Environ Sci Pollut Res DOI 10.1007/s11356-014-3308-7

RESEARCH ARTICLE

Characterization and cadmium-resistant gene expression of biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11 Jaya Chakraborty & Surajit Das

Received: 3 March 2014 / Accepted: 7 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11 was isolated from coastal marine sediment of Paradeep Port, Odisha, East Coast, India, which resisted up to 1,000 ppm of cadmium (Cd) as cadmium chloride in aerobic conditions with a minimal inhibitory concentration of 1,250 ppm. Biomass and extracellular polymeric substances (EPS) secreted by the cells effectively removed 58.760±10.62 and 29.544±8.02 % of Cd, respectively. The integrated density of the biofilm-EPS observed under fluorescence microscope changed significantly (P≤0.05) in the presence of 50, 250, 450, 650 and 850 ppm Cd. ATR-FTIR spectroscopy showed a peak at 2,365.09/cm in the presence of 50, 250, 450 and 650 ppm Cd which depicts the presence of sulphydryl group (–SH) within the EPS, whereas, a peak shift to 2,314.837/cm in the presence of 850 ppm Cd suggested the major role of this functional group in the binding with cadmium. On exposure to Cd at 100, 500 and 1,000 ppm, the expression profiles of cadmium resistance gene (czcABC) in the isolate showed an up-regulation of 3.52-, 17- and 24-fold, respectively. On the other hand, down-regulation was observed with variation in the optimum pH (6) and salinity (20 g l−1) level. Thus, the cadmium resistance gene expression increases on Cd stress up to the tolerance level, but an

Responsible editor: Robert Duran Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-3308-7) contains supplementary material, which is available to authorized users. J. Chakraborty : S. Das (*) Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela, Odisha 769 008, India e-mail: [email protected] S. Das e-mail: [email protected]

optimum pH and salinity are the crucial factors for proper functioning of cadmium resistance gene. Keywords Cadmium resistance . Marine bacteria . Pseudomonas aeruginosa . Gene expression . czcABC gene

Introduction Cadmium (Cd) is one of the non-essential highly toxic metals having a specific gravity of 8.65 and atomic weight of 112.411. Due to industrial contamination, it is present ubiquitously, accumulated in the ecosystem and reported to have carcinogenic characteristics (Saluja et al. 2011). The sources of cadmium pollution include industries, such as those producing chlor-alkali, paints, electroplating, copper alloys, pulp and paper, alkaline batteries, mining, fertilizer and zinc refining. Burning of fossil fuels and sewage sludge is also another major source of cadmium in the environment (USEPA 2000). The presence of cadmium in the environment is most universally in 2+ oxidation state in salts, such as cadmium oxide, chloride, sulphide or sulphate (Bortman et al. 2003). Cadmium causes toxicity by mimicking other metals of 2+ oxidation state like calcium and zinc in biological systems (Sutoo et al. 1990). The toxic effects of cadmium on higher organisms are widespread. Cadmium acts as a catalyst in reactive oxygen species formation causing oxidative stress altering body metabolism. It activates free radical formation and inhibits glutathione peroxidase formation leading to reduced defence against lipid peroxidation (Congui et al. 2000; Novelli et al. 2000). With increase in cadmium concentration (greater than ten milligrams) in the body, damage occurs to the kidneys, cardiac tissue and bones (Longe 2005). The toxic effect of cadmium in microorganisms leads to disruption of protein function by binding to sulphydryl groups of different enzymes, while its binding to nucleotides leads to

