Characterization and identification of newly isolated Acinetobacter baumannii strain serdang 1 for phenol removal Z. H. M. Yadzir, M. Y. Shukor, M. S. Nazir, and M. A. Abdullah Citation: AIP Conference Proceedings 1482, 223 (2012); doi: 10.1063/1.4757470 View online: http://dx.doi.org/10.1063/1.4757470 View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1482?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Fermentative hydrogen production by newly isolated Clostridium perfringens ATCC 13124 J. Renewable Sustainable Energy 6, 013130 (2014); 10.1063/1.4863085 Sequential chemical extraction for a phosphogypsum environmental impact evaluation AIP Conf. Proc. 1529, 52 (2013); 10.1063/1.4804081 Performance of mesoporous organosilicates on the adsorption of heavy oil from produced water AIP Conf. 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R., Malaysia 2 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, 43400, Selangor, Malaysia * Corresponding author. Tel.: +605-3687636; Fax: +605-3656176 E-mail address: [email protected] Abstract. A new indigenous bacterial strain from Malaysian soil contaminated with petroleum waste had been successfully isolated, characterized and identified for phenol removal. The gram negative bacteria showed 98% identity with Acinetobacter baumannii based on BiologTM Identification System and the determination of a partial 16S ribosomal RNA (rRNA) sequence. The isolate clustered with species belonging to Acinetobacter clade in a 16S rDNA-based neighbour-joining phylogenetic tree. Keywords: Acinetobacter baumannii, phenol, phylogenetic tree analysis, 16S ribosomal RNA PACS: 87.17.Uv INTRODUCTION Microorganisms can utilize phenol as carbon and energy sources, for growth, cell maintenance and cometabolism of other less degradable substances [1,2]. Bacteria plays major role in the degradation of phenol in the ecosystem. Large numbers of phenol-degrading bacteria, fungi and yeast have been isolated, characterized and identified, which include Acinetobacter calcoaceticus [3], Pseudo-monas putida [4], Graphium and Fusarium species [5], Candida oleophila [6] and Trichosporon montevideense [7]. Various Acinetobacter strains are capable of degrading xenobiotic compounds such as phenol [8], zearelenone [9], trinitrotoluene [10], 4-hydroxy benzoate [11] and chlorinated biphenyls [12]. Others have reported degradation of lignin [13], amino acids [14], oil [15] and heavy metals removal [16]. Several strains of Acinetobacter produce extracellular degradation of polysaccharides [17], wax esters, polyhydroxyalkalonic acids and cyanophycin [18,19]. A bioluminescent reporter strain, Acinetobacter sp. DF4, has been constructed showing sensitive bioluminescence response to phenol concentrations ranging from 5 to 100 ppm [20]. The objectives of this study were to characterize and identify newly-isolated, indigeneous bacteria from Malaysian soil contaminated with petroleum waste for possible use in the removal of hazardous materials such as phenol. MATERIALS AND METHODS Phenotypical and Biochemical Characterization Bacterial Isolates The bacterial strain was isolated from soil contaminated with petrol wastes from a car workshop in Serdang, Selangor, Malaysia. The strain was initially assigned Isolate Serdang 1. The morphology was observed by Gram staining and microscopy, and the shape, elevation and pattern of colonies were observed and recorded. Bacteria capable of utilizing phenol as a sole carbon source was isolated by enrichment in mineral salts medium containing 500 mg/L phenol as carbon source. The mineral medium constituents were (g/L): 1.0 (NH4)2SO4 (R&M Chemical, China), 0.25 MgSO4.7H20 (Merck, Germany), 0.25 KH2PO4 (Merck, Germany), 0.07 CaCl2.2H2O (Merck, Germany), 0.1 yeast extract, 0.5 phenol (Hamburg Chemical, Germany). The isolation of microbial strains was done by plating technique as prescribed in APHA 9215 [21]. International Conference on Fundamental and Applied Sciences 2012 AIP Conf. Proc. 1482, 223-228 (2012); doi: 10.1063/1.4757470 © 2012 American Institute of Physics 978-0-7354-1094-7/$30.00 223 to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: This article is copyrighted as indicated in the article. Reuse of AIP content is subject 111.68.103.233 On: Thu, 10 Apr 2014 06:24:44 Gram staining A microbial suspension was transferred to a glass microscope slide, passed through a flame to affix the cells. The smear was applied with crystal violet dye (Fluka, Switzerland) as the primary stain, which stained the cells blue-purple. The slide was washed with distilled water and the specimen should appear blue-purple. The slide was then flooded with Gram’s iodine (Ajax Chemical, Australia), to increase the affinity of the primary stain to the bacterial cells. The