Isolation, characterization and molecular

0 downloads 0 Views 366KB Size Report
INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 5, 2013 ..... World Journal of Microbiology and Biotechnology 20, pp 881-886. 31.

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 5, 2013 © Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article

ISSN 0976 – 4402

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

Pandit R.J.1, Patel B1, Kunjadia P.D2, Nagee A 1 1- Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences, Anand, Gujarat, India 2-P.G. Department Pharmaceutical Sciences, Sardar Patel University, Gujarat, India) [email protected] doi: 10.6088/ijes.2013030500037

ABSTRACT In recent years, heavy metal pollution has become one of the most serious environmental problems. The pollution of environment with toxic heavy metals is spreading throughout the world along with industrial progress. Presence of heavy metals even in traces is toxic and detrimental to all living organisms. Moreover it cannot be degraded in to simpler harmless compounds like other pollutants including, diverse range of organics, poly aromatic hydrocarbons (PAHs), organochlorines, dyes etc. The south Gujarat area is “Toxic Hotspot” in this state. In the presence study we have isolated and characterized six heavy metal resistant bacteria from industrial effluents from Amala Khadi, Ankleshwar (Gujarat). Initially total of 40 isolates were screened on Nutrient Agar plates containing different heavy metals Cr6+, Cu2+, Cd2+, Ni2+ at concentration 50 ppm in their salt form. Isolates were selected based on high level of heavy metal resistance and it’s morphological and biochemical characterization was done. Identification based on 16S rRNA gene sequencing identified Comamonas testosteroni (JX028142), Bacillus sp. (JX028143), Aeromonas hdrophila (JX028144), Exibacterium profundum (JX028145), Bacillus cereus (JX028146) and Exiguobacterium sp. (JX028147). Isolates showed high degree of resistance to heavy metals under investigation, ranging from 25-300 ppm. These isolates can further be used for bioremediation of heavy metals form industrial effluent. Keywords: Environment, Heavy metals, Pollution, Wastewater. 1. Introduction Increasing population and industrialization results in pollution of water bodies, especially with heavy metals and other organic compounds which are recalcitrant. Water pollution is the contamination of water bodies e.g. lakes, rivers, oceans, aquifers and groundwater. It occurs when pollutants are discharged directly or indirectly into water bodies without adequate treatment to remove detrimental compounds. Among the pollutants which are discharged in to water bodies, heavy metals are of most concern because other pollutant may be degraded by some microorganisms but metals cannot be degraded. Some heavy metals are purely toxic with no known role (Shi et al., 2002), other metals are essential for life at low concentration but become toxic at high concentrations (Badar et al., 2000; Gortet et al., 1999; Gadd 1992; Franke et al, 2003). Out of 17 most important heavy metals Fe, Cu, Mo, Mn are classified to have low toxicity , Zn, Ni, V, Co, W, Cr are categorized to have average toxicity, while As, Ag, Sb, Cd, Hg, Pd, U are grouped in highly toxic heavy metals (Neis, 1999).