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single-strand breaks in cellular DNA (Mitra and Berstein 1978; Cunningham and Lundie 1993). These intoxicating effects lead to prolonged lag phase, decreased growth rate, lower cell density or death of microorganisms (Wang et al. 1997; Deb et al. 2013). Cadmium affects microbial cells by thiol-binding, protein denaturation and membrane damage, thus losing their protective function (Nies 1999). However, these can be combated by evolution of various resistance mechanisms which include a common plasmid-encoded heavy metal efflux pump. This specifically intakes and effluxes undesirable divalent cations through the cell membrane (Nies 1992). In gram-negative bacteria, cadmium is detoxified by RND-driven systems like Czc, which is mainly a zinc exporter (Nies and Silver 1989; Nies 1995), and Ncc, which is mainly a nickel exporter (Schmidt and Schlegel 1994). The product of genes czcA, czcB and czcC forms the membranebound cation efflux protein complex czcABC. It includes three subunits—CzcA, located in the cytoplasmic membrane as a cation/proton antiporter, CzcB, in the periplasm belonging to the membrane fusion protein, and CzcC, an outer membrane protein. They respectively transport the cations across the cytoplasmic membrane, periplasmic space, and the outer membrane, resulting in the decrease of intracellular cations. Whereas, in gram-positive bacteria, the primary resistance mechanism is by the cadmium exporting P-type ATPase, the CadA pump from Staphylococcus aureus (Nucifora et al. 1989). Alternatively, heavy metals can also be sequestered by adsorption to the cell wall or by binding to detoxifying ligands, proteins or polymers (Monachese et al. 2012). Microbial mediated precipitation of heavy metals such as insoluble sulphides, carbonates, phosphates and hydroxides can also reduce the bioavailable concentration of the toxic ions (Aiking et al. 1984). Besides, microbial biofilms, encapsulated in a hydrated extracellular polymeric substance (EPS), have shown a great promise in biosorption of toxic metals. BiofilmEPS is a rich matrix of polysaccharides, proteins and nucleic acids (Mangwani et al. 2014). These substances are high molecular weight compounds secreted by microorganisms into their environment and consist of glucose, fructose, mannose, pyruvate, fucose, mannuronic and guluronic acid complexes (Brisou 1995). The structure of EPS is complex, and its content in biofilms varies with environmental factors, nutrient availability and physicochemical conditions (Renner and Weibel 2011). Marine sediments and coastal waters are often contaminated with a variety of toxic compounds which is an environmental hazard (Daane et al. 2001). Moreover, marine environments are one of the most adverse environments owing to their varying nature of temperature, pH, salinity, sea surface temperature, currents, precipitation regimes and wind patterns. These constant environmental variations make the marine microorganisms more suitably adapted to these adverse

conditions for their better utilization in bioremediation of heavy metals (Dash et al. 2013). Activity by the marine microorganisms has been recognized as one of the most significant and efficient approach for removing toxic metals from the environment (De et al. 2008; Zhou et al. 2013). The complex interaction of metal cations with bacterial cell walls and onto biofilm-EPS is able to control the mobility, speciation and bioavailability of metals (Ueshima et al. 2008). Previous studies on metal adsorption onto bacterial biomass have involved cells with little or no EPS present (Costerton et al. 1995; Davey and O’Toole 2000; Flemming and Wingender 2001; Allesen-Holm et al. 2006). Recently, many findings relating to the metal-EPS binding have demonstrated the removal of toxic metals (Teitzel and Parsek 2003). In a previous report, Pseudomonas aeruginosa E1 with cadmium ion resistance and biosorption ability was isolated from heavy metal-polluted sites and showed resistance up to 366 ppm (2 mM) (Zeng et al. 2012). To analyze the effect of different environmental parameters on the resistance mechanism of bacteria, relevant gene expressions at transcriptional level have been investigated in the present study in response to cadmium stress, pH and salinity. The present study reports efficient cadmium removal by a biofilm-forming cadmiumresistant marine bacterium with the help of cellular biomass and EPS with deeper insights into the expression pattern of cadmium resistance gene under different environmental parameters.

Materials and methods Sampling and isolation of cadmium-resistant bacteria Sediment samples were collected from the Odisha coast of Bay of Bengal (N 20° 17.542′ and E 86° 42.996′) in autoclaved centrifuge tubes (Tarsons, India), and cadmiumresistant bacteria were enumerated. The samples were serially diluted and plated onto seawater nutrient (SWN) agar medium (peptone 5 g, yeast extract 3 g, agar 15 g, aged seawater 500 ml, pH 7.5±0.1) supplemented with varying concentrations of Cd as CdCl 2 (Merck, India) (100–500 ppm). Cadmium-resistant marine bacteria were enumerated, and several single colonies were picked up based on distinct colony morphology (Sinha and Mukherjee 2008). Minimal inhibitory concentration (MIC) of cadmium was determined for the isolates by micro-broth dilution technique (Clinical and Laboratory Standards Institute 2006), and an isolate (JP-11) showing highest cadmium resistance was gram-stained using Gram staining kit (HiMedia, India) and selected for further study.