smear was then discolored by 95% ethyl alcohol (Hamburg Chemical, Germany), rinsed with distilled water, before counterstained with safranin (Ajax Chemical, Australia), which stained those bacteria that were decolorized in the previous step. The slide was blotted with bibulous paper and examined under the light microscope (Olympus) at 100x magnification with an oil immersion. The morphology of the cell was also noted. micropipettor. The ID Microplate was incubated at room temperature for 16 to 24 hours, and placed in the reader for analysis. BiologTM Microstation Identification was carried out using the pre-loaded ID Database on a computer which identifies bacteria to the species level. Molecular Characterization of PhenolDegrading Bacteria Genomic DNA Extraction Cells were harvested from 1.5 ml culture by centrifugation (Eppendorf, North America) at 13,000 rpm for 2 minutes and the supernatant was discarded. Genomic DNA was extracted by alkaline lysis using Wizard® Genomic DNA Purification KitTM (Promega, USA). The DNA pellet obtained was used as a template in Polymerase Chain Reaction (PCR). The DNA pellet was stored at 4°C. Oxidase and Catalase tests DNA Quantification and Purity Oxidase test was conducted to test for the presence of cytochrome C oxidase, a component of electron transport chain. A separate and well-grown colony from a culture plate of 24 hour old was spread on to a reaction zone of an oxidase strip (Medvet, South Australia) and the colour change was observed for up to 5 sec. A deep blue/violet color indicates positive reaction, and a no color change indicates no reaction. Catalase test was carried out to check the presence of catalase enzyme as previously prescribed [22]. Culture streak of 24 hour old was applied with three or four drops of 3% (v/v) hydrogen peroxide to the entire culture surface to check for the presence or absence of bubbling or foaming. Bubble formation indicates presence of catalase. The concentration and purity of the extracted DNA was determined by a spectrophotometer (Beckman, USA) [23]. BiologTM Identification System BiologTM system uses a 96 well microplate having different carbon source in each well as described in the Microlog Users Manual (Biolog Inc., Hayward, CA). The microplates are specific for Gram positive and Gram negative isolates. The test inoculum was prepared from overnight culture of bacterial Isolate Serdang 1 on nutrient agar plate. The bacterial colonies were picked using sterile cotton swab and resuspended into inoculation fluid. The bacterial suspension was adjusted to 61% turbidity using the BiologTM Turbidimeter (Biolog, Inc) according to the manufacturer’s recommendation. One hundred and fifty μl of the suspension was dispensed into all wells of the BiologTM ID Microplates using the 8-channel Amplification of Genomic DNA by PCR PCR was performed by incubating the samples at three temperatures in a thermal cycler (MJ Research Inc., USA) corresponding to three steps in an amplification cycle- denaturation, annealing and extension. To amplify the 16s rRNA region, PCR was performed using the 16s universal primer [24]. Following these cycles, a prolonged extension at 72°C for 10 min was used [23]. Detection of PCR Products DNA amplified was separated using agarose gel electrophoresis. The agarose gel at 50-70°C was poured into the gel tray and the comb was inserted. Seven microliters of amplified PCR products were premixed with 1 μl of 6X loading dye (MBI Fermentas, Lithuania) on a piece of parafilm and loaded into the gel well. Gene Ruler TM 1kb DNA marker (MBI Fermentas, Lithuania) was used as a standard. The DNA was electrophoresed at 70 V supplied from a Power Supply EPS 600 (Pharmacia, Biotech) for 1 hour and 30 min. After electrophoresis, the gel was removed, stained with 0.5 μg/ml ethidium bromide for 15 min and then destained with distilled water. The bands on the gel were visualized on an ultraviolet light trans-illuminator and photographed using the Bio 224 to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: This article is copyrighted as indicated in the article. Reuse of AIP content is subject 111.68.103.233 On: Thu, 10 Apr 2014 06:24:44 Imaging System (Syngene,UK). in Gene Snap Program Purification of desired PCR Product & Plasmid vector The QIAquick Gel Extraction (QIAGEN, Germany) was used to extract and purify the DNA fragment from agarose gel, according to the manufacturer’s instruction. The DNA fragment band was excised from the agarose gel with a clean, sharp scalpel. TOPO TA Cloning® provides a highly efficient, one step cloning strategy for direct insertion of Taq polymerase-amplified PCR products into a plasmid vector. It was supplied in linear form with single 3’thymidine (T) over hanged at both ends of the TA