Received on January 2013 Published on April 2013

1689

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

Bioremediation is the use of microorganisms to remove pollutants. 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. Cu (II), Cd (II), Ni (II), Cr (IV) are toxic to the environment and can be removed by use of microorganisms that are resistant to heavy metal. Tolerance and removal of toxic metal ions have been studied in bacteria (Silver and Phung, 2005; Deveci et al., 2004; Yilmaz 2003; Volesky and Holan, 1995), cyanobacteria (Wang et al., 2005 and Inthorn et al., 1996), algae (Feng and Aldrich, 2004 and Davis et al., 2003) and fungi (Pas et al., 2000 and Holan and Volesky, 1995). Among bacteria participating in polluted environment communities those genera predominate, which are known to be involved in biodegradation of organic pollutants. They often belong to the genus Pseudomonas, Comamonas or Acinetobacter (Kupka and Sevcik, 1995; Barberio and Fani, 1998; Lyamlouli et al., 2011), all of these being Gramnegative bacteria. However, in environments contaminated not only with organic pollutants but also with heavy metals, species diversity and metabolic activities of the microorganisms are reduced, and the metal-tolerant bacterial populations are developed (Knotek et al., 2003) with species of Pseudomonas and/or acidophilic bacteria predominating (Babich and Stotzky, 1985 and Dopson et al., 2003). The “Golden Corridor” of the Indian state of Gujarat is industrial area of southern Gujarat includes Ankleshwar, Nandesari and Vapi. These areas hold large numbers of industries related to chemical, dyes, textile, pesticides, pharmaceutical etc. They produce a heavy metal containing waste and part of it contaminate water bodies nearby and then flow in to main stream. It is responsible for the heavy metal toxicity in the aquatic flora and fauna and soil pollution. Aim of the present study was to isolate heavy metal resistant bacteria and its characterization. It can be further used for removal of heavy metal present in the industrial effluent from these areas. 2. Materials and methods 2.1 Collection of samples Industrial effluent samples were collected from Amravati Khadi, Ankleshwar (Gujarat, India), in sterile bottles from five different sites in the month of Jan, 2012. Samples were transfer aseptically and processed immediately in laboratories. 2.2 Isolation of Cr, Cd, Cu and Ni heavy metal resistant bacteria For isolation, the samples were diluted in sterile phosphate buffer saline solution and 200µl form each dilution was plated on Nutrient Agar plates supplemented with individual heavy metals Cr, Cd, Cu, and Ni in the form of their salts, K2Cr2O7, CdCl2, CuCl2 and NiSO4 respectively at concentration 50ppm. The plates were incubated at 37º C for 24-48 hours. The bacteria that grew were sub-cultured and obtained in form of pure culture on Nutrient agar. For further screening of multi metal resistant bacteria, the isolates were inoculated on Nutrient agar media containing 50ppm of all four metals and incubated at 37º C for 24 h. Isolates grow on these plates were transferred on nutrient agar plate and subsequently purified. The isolates were partially identified according to Bergey’s Manual of Systematic Bacteriology (Bergey and Holt, 1989).

Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1690

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

2.3 Determination of MIC of heavy metals Heavy metal ion resistance study was done by determining the Minimal Inhibitory Concentration (MIC) of the metals ions in nutrient broth media. Analytical grade K2Cr2O7, CuSO4, CdCl2 and NiSO4 were dissolved in sterilized deionised water to form desired stock solutions. Individual isolates were inoculated in 10 ml sterile nutrient broth containing 100, 200, 300, 400 and 500 ppm of each metal individually. Tubes were incubated at 37º C for 24 h. After 24 h growth of bacteria was measured at 600 nm (ELICO, BL198) using nutrient broth containing equal amount of metal as blank. 2.4 Determination of optimal temperature and pH for growth The optimal growth conditions with reference to pH and temperature were determined. For pH the isolates were inoculated into NB medium with different pH values 5, 6, 7, 8, 9 set using 1.0M NaOH or 1.0M HCl and incubated at 37º C for 24 h. For temperature isolates were inoculated into NB medium and incubated at different temperature i.e. 10º C, 28º C, 37 º C and 55º C for 24 h. Absorbance was measured at 600 nm to determine the growth. 2.5 Growth studies Growth studies of bacterial isolates was studied in 250 ml flasks containing 50 ml NB medium supplemented with all four metlas Cd, Ni, Cu (100ppm) and Cr (25ppm). Flasks were inoculated with 100 µl of overnight culture and agitated on a rotary shaker at 150 rpm. Growth was monitored as a function of biomass by measuring the absorbance at 600 nm using spectrophotometer at different time interval 2, 4, 6, 8, 10, 24, 48 and 72 h. 2.6 Isolation of genomic DNA and 16S rRNA gene amplification Genomic DNA was isolated and 16S rRNA gene was amplified by using the universal primers, F27 5’AGAGTTTGATCMTGGCTCAG3’ and R1492 5’TACGGYTACCTTGTTACGACTT3’ (Sigma, INDIA). The PCR reaction mixture (25 μL) contained bacterial genomic DNA (50 ng), 1X Taq buffer, 1.0 U Taq polymerase (Bangalore Genei), 1.5 mM MgCl2, 200 μM dNTPs, and 0.5 μM of each primer. PCR amplification was carried out in a thermocycler (NYX TECH, A6 825VAC). PCR condition was as follow. 94° C for 5 min (initial denaturation), followed by 40 cycles of 94° C for 1 min (denaturation), 36° C for 1 min (annealing), 72°C for 1 min (extension) and final extension 72°C for 10 min. Amplified products were checked on agarose gel electrophoresis and sent to Xcelris Labs, Ahmadabad for partial sequencing. Sequence analysis was performed using the sequence alignment software BLASTn with the NCBI database (National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The nucleotide sequences were deposited in GeneBank. Alignments of sequences were carried out with ClustalW software and phylogenetic tree was constructed using the neighbour-joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Neighbour- Joining method. The analysis involved 6 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 632 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.

Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1691

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

3. Results and Conclusion 3.1 Isolation of Cr, Cd, Cu and Ni heavy metal resistant bacteria Initially 40 isolates which can tolerate 50 ppm of different metal concentration were isolated. After growing on nutrient agar containing all 4 metals at 50ppm concentration six best isolates were taken for further studies. Isolates were maintained on Nutrient agar slants at 4°C for further characterization and were assigned codes BP01 to BP06. Out of 6 isolates, 4 belong to gram positive rod and 2 was gram negative rod. Phenotypic studies results have been depicted in the Table 1 and biochemical characterization results have been summarized in Table 2. Biochemical study shows that the isolates were sugar fermentor. Isolates were positive for nitrate reductases, catalase and dehydrogenase activity and also positive for oxidase. Except bacterium identified as Bacillus sp., remaining liquefy gelatine. None of the isolates was producing ammonia. Except BP04 and BP06 remaining gives Methyl-red test positive. 3.2 Determination of MIC Six isolates showed high degree of resistance to all the heavy metals except Cr. Isolates were resistant to 4 heavy metals under investigation, ranging from 25-300 ppm. Graphs show the growth of all the bacteria in presence of Cu, Cd, Cr and Ni. All the isolates tolerate Ni2+, Cd2+ and Cu2+ upto 200ppm except BP01 which was resistant to Cd2+ upto 100ppm and BP02 which show maximum Cu resistant, 400ppm. BP01 was most resistant to Cr6+ that is 300 ppm in compare to BP02, BP03 and BP04 which were shows only 25ppm tolerance. BP05 and BP06 were resistant to Cr6+ upto 200 ppm Table3. Results clearly indicate that isolates have prominent metal resistance capability. 3.3 Determination of optimum growth condition, pH and temperature Optimum growth condition was determined in order to large scale biomass production for further application. The bacteria showed maximum growth at pH 7 and 8 and very moderate growth were observed at pH 5. That mean acidic pH retard the growth. Graph also shows that BP01 and BP05 were comparatively fast growing than other isolates Fig1. Effect of temperature was observed by incubating the N broth containing bacteria at different temperatures. The maximum growth was observed at 28º C and 37 º C. Somewhat equal growths were observed at both temperatures. Very low and high temperature i.e. 10º C and 55º C were growth inhibitory that is no growth was observed at these temperatures Fig2. Growth studies will helps when these bacteria are used for bioremediation purpose.

Figure 1: Effect of pH on growth of isolates.

Figure 2: Effect of pH on growth of isolates.

Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1692

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

3.4 Growth kinetics studies Growth curves for each of isolated strains were studied in the presence of metals. Graphs shows that growth was not significantly affected in the presence of all four metals tested. Presence of Cd, Ni and Cu at 100ppm concentration and Cr at 25ppm together was not much effective for the isolates. This furthermore indicates that isolates are fined resistant to tested metals. Growth rate of isolates was little slower in presence of metal than that of control such type of results also have been reported earlier (Pal et al., 2004; Edward et al., 2006) but not totally stall the growth.