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Molecular identification of the isolate The cadmium-resistant marine bacterium JP-11 was identified by 16S ribosomal RNA (rRNA) gene sequencing. DNA was extracted from the cells grown in SWN broth medium using bacterial genomic DNA extraction kit (QiaGen, Germany), and the amplification reaction was performed using the universal 16S recombinant DNA (rDNA) primers of 27F (5′AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-ACGG CTACCTTGTTACGA-3′) (Sigma-Aldrich, USA) in a thermal cycler (Bio-Rad, USA) (Dash et al. 2014). Polymerase chain reaction was performed in 50-μl volume containing 2 mM MgCl2, 2.5 U Taq polymerase (Sigma-Aldrich, USA), 100 μM of each dNTP, 0.2 μM of each primer and 3 μl template DNA. The PCR programme used was an initial denaturation at 96 °C for 5 min followed by 30 cycles of 95 °C for 15 s, 49 °C for 30 s and 72 °C for 1 min and a final extension at 72 °C for 10 min. Amplified DNA was purified by PCR purification kit (Sigma-Aldrich, USA) following the manufacturer’s instructions and finally quantified by BioPhotometer Plus (Eppendorf, Germany). The sequencing reactions (Sanger method) were carried out by Xcelris Genomics, Ahmedabad, India. Sequence data was compiled, and consensus sequence was obtained by using BioEdit (7.0.5.3) programme (Hall 1999) and examined for sequence homology with the archived 16S rDNA sequences from GenBank at www.ncbi.nlm.nih.gov/nucleotide, employing the BLASTN (Altschu et al. 1990). Multiple alignments of sequences were performed with ClustalX (1.83) (Thompson et al. 1997). A phylogenetic tree was constructed using the neighbour-joining DNA distance algorithm (Saitou and Nei 1987) using MEGA 5. The resultant tree topology was evaluated by bootstrap analysis (Felsenstein 1985) of neighbour-joining data sets based on 1,000 resamplings. GenBank submission The partial sequence of 16S rRNA gene of JP-11 was submitted to NCBI GenBank, and the assigned accession number is KC771235. Cell growth in the presence of cadmium and different carbon and nitrogen sources Freshly grown overnight culture of JP-11 in SWN broth medium was used as the inoculum for analysis of growth pattern. A 0.5 ml of cells (1 % inoculum) was inoculated in SWN broth medium supplemented with cadmium below (500 and 1,000 ppm) and above (2,500 ppm) the MIC value (1,250 ppm) and incubated at 37 °C with shaking at 200 rpm. The culture in SWN broth without Cd supplementation was used as a positive control. Growth pattern was monitored by measuring the optical density at 630 nm