Cloning vector and topoisomerase 1 enzyme covalently bound to the vector. TOPO Cloning Ligation Reaction Six microliters of TOPO Cloning reaction mixture (Fresh PCR product : Salt solution : pCR® 2.1-TOPO vector = 4 : 1 : 1 μl) was prepared. The solution was mixed gently and incubated for 5 min at room temperature to prevent the reduction of transformation and cloning efficiency. Fermentas, Lithuania). The restriction digestion reaction of 10 μl total volume (Sterile deionized water : Restriction endonuclease Buffer : Restriction endonuclease (BamHI) : Restriction endonuclease (XhoI) : Recombinant plasmid = 1 : 2 : 1 : 1 : 5 μl ) was prepared. The solution was mixed gently by repeated pipeting, followed by incubation at 37°C for 2 hours. The bands on the gel were visualized on an ultraviolet light trans-illuminator and photographed by BioImaging System in Gene Snap Program (Syngene,UK). Automated DNA Sequencing The isolated plasmid DNA was subjected to automated sequencing on both strands using forward and reverse universal primers. The sequencing was carried out by First Base Laboratories Sdn Bhd, Malaysia. ABI PRISM BIGDYETM and Amersham Pharmacia Biotech DYEnamic ET Terminator Chemistry were used for the sequencing reactions. The ABI PRISM® 377-96 DNA Sequencer automatically analyses DNA molecules labeled with multiple fluorescent dyes by the BIGDYETM or DYEnamic ET Terminator sequencing reactions. The samples were loaded onto vertical 5% Long Ranger DNA (BioWhittaker Molecular Applications) sequencing gels. TOP 10 One Shot Chemical Transformation DNA Sequence Homology Analysis Twenty μl chemical competent cells of E.coli TOP 10 One Shot (Invitrogen, USA) were thawed on ice. Six microliters of TOPO Cloning ligation reaction mixture was added to a vial containing E.coli TOP 10 One Shot cells and mixed. The mixture was incubated on ice for 30 min. The cells were heat shocked for 1 min at 42°C without shaking. The tube was then incubated on ice for 2 min. The plates were incubated at 37°C for 16 hours. After incubation, blue and white colonies were observed. Plasmid Extraction Plasmid extraction was performed according to the protocol as described by GeneJETTM Plasmid Miniprep Kit (MBI Fermentas, Lithuania). The plasmid DNA was stored at -20°C until use. Analysis of Positive Clones by Restriction Endonuclease (RE) Analysis The presence of the inserted gene in the plasmid was confirmed by digesting the extracted plasmid with restriction endonucleases- BamHI and XhoI (MBI The homology of the PCR amplified DNA fragment was analyzed and compared with Genbank database using the BLASTN 2.0.11 programs of the National Center for Biotechnology Information, which can be accessed at http://www.ncbi.nlm.nih.gov/ Genbank/. Phylogenetic Analysis A multiple alignment of 22 16S rRNA gene sequences which closely matches Phenol-degrading strain was retrieved from GeneBank and was aligned using clustal-W [25] with the PHYLIP (phylogeny inference package) output option. The aligment was visually checked for any obvious mis-alignments. Alignment positions with gaps were excluded from the calculations. A phylogenetic tree was constructed using PHYLIP, version 3.573 (J.Q. Felsenstein, PHYLIP-phylogeny inference package, version 3.573, Department of Genetics, University of Washington, (http://evolution.genetics.washington.edu/phylip.html), with Bacillus strain as the out group in the cladogram. 225 to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: This article is copyrighted as indicated in the article. Reuse of AIP content is subject 111.68.103.233 On: Thu, 10 Apr 2014 06:24:44 Batch experiment Culture Media The medium used in all experiments was as described before [26]. The medium was autoclaved (Hirayama, USA) at 121°C for 15 minutes. Phenol solution was filter sterilized using 0.2 μm syringe filter (Sartorius, Germany) and added to the sterilized medium at the beginning of the experiment. For the preparation of phenol solid medium in plates, 20 g/l of agar was added. Bacterial Stock and Working Culture Well-defined colonies of all pure bacterial isolates were transferred to a phenol medium slant and preserved at 4°C in refrigerator for future use. For culture library collection, pure culture was transferred into Eppendorf tubes containing 20% (v/v) glycerol and preserved at -20°C for future use. Single colony was transferred into a universal bottle containing 5 ml liquid phenol medium at 500 mg/l phenol concentrations. Cultures were incubated at 30°C for 48 hours at 100 rpm. One percent of bacterial suspension (absorbency for concentration A600= 1.0) was inoculated into a 250 ml Erlenmeyer flask (Pyrex) containing 50 ml liquid phenol medium and incubated on a rotary shaker (100 rpm) at 30°C. The cultures were used