2011

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medium used for enumeration (Nair et al., 1993). In this study, different heavy metals, which are known to be toxic viz., mercury, chromium, cadmium and zinc, were used. A Majority of the isolates were observed to be gram negative. Gram positive isolates accounted for only 20%. All of them were motile and possessed spores which are common characteristics of the marine bacterioplanktons (Brynhildsen et al., 1988). Bacteria are generally the first organisms to be affected by discharges of heavy metals into the environment, resulting in an increase of metal resistant bacteria in these environments (Trevors, 1987; Jain, 1990; Silver, 1996; Nair et al., 1993). Duxbury (1986) reported that heavy metal resistance was pronounced in gram negative bacteria. A high level of resistance was expressed against chromate (50.94%) suggesting that there might be chromate contamination in the area (Clausen.2000). MJ De Souza, 2007 reported that these resistant isolates might have only a fewer enzyme expressions when compared with the sensitive ones. In aquatic habitats, metals can be bound and removed from the water by organic sediments, which effectively reduces the total metal ion concentration in solution.Zinc is a bioessential micronutrient (Brynhildsen et al., 1988) which could have been the reason for tolerance to Zn at 700 ppm. Bacteria are a potentially important source of metal accumulation in filterfeeding mollusk like limpets as ingestion of bacteria contributed up to 17% of Zn accumulation (Qiu et al., 2001). Interestingly, about 13.20% of the isolates were resistant to 250 ppm of cadmium salt. This aspect is perhaps reflected at higher trophic levels too. The main reason for the high levels of cadmium observed in petrels and skuas was due to movement of Cd up the aquatic food chain as relatively high levels was noted in krill (Nygaerd et al., 2001).In general there is a sharp rise in resistant bacteria capable of tolerating very high concentration of metal mercury in the coastal environment of India and was irrespective of the current levels of pollution (Ramaiah and De, 2003). However, in this study resistance to lower concentration of Hg was observed (11.53%) suggesting that the contamination of this metal in these waters could be low. These isolates are of interest for molecular characterization of mechanisms for resistance to multiple metals and hold promise for bioremediation of toxic heavy metals, including in environments that are contaminated by several metals. 5. Conclusion The present study revealed the capacity of bacterial isolates to tolerate and grow with different concentrations of mercury, chromium, cadmium and zinc. The metal concentration can be an important environmental factor regulating tolerance to the metal. The native isolates, which tolerated high concentration, can be effective in remediation strategies for ecosystem polluted with metals. 6. References 1. Ackerley, D.F., Gonzalez, C. F., Park. C. H., Blake. R., Keyhan. M. and Matin. A. (2004). Chromatereducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli. Applied Environment Microbiology, 70: pp 873882. 2. Brynhildsen. L., Lundgren. B.V., Allard. B. and Rosswall. T. (1988). Effects of glucose concentrations on cadmium, copper, mercury and zinc toxicity to a Klebsiella spp., Applied Environmental Microbiology, 54: pp 16891691.


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