Figure 3: Growth of isolates in presence of Cd, Ni, Cu (100ppm) and Cr (25ppm). C= Contorl, T= Test. 3.5 16S rRNA gene amplification and sequencing Genomic DNA was isolated and visualized as a sharp band on 1.0% agarose gel fig 4. About1.5kb 16S rRNA gene was amplified with universal primers F27 and R1492. Amplified products were run in 1.0 % agarose gel electrophoresis and observed on in UV light as seen in Fig5. The Amplified DNAs were sequenced and isolates were identified by Gene Bank database by using BLAST search programme and sequences were submitted to NCBI (Table 4). Isolates were identified as Comamonas testosteroni, Bacillus sp., Aeromonas hdrophila, Exibacterium profundum, Bacillus cereus and Exiguobacterium sp.. Unrooted NeighbourJoining Phylogenetic tree clearly forms two separate clades for gram +ve and gram –ve bacteria. Many other researchers have isolated and study the heavy metal resistant competence and also use such bacteria for the bioremediation on heavy metals. Among these isolates Exiguobacterium sp. and Exibacterium profundum are more interesting because many researchers have studied characteristics of resistance to extreme conditions such as high/low temperature, alkaline environment, and high concentrations of salts of this group (Crapart et al., 2007; Kasana et al., 2007; La Duc et al., 2007; Vishnivetskaya et al., 2007; Joshi et al., 2008 and Ponder et al., 2008). Exiguobacterium sp. are not only resistant to conditions stated earlier but can also degrade some xenobiotic compounds (Lopez et al., 2005; Edlund et al., 2008 and Sivaprakasam et al., 2008). Aeromonas hdrophila is commonly found in aquatic environment (Matyar et al, 2007; Martinez- Mucia et al., 2008) and many researchers have studied its heavy metal and antibiotic resistant competence (Rama Chandra and Monika Sankhwar 2011 and Odeyemi et al., 2012). Bacillus sp. are also commonly found in industrial effluent and many researchers have isolated and studied heavy metal bio sorption competence of this group (Quing et al., 2007; Khosro et al., 2011; Pandey et al., 2011 and Murtyet al., 2012). Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1693

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

Table 1: Gram’s and colony characterization of isolates Isolate

Colony Colour

BPO1 BPO2 BPO3 BPO4 BPO5 BPO6

Reddish-white Creamy Buff color Orange Off white Orange

Gram’s staining Gram-ve, rod Gram+ve, rod Gram-ve, rod Gram+ve, rod Gram+ve, rod Gram+ve, rod

Shape/size

Elevation

Surface

Round- small Irregular- large Round- medium Round- medium Irregular- large Round- medium

Convex Flat Convex Raised Flat Raised

Smooth Rough Smooth Glossy Rough Glossy

Table 2: Biochemical characterization of isolates. Note: + Positive; - Negative Test/ Isolate

BPO1

BPO2

BPO3

BPO4

BPO5

BPO 6

Carbohydrates fermentation test Glucose

-

+

+

+

+

+

Sucrose

-

+

+

+

+

+

Maltose

+

+

+

+

+

+

Mannitol

+

+

+

+

_

+

Citrate utilization

+

_

+

_

+

_

Methyl red test

+

+

+

_

+

_

V-P test

_

+

_

_

+

_

Nitrate reduction

+

+

+

+

+

+

Catalase

+

+

+

+

+

+

Oxidase

-

+

+

-

+

-

Starch hydrolysis

-

+

+

+

+

+

Gelatine liquefaction

+

_

+

+

+

+

Ammonia production

_

_

_

_

_

_

Dehydrogenase

+

+

+

+

+

+

Table 3: MIC value of four metals for isolates. MIC (ppm) Isolate BP01 BP02 BP03 BP04 BP05 BP06

Cu2+ 200 400 200 200 200 200

Cd2+ 100 200 200 200 200 200

Cr6+ 300 25 25 25 200 200

Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

Ni2+ 200 200 200 200 200 200

1694

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

01

02

03

04

05

06

Figure 4: Bands of genomic DNA on 1% agarose gel.