(OD630) at regular intervals of 30 min (Dash et al. 2014). One percent inoculum was inoculated in Minimal Broth Davis (MBD) (HiMedia, India) medium containing different carbon sources (1 % w/v) viz. glucose, galactose, raffinose, trehalose, sucrose, L-arabinose, glycerol, mannitol, arabitol, erythritol, α-methyl-D-glucoside, rhamnose, cellobiose, esculin, citrate, malonate and sorbose, and different nitrogen sources (1 % w/ v), viz. yeast extract, potassium nitrate and urea supplemented with cadmium (1,000 ppm) in test tubes and incubated at 37 °C with shaking at 200 rpm, and growth (OD630) was monitored after 24 h. Determination of optimum pH and salinity levels One percent inoculum was grown in MBD medium supplemented with 1 % glucose (w/v) at a pH range of 4, 5, 6, 7, 8, 9 and 10, respectively, and salinity range of 5, 10, 15, 20 and 25 g/l, respectively, in the presence of cadmium (1,000 ppm) and incubated in an orbital shaker at 200 rpm/min at 37 °C. The growth pattern was monitored by measuring the optical density at 630 nm (OD630) at regular intervals of 30 min. Tolerance to metals and antibiotic resistance The metals used in this study were chromium (Cr2+), arsenic (As3+), mercury (Hg2+), lead (Pb2+), nickel (Ni+2) and zinc (Zn2+) as the respective metal salts. The stock solution of each metal was 1,600 ppm except mercury which was 500 ppm. Antibiotics tested were amikacin 30 μg (AMK), amoxicillin 30 μg (AMX), vancomycin 30 μg (VA), neomycin 30 μg (N), ampicillin 10 μg (AMP), chloramphenicol 30 μg (C), norfloxacin 10 μg (NX), ciprofloxacin 5 μg (CF) and azithromycin 15 μg (AZM) (HiMedia, India). MIC of the isolate was determined for other metals following CLSI guidelines by micro-broth dilution technique (Clinical and Laboratory Standards Institute 2006). Briefly, the bacterial isolate was enriched in Luria Bertani broth medium (casein enzymic hydrolysate 10.0 g, yeast extract 0.5 g, NaCl 10.0 g and distilled water 1,000 ml, pH 7.5±0.2). Stock solution of the metals was prepared by dissolving the metal containing salt in the autoclaved Mueller Hinton Broth (MHB) and MBD media (HiMedia, India) supplemented with 1 % glucose. MIC was determined by using MHB and MBD media (double diluted) containing various concentrations of the metal serially diluted in a micro-titer plate. To each dilution, 10 μl of 0.5 McFarland culture was added, and the plate was incubated at 37 °C for 24 h. Media without the inoculums was taken as the negative control, and media without cadmium supplementation with the presence of inoculum was taken as the positive control. After incubation, the bacterial growth was monitored by measuring the turbidity of the culture by a micro-titer plate reader (OD630) (Victor X3 2030 multilabel reader, Perkin Elmer). MIC was determined as the lowest concentration of

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compound at which the visible growth of the isolate was completely inhibited. The antibiotic susceptibility testing of the isolates was performed by Kirby Bauer’s disc diffusion technique (Bauer et al. 1966) using commercially available antibiotic discs (HiMedia, India).

Biofilm formation and cadmium removal The ability of JP-11 to form biofilm was studied by tube assay (Mack et al. 1994; O’Toole et al. 2000) with slight modifications (Jain et al. 2013) both in the presence and absence of cadmium. The isolate from fresh SWN agar plate was inoculated in MBD medium supplemented with 1 % glucose in the presence of 100, 500 and 1,000 ppm of Cd as CdCl2 and incubated for 48 h at 37 °C in static condition. After incubation, the content of each tube was washed with phosphatebuffered saline (PBS, pH 7.3) to remove the free-floating planktonic bacteria. The tubes were then stained with 0.1 % (w/v) crystal violet solution. Excess stain was washed off thoroughly with 95 % ethanol, and the tubes were kept for drying. Biofilm formation in tubes was then observed visually. Formation of biofilm was confirmed with the presence of attachment (visible film) on the wall and bottom of the tube. Similarly, the overnight grown culture was diluted 100 times, and individual wells of sterile, polystyrene, 96-well flat bottom micro-titer plates (Tarsons, India) were filled with 200-μl aliquots of the above diluted culture and broth without culture (control). The plates were incubated for 48 h at 37 °C to observe biofilm formation. After incubation, the content of each well was gently removed by slightly tapping the plates, and the wells were washed with PBS (at pH 7.3) to remove the free-floating planktonic bacteria. The plates were then stained with 0.1 % (w/v) crystal violet solution. Excess stain was washed off thoroughly with 95 % ethanol, and the plates were kept for drying. Optical density (OD595) of the wells was determined with a micro-plate reader (Victor X3 2030 multilabel reader, Perkin Elmer). Optical density (OD595) values >0.01 were considered as an index of attachment to the surface and biofilm formation (Jain et al. 2013). The experiment was performed in triplicates, and the mean±SE OD value is presented. Cells of JP-11 were grown in 250-ml shake flasks containing 50 ml of cadmium-supplemented medium (1,000 ppm Cd) inoculated with 0.5 ml of cells (1 % inoculum). Flasks were incubated at 37 °C for 48 h and agitated at 200 rpm in a shaker bath. Optical density at 630 nm (OD630) was monitored. The culture was transferred to centrifuge tubes and centrifuged at 12,000 rpm for 30 min. The supernatant was taken and analyzed by atomic absorption spectrophotometer (Perkin-Elmer AAnalyst™ 200) at 228.8 nm with a cadmium lamp to determine the residual Cd concentration. A standard solution of CdCl2 (Merck) was used in this experiment.