as inoculum for experiments after leaving overnight (~15 hours). Bacterial Growth Determination Bacterial growth population was determined using a serial dilution technique to enumerate the colonyforming unit (CFU). The suspensions (100μl) were spread onto phenol media agar and mixed by rotation. The plates were incubated for 48 hours at 30°C and the resulting colonies were counted. The exponential increase in cell density after inoculation is measured as a function of time and analyzed to obtain the specific growth rate (μ), for that particular substrate concentration as represented by the equation : dX / dt = µX (1) where X is cell density (CFU/ml), and t is time (day). Phenol Concentration Determination Colorimetric method based on 4-aminoantipyrine (4-AAP) was used to determine phenol concentrations using phenol solution as a standard [21]. After 15 minutes, the absorbance was read at 510 nm using spectrophotometer. The amount of phenol concentrations was estimated from the standard phenol curve. This method determines phenol and ortho- and meta-substituted phenols. RESULTS AND DISCUSSION Morphological and Physiological Characterization The colonies of bacterial strain Isolate Serdang 1 were observed as creamy colonies, round in shape with either smooth or irregular edges. The cells were cocci in shape and formed chains of 3-4 bacterial cells per chain. The pink-red colour from gram-staining indicated that it was Gram-negative. The formation of bubbles in the Catalase test may suggest the evolution of oxygen and the presence of catalase. However, the results with oxidase test strip suggest that the Isolate Serdang1 did not produce cytochrome c oxidase. It could mean that it lacks cytochrome c oxidase but not that it lacks an electron transport chain. Oxidase test aids in differentiation among members of the genera Pseudomonas, which are oxidase positive, and Enterobacteriacea, which are oxidase negative. Gram-negative E.coli is another example of Enterobacteriacea with an oxidase negative, but having an electron transport chain that contains other cytochromes in its cytoplasmic membrane [22]. Oxidase enzymes play a vital role in the electron transport system during aerobic respiration. Cytochrome c oxidase, an enzyme found in many electron transport chains including those of eukaryotes, catalyzes the oxidation of a reduced cytochrome by molecular oxygen, resulting in the formation of hydrogen peroxide or water [27,28]. The physiological characteristics of Isolate Serdang 1 were determined by Biolog TM Identification System, which can identify isolate to the species level and show the metabolic capabilities of the isolate. The system tests the ability of microorganisms to oxidize a panel of 95 different carbon sources. Isolate Serdang 1 showed similar characteristics to Acinetobacter baumannii at 99% probability, with similarity index of 0.712 at 24 hours. The identification was accepted as correct based on similarity index of the genus and species name as listed in the system database and if the assigned identity matched the genus and species of the reference ATCC strain [29]. Molecular Characterization The intensity of absorbance of the DNA solution at wavelength 260 nm and 280 nm was used as a measure of DNA purity [23]. DNA absorbs UV light at 260 nm and protein absorbs UV light at 280 nm. 226 to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: This article is copyrighted as indicated in the article. Reuse of AIP content is subject 111.68.103.233 On: Thu, 10 Apr 2014 06:24:44 Pure sample of DNA has the 260/280 ratio at 1.7 to 2.0 and is relatively free from protein contamination. In this study, the 260/280 ratio at 1.7 was recorded, suggesting that pure DNA was obtained. The recombinant plasmids were isolated and digested with BamHI and XhoI which produced two bands, the vector (~3.9 kbp) and the insert (~1.5 kbp). Based on gel electrophoresis analysis as shown in Figure 1, ~1.5 kbp band was seen, corresponding to the 16s rRNA region of the complete genomic DNA. Lane 1 Lane 2 Lane 3 ~3.9 kbp ~1.5 kbp strictly aerobic and tend to be paired cocci, rather than monoflagellate rods. They can use various carbon sources for growth and can be cultured on relatively simple media [30]. Based on cell shape, absence of flagella, G+C content of DNA and nutritional properties, these organisms are initially classified in the genus Moraxella but now known more as Acinetobacter [31]. The Acinetobacter baumannii– Acinetobacter calcoaceticus complex is the species most commonly isolated from clinical specimens and found as part of the normal skin, throat and rectal flora as well as in food and body lice. It colonises patients in Intensive Care Units and contaminates inanimate hospital surfaces and devices