01

02

03

04

05

Ladder 06

Figure 5: 16S rRNA gene PCR Product with 100bp ladder.

Figure 6: Neighbour-Joining phylogenetic tree of 16S rRNA gene sequences showing the positions of isolates. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Figure 6 shows Neighbour-Joining (N-J) phylogenetic dendrogram of 16S rRNA gene sequences by using MEGA5. Bacillus and Exiguobacterium both are gram positive and forms a separate clad than Comamonas and Aeromonas which are gram negative. Table 4: Name and Gene Bank accession numbers of isolates Isolate BP01 BP02 BP03 BP04 BP05 BP0

Name of bacteria Comamonas testosteroni Bacillus sp. Aeromonas hdrophila Exiguobacterium profundum Bacillus cereus Exiguobacterium sp.

Accession number JX028142 JX028143 JX028144 JX028145 JX028146 JX028147

4. Conclusion The industrial effluents are rich source of heavy metals, it is likely that bacteria reside in it must be resistant to heavy metals. The present study was initiated with the aim to isolate and identify four heavy metal, Cu, Cr, Cd and Ni resistant bacteria from industrial effluent contaminated sites of Ankleshwar, Gujarat. We have isolated and characterized 6 isolates and they were identified to Comamonas testosteroni, Bacillus sp., Aeromonas hdrophila, Exibacterium profundum, Bacillus cereus and Exiguobacterium sp.. All the isolates show elevated level of resistant to Cu, Cr, Cd and Ni. Growth kinetic study in the presence of Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1695

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

heavy metals also shows that growth rate of isolates was not much affected by the presence of metals. The overall study shows that isolates have high capacity to tolerate metals under investigation and hence, these isolates can be used in bioremediation of effluents from industries handling heavy metals. 5. References 1. Babich, H., and Stotzky, G., (1985), Heavy metal toxicity to microbe-mediated ecologic processes: a review and potential application to regulatory policies, Environmental Research, 36(1), pp 111–137. 2. Badar, V., Ahmed, N., Beswick. A.J., Pattanapipitpaisal, P., and Macaskie, L.E., (2000), Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan, Biotechnology letters, 22, pp 829-836. 3. Barberio, C., and Fani, R., (1998), Biodiversity of an Acinetobacter population isolated from activated sludge, Research in Microbiology, 149, pp 665-673. 4. Crapart S., Fardeau, M.L., Cayol, J.L., Thomas, P., Sery, C., Ollivier, B., and Blans, Y.C., (2007), Exiguobacterium profundum sp. nov., a moderately thermophilic, lactic acid-producing bacterium isolated from a deep-sea hydrothermal vent, International Journal of Systematic and Evolutionary Microbiology, 57(2), pp 287–292. 5. Davis, T.A., Volesky, B., and Mucci, A,, (2003), A review of the biochemistry of heavy metal biosorption by brown algae, Water Research, 37(18), pp 4311-4330. 6. Deveci, H., Akcil, A., and Alp, I., (2004), Bioleaching of complex zinc sulphides using mesophilic and thermophilic bacteria: comparative importance of pH and iron, Hydrometallurgy, 73, pp 293-303. 7. Bergey, D.H., and Holt, J.G., (1989), Bergey's Manual of Systematic Bacteriology, Springer. 8. Kupka, D., and Sevcik, I., (1995), Biosorption and Bioremediation, Czech Society for Biochemistry and Molecular Biology, Prague, pp 4–5. 9. Dopson, M., Baker-Austin, C., Koppineedi, P.R., and Bond, P.L, (2003), Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic microorganisms, Microbiology, 149, pp 1959–1970. 10. Edlund, A., and Jansson, J.K, (2008), Use of bromodeoxyuridine immunocapture to identify psychrotolerant phenanthrene-degrading bacteria in phenanthrene-enriched polluted Baltic Sea sediments, FEMS Microbiol Ecology, 65, pp 513–525. 11. Edward, R.C., Anbazhagan, K., and Selvam, G.S., (2006), Isolation and characterization of a metal resistant Pseudomonas aeruginosa strain. World Journal of Microbiology and Biotechnology, 22, pp 577- 586. 12. Feng, D., and Aldrich, C., (2004), Adsorption of heavy metals by biomaterials derived from the marine alga Ecklonia maxima, Hydrometallurgy, 73(1-2), pp 1-10. Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1696