To study the removal rate by EPS, the culture was grown for 48 h in cadmium-supplemented medium (1,000 ppm Cd) at 37 °C and then centrifuged at 12,000 rpm for 60 min at 4 °C. The cell supernatant was extracted overnight with equal volumes of chilled ethanol (99.9 %). The EPS synthesized was dried and then treated with 50 ml of cadmium solution (1,000 ppm). The mixture was kept in a rotary shaker for 48 h at 37 °C. The next day, supernatant was taken and the residual Cd concentration was determined by atomic absorption spectrophotometer (Perkin-Elmer AAnalyst™ 200) at 228.8 nm with a cadmium lamp. Fluorescence microscopy of EPS in the presence of cadmium Staining with acridine orange followed by fluorescence microscopy (Olympus, IX71, Japan) was performed to monitor the EPS architecture of JP-11 grown on glass slides under various Cd concentrations. Cells were grown in MBD medium with 0.2 % glucose, supplemented with different concentrations of Cd (50, 250, 450, 650 and 850 ppm). The grown cells were harvested from their mid-log phase (OD630 =0.35– 0.4) by centrifugation at 8,000 rpm at 4 °C for 10 min and washed twice with PBS. The cell pellet was resuspended in MBD tubes supplemented with different concentrations of Cd (50, 250, 450, 650 and 850 ppm) containing glass slides to provide the base for biofilm formation and EPS synthesis. The tubes were incubated at 37 °C for 48 h under static condition. After 48 h, the slides were removed gently, washed with PBS and then stained with 0.02 % acridine orange for 5 min and washed again with PBS. A thin cover slip was placed over the films and mounted upside down over the objective lens of the fluorescence microscope. About ten images were collected randomly from different points in order to get the significant data. The integrated density and surface plots were quantified by ImageJ analysis to detect architectural change of EPS in different concentrations of cadmium. The corrected total cell fluorescence (CTCF) was calculated by using the formula: CTCF=integrated density−(area of selected cell * mean fluorescence of background readings). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) ATR-FTIR spectroscopy was used to elucidate the chemical binding environment of Cd in association with the biofilmEPS from the bacterial isolate P. aeruginosa JP-11 following Chakravarty and Banerjee (2012). EPS treated with different concentrations (50, 250, 450, 650 and 850 ppm) of cadmium (as CdCl2) was analyzed in the experiment. Analyses were performed in an open atmosphere using the ATR-FTIR spectrophotometer (Bruker, Germany), with a diamond ATR objective. FTIR spectra were collected from 400 to 4,000/cm. Background spectra of water were collected prior to

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measurement of the treated EPS samples and subtracted from the test samples. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) Ten millilitres of broth culture of JP-11 was grown in Erlenmeyer flask supplemented with Cd (1,000 ppm) and was centrifuged at 8,000 rpm for 5 min at 4 °C. The cells were washed twice with 0.1 M PBS (15 mM phosphate buffer, 138 mM NaCl, 2.7 mM KCl, pH 7.4) and fixed overnight in 2 % glutaraldehyde (prepared in 0.1 M PBS). The cells were washed with PBS and distilled water prior to dehydration through an ethanol series (10 % to absolute), held at each concentration for 30 min. Samples were placed on a brass stub, sputter-coated with platinum and examined by SEM (JEOL T-330, scanning electron microscope, Germany). EDS was carried out to detect cadmium that was either adsorbed to the cell surface or entrapped in the EPS (Guibaud et al. 2012).