as well as wounds, including war injuries. Acinetobacter has also emerged as a cause of nosocomial outbreaks and is characterised by increasing antimicrobial multiresistance [30]. Kinetics of Microbial Cell Growth and Phenol Removal FIGURE 1. Restriction Endonuclease (RE) analysis of the extracted plasmid of positive transformant colony. Lane 1: DNA ladder marker; Lane 2: White colony: Vector (~3.9 kbp) with released PCR products (~1.5 kbp) Lane 3: Blue colony: Vector (~3.9 kbp) without released PCR products. This analysis confirmed that the clone carrying the gene as an insert, has been obtained. The plasmid vector contained a gene (ampR) coding for resistance to the antibiotic ampicillin. The host bacterium will not be able to grow on the test medium, containing ampicillin, unless the vector had transferred the ampicillin-resistance gene. The PCR product was sequenced using M13 forward and reverse universal primers on both strands and the complete sequence revealed that this gene consists of 1501 nucleotides (data not shown). Based on a BLASTN search of GenBank, the complete sequences of these bacteria share 98% similarity with Acinetobacter baumannii. This is also confirmed by a neighbour-joining phylogenetic tree based on the alignment of 16S rRNA gene sequence of Isolate Serdang 1 with 16S rRNA sequence of the 22 described Acinetobacter type strains available in GenBank databases and rooted by using Bacillus. Based on earlier results, the Isolate Serdang 1 has been re-designated Acinetobacter baumannii Serdang1 and the complete sequence has been submitted to GeneBank database under the accession number EF525671. Acinetobacter spp. are ubiquitous and can be found in water, soil and living organisms. These are Gram-negative bacteria, oxidase-negative, non-motile, The batch kinetic profile shows the variation of phenol and cell density versus time as depicted in Figure 2. A decrease in phenol concentration was concomitant with an increase in cell growth suggesting that phenol is being assimilated for energy and growth. There were two phases observed: - the first phase (0-5 days) when cells began to divide and entered a logarithmic phase with lower phenol removal rate at 48 mg L-1 day-1; and the second phase when cells were in stationary phase (5-7 days) and the phenolremoval rate was 130 mg L-1 day-1. The maximum cell density on day 5 was 8.9 log CFU/ml corresponding to 66 x 10-7 CFU/ml and the cell specific growth rate was 0.560 day-1. In this preliminary study, the time needed for complete phenol removal at 500 mg/L initial concentration was 7 days, while others have reported 5 days with Acinetobacter sp. strain W-17 [32]. This shows the need for identification of important design parameters for further optimization of phenol removal. FIGURE 2. Cell growth profile of Acinetobacter baumannii Serdang 1 at basic conditions; 30°C, pH 7, 100 rpm on orbital shaker in basic medium with 500 mg/L phenol. 227 to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: This article is copyrighted as indicated in the article. Reuse of AIP content is subject 111.68.103.233 On: Thu, 10 Apr 2014 06:24:44 CONCLUSION A new indigenous phenol-degrading bacteria strain from Malaysian soil had been successfully isolated, characterized and identified. Based on a BLASTN search of GenBank, the complete sequences of bacteria shared 98% similarity with Acinetobacter baumannii. This is confirmed by BiologTM Identification System, sequence alignment and phylogenetic tree analysis. The Isolate Serdang 1 has been redesignated Acinetobacter baumannii Serdang1 and the complete sequence submitted to GeneBank database under the accession number EF525671. ACKNOWLEDGMENTS The authors would like to thank Universiti Teknologi PETRONAS for the scholarship to Zailatul Hani Mohamad Yadzir and the research facilities, and Universiti Putra Malaysia for the research facilities to carry out microbial and molecular work. REFERENCES 1. P.M. van Schie, L.Y. Young, Bioremed. 4, 1-18, (2000) 2. G. Cornelissen, D.T.H.M. Sijm, Chemosp. 33, 817-830, (1993) 3. Y. Zhan, Y. Yan, W. Zhang, M. Chen, W. Lu, S. Ping, M. Lin, Research in Microbiology,163, 36-43 (2011) 4. P. C. Ignacio, B. Judith, D. Katrin, dos S. Vitor, W. Christoph, Appl. Microb. Biotechnol., 93, 2279-90 (2012) 5. K. M.Basha, A. Rajendran, and V. T.Velu, Asian J. Exp. Biol. Sci, 1, 219-234 (2010) 6. C. Amaral, M.S. Lucas, A. Sampaio, Int. J. Biodet Biodegrad., 68, 45-50 (2012 ) 7. H.Liu, Q.J.Yu, G.Wang, F.Ye, Y.Cong, Proc. 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Lett. 24, 1295-1297 (2002) 228 to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: This article is copyrighted as indicated in the article. Reuse of AIP content is subject 111.68.103.233 On: Thu, 10 Apr 2014 06:24:44