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

13. Franke, S., Grass, G., Rensing, C., and Nies, D.H., (2003), Molecular analysis of the copper- transporting efflux system cusCFBA of Escherichia coli, Journal of Bacteriology, 185(13), pp 3804-3812. 14. Gadd, G.M., (1992), Metals and microorganisms: A problem of definition, FEMS Microbiology Letters, 100, pp 197-204. 15. Holan, Z.R., and Volesky, B., (1995), Accumulation of cadmium, lead, and nickel by fungal and wood biosorbents, Applied Biochemistry and Biotechnology, 53, pp 133146. 16. Hu, Q., Dou, M.N., Qi, H.Y., Xie, X.M., Zhuang, G.Q., Yang, M, (2007), Detection, isolation, and identification of cadmium-resistant bacteria based on PCR-DGGE, Journal of Environmental Sciences, 19, pp 1114–1119. 17. Inthorn, D., Nagase, H., Isaji, Y., Hirata, K., and Miyamoto, K., (1996), Removal of cadmium from aqueous solution by the filamentous cyanobacterium Tolypothrix tenuis, Journal of Fermentation and Bioengineering, 82(6), pp 580-584. 18. Joshi, A.A., Kanekar, P.P., Kelkar, A.S., Shouche, Y.S., Vani, A.A., Borgave, S.B., and Sarnaik, S.S., ( 2008), Cultivable bacterial diversity of alkaline Lonar lake, India. Microbial Ecology, 55(2), pp163–172. 19. Kasana, R.C., and Yadav, S.K., (2007), Isolation of a psychrotrophic Exiguobacterium sp. SKPB5 (MTCC 7803) and characterization of its alkaline protease, Current Microbiology, 54(3), pp 224–229. 20. Khosro, I., Mohammad reza majid, K.P, and Alireza, M., (2011), Bioremediation of toxic heavy metals pollutants by Bacillus spp. isolated from Guilan bay sediments, north of Iran, International Conference on Biotechnology and Environment Management (IPCBEE),18, pp 67-71. 21. Knotek-Smith, H.M., Deobald, L.A., Ederer, M., and Crawford, D.L, (2003), Cadmium stress studies: media development, enrichment, consortia analysis, and environmental relevance, Biometals, 16, pp 251–261. 22. La Duc, M.T., Dekas, A., Osman, S., Moissl, C., Newcombe, D., and Venkateswaran, K., (2007), Isolation and characterization of bacteria capable of tolerating the extreme conditions of clean room environments, Applied and Environmental Microbiology, 73(8), pp 2600–2611. 23. Lopez, L., Pozo, C., Rodelas, B., Calvo, C., Juarez, B., Martinez-Toledo, M.V., and Gonzalez-Lopez, J., (2005), Identification of bacteria isolated from an oligotrophic lake with pesticide removal capacities, Ecotoxicology, 14(3), pp 299–312. 24. Lyamlouli, L., Kharbouch, K., Moutaouakkil, A., and Blaghen, M., (2011), Study of multi-resistant to heavy metals, antibiotics and some hydrocarbons of bacterial strains isolated from an estuary basin, Journal of environmental chemistry and toxicology, 3(9): pp 229-232.

Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1697

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

25. Martinez-Murcia, A.J., Saavedra, M.J., Mota, V.R., Maier, T., Stackebrandt, E., and Cousin, E., (2008), Aeromonas aquariorum sp. nov., isolated from aquaria of ornamental fish, International Journal of Systematic and Evolutionary Microbiology, 58, pp1169 – 1175. 26. Matyar, F., Kaya, A., and Dincer, S., (2007), Distribution and antibacterial drug resistance of Aeromonas spp. from fresh and brackish waters in southern Turkey, Annals of Microbiology, 57, pp 443 - 447. 27. Murthy, S., Bali, G., and Sarangi, S.K, (2012), Lead biosorption by a bacterium isolated from industrial effluents, International Journal of Microbiology Research, 3, pp.-196-200. 28. Nies, D.H, (1999), Microbial Heavy Metal resistances, Applied Microbial biotechnology, 51 pp 730-750. 29. Olumide, A.O., Ahmad, A., and Gires, U, (2012), Antibiotics resistance and putative virulence factors of Aeromonas hydrophila isolated from estuary, Journal of Microbiology, Biotechnology and Food Science, 1 (6), pp 1339-1357. 30. Pal, A., Choudhuri, P., Dutta, S., Mukherjee, P.K., and Paul, A.K, (2004), Isolation and characterization of nickel-resistant micro flora from serpentine soils of Andaman, World Journal of Microbiology and Biotechnology 20, pp 881-886. 31. Pandey, S., Shah, P., Biswas, S., and Maiti, T.K., (2011), Characterization of two metal resistant Bacillus strains isolated from slag disposal site at Burnpur, India, Journal of Environmental Biology, 32, pp 773-779. 32. Pas M., Milacic R., Draslar K., Pollak N., and Raspor P, 2004, sorption of Chromium (III) and chromium (VI) compounds in yeast cells structure, Biomaterials, 17, pp 2533. 33. Ponder, M.A., Thomashow, M.F., and Tiedje, J.M., (2008), Metabolic activity of Siberian permafrost isolates, Psychrobacter arcticus and Exiguobacterium sibiricum, at low water activities, Extremophiles, 12(4), 481–490. 34. Rama Chandra, and Monica, S, (2011), Influence of lignin, pentachlorophenol and heavy metal on antibiotic resistance of pathogenic bacteria isolated from pulp paper mill effluent contaminated river water, Journal of Environmental biology, 32, pp 739745. 35. Saitou, N., Nei, M., (1987), The Neighbor-Joining method: a new method for reconstructing phylogenetic trees, Molecular Biology and Evolution, 4, pp 406–425. 36. Shi, W., Becker, J., Bischoff, M., Turco, R.F., and Konopka, A.E., (2002), Association of microbial community composition and activity with Lead, chromium, and hydrocarbon contamination, Applied Environ mental Microbiology, 68(8), pp 3859-3866.

Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1698

Isolation, characterization and molecular identification of heavy metal resistant bacteria from industrial effluents, Amala-khadi- Ankleshwar, Gujarat

37. Silver, S, and Phung le, T., (2005), A bacterial view of the periodic table: Genes and proteins for toxic inorganic ions, Journal of Industrial Microbiology and Biotechnology, 32, pp 587–605. 38. Sivaprakasam, S., Mahadevan, S., Sekar, S., Rajakumar, S., (2008), Biological treatment of tannery wastewater by using salt-tolerant bacterial strains, Microbial Cell Factories, 7, pp15. 39. Vishnivetskaya, T.A., Siletzky, R., Jefferies, N., Tiedje, J.M., and Kathariou, S., (2007), Effect of low temperature and culture media on the growth and freezethawing tolerance of Exiguobacterium strains. Cryobiology, 54(2), pp 234–240. 40. Volesky, B. and Holan, Z.R., (1995), Biosorption of heavy metals, Biotechnology Progress, 11, pp 235-250. 41. Wang, W.X., Dei, R.C.H, and Hong, H, (2005), Seasonal study on the Cd, Se, and Zn uptake by natural coastal Phytoplankton assemblag, Environmental Toxicology and Chemistry, 24(1), pp 161-169. 42. Yilmaz, E.I, (2003), Metal tolerance and biosorption capacity of Bacillus circulans strain EB1, Research in Microbiology, 154, pp 409-415.

Pandit R.J et al., International Journal of Environmental Sciences Volume 3 No.5, 2013

1699

Suggest Documents