cDNA of each sample was used as template for qRT-PCR of czcABC gene. Each mixture (final volume 25 μl) contained 12.5 μl of SYBR Green Real-Time PCR Master Mix (Sigma), 1 μl of czcABC forward primer (10 mM), 1 μl of czcABC reverse primer (10 mM), 5 μl of cDNA and 5.5 μl of nucleasefree water. The real-time PCR was carried out with the realtime PCR detection system (Eppendorf, Germany) under the following conditions: 1 cycle of 95 °C for 30 s, then 40 cycles of 95 °C for 15 s, 53 °C for 30 s, 72 °C for 30 s, followed by the final extension at 72 °C for 7 min. Three parallel measurements for each cDNA sample from independent RNA isolation were detected. The gene expression ratio was recorded as the fold difference in quantity from the treated samples (100, 500 and 1,000 ppm Cd stress) versus the control samples (0 ppm Cd stress), different pH values (5, 7) versus optimum pH (6) and different salinity values (5, 10, 15 g/l) with respect to optimum salinity (20 g/l). The house keeping gene 16S rRNA was used for normalization of the sample variation. The primer for 16S has a sequence of forward primer 5′GTCTTC GGATTGTAAAGCAC3′ and reverse primer 5′GCTACACA AGGAAATTCCAC3′ (Zeng et al. 2012).

Genomic DNA isolation and PCR amplification of czcABC gene Genomic DNA was isolated using protocol of Sambrook et al. (1989). The conserved region of czcABC genes of cadmium resistance was amplified by the primers, F1-czcABC 5′TCCT CAAATCCGAACTGGGC3′ and F2-czcABC 5′GCTCGATG GCGAATTGGATG3′. The primers were designed with NCBI Primer Blast and then synthesized by Sigma-Aldrich, USA. The amplification reaction was performed in a total volume of 25 μl by using a thermal cycler (Bio-Rad, USA). The PCR mixture contained 1 U/μl Taq polymerase (Sigma), 1× enzyme buffer, 200 μM of each dNTP (Sigma), 1.25 mM MgCl2, and 0.5 μM of each primer. The genes were amplified with an initial denaturation of 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 53 °C for 90 s, 72 °C for 2 min followed by a final extension of 72 °C for 10 min. The PCR products were analyzed using gel electrophoresis (1.5 %) and visualized in Gel Documentation System (Bio-Rad, USA). Total RNA preparation, cDNA synthesis and qRT-PCR Total cellular RNA was extracted using the GeneJET RNA purification kit (Fermentas) according to the protocol provided by the manufacturer under various concentrations of cadmium, i.e., 100, 500 and 1,000 ppm, pH of 5, 6 and 7 (in 1,000 ppm Cd) and salinity of 5, 10, 15 and 20 g/l (in 1,000 ppm Cd). RNA was quantified at OD260 and OD280 by a NanoDrop spectrophotometer (Eppendorf), which served as the template to synthesize complementary DNA (cDNA) with reverse transcriptase (Fermentas) and random primers (Fermentas).

Results Characterization of the cadmium-resistant marine bacterium The potent isolate has been identified by 16S rRNA gene amplification and sequencing. The phylogenetic tree (Fig. 1) based on 16S rRNA gene sequence was constructed, and the isolate was identified as P. aeruginosa JP-11. It resisted up to 1,000 ppm of cadmium (Cd) as cadmium chloride in aerobic conditions with a MIC of 1,250 ppm. Gram staining showed that this isolate was a gram-negative long rod. The growth pattern of the isolate P. aeruginosa JP-11 was characterized in SWN broth supplemented with various concentrations of CdCl2 (500, 1,000 and 2,500 ppm) with no CdCl2 as control (Fig. 2). The result indicated that the isolate showed a profound growth pattern in the presence of various concentrations of CdCl2. However, the isolate entered into the log phase at a much lesser time in the media without the presence of Cd in comparison to the media supplemented with Cd. The highest concentration of cadmium up to which the isolate could resist and grow was 1,000 ppm. It did not show any growth at 2,500 ppm of CdCl2 concentration. The isolate P. aeruginosa JP-11 supplemented with cadmium at its respective highest resistance concentration (1,000 ppm) grown in different carbon sources showed different patterns of sugar utilization. The carbon sources in which the isolate showed profound growth (OD630 ≥0.41) were glucose, galactose, raffinose, trehalose, sucrose, Larabinose, glycerol and rhamnose, respectively. Slow growth

Environ Sci Pollut Res Fig. 1 Phylogenetic tree for 16S rRNA gene sequences of JP-11 which has been identified as Pseudomonas aeruginosa. The tree was constructed using the neighbour-joining DNA distance algorithm. The resultant tree topologies were evaluated by bootstrap analysis of neighbourjoining data sets based on 1,000 resamplings

(OD630 ≤0.40) was observed in mannitol, arabitol, erythritol, α-methyl-D-glucoside, cellobiose, esculin, citrate, malonate and sorbose. Among the three nitrogen sources tested for growth of the isolate in the presence of cadmium, better growth was observed in yeast extract with respect to potassium nitrate and urea. Optimum pH and salinity for the growth of this isolate were determined. The growth of bacteria at different pH showed that the optimum pH was 6. Similarly, growth at different salinity levels showed that the optimum salinity level of P. aeruginosa JP-11 was 20 g/l (Fig. S1, supplementary material)

Metals and antibiotic resistance of P. aeruginosa JP-11 P. aeruginosa JP-11 was found to tolerate various concentrations of the heavy metals tested with MHB (nutrient rich) and MBD (nutrient deficient) media (Table 1). In both the culture media, the tolerance level of metals used was found to be nearly the same. It could tolerate chromium (Cr2+) (200± 5.77 ppm), arsenic (As3+) (198±5.29 ppm), mercury (Hg2+) (10±1.15 ppm), lead (Pb2+) (400±5.77 ppm), nickel (Ni2+) Fig. 2 Growth of P. aeruginosa JP-11 in the presence of different Cd2+ concentrations. The test concentrations include 500, 1,000 and 2,500 ppm of Cd2+. SWN broth without Cd supplementation was used as positive control, while the uninoculated seawater nutrient broth was used as negative control during this experiment

(170±8.66 ppm) and zinc (Zn2+) (900±5.77 ppm). The isolate was found to resist various antibiotics which included amikacin 30 μg (AMK), vancomycin 30 μg (VA), tetracycline 30 μg (TE), ciprofloxacin 5 μg (CF), amoxicillin 30 μg (AMX), ampicillin 10 μg (AMP) and neomycin 30 μg (N). The isolate was susceptible to chloramphenicol 30 μg (C), norfloxacin 10 μg (NX) and azithromycin 15 μg (AZM).

Biofilm formation and cadmium removal The isolate P. aeruginosa JP-11 showed potential biofilm formation after 48 h in glass tube assay as well as micro-titre plate assay in MBD medium supplemented with glucose in the presence of different concentrations of cadmium (Fig. S2a, supplementary material). An increase in biofilm formation was observed at 48 h of incubation with increase in cadmium concentration as indicated by the (OD595) values (Fig. S2b, supplementary material). Cadmium removal study by the biomass as well as the EPS extracted after 48 h indicated that the percentage of cadmium trapped in the biomass and EPS of the isolate P. aeruginosa JP-11 was 58.76±10.62 and 29.54± 8.02 %, respectively, (Table 2).

Environ Sci Pollut Res Table 1 Effect of other heavy metals on the growth of P. aeruginosa JP-11

Toxic metals

Resistant concentration

Chromium Arsenic Mercury Lead Nickel

200±5.77 ppm 198±5.29 ppm 10±1.15 ppm 400±5.77 ppm 170±8.66 ppm

Zinc

900±5.77 ppm

Fluorescence microscopy of EPS in the presence of cadmium To study the impact of Cd in biofilm-EPS formation, different concentrations of Cd were used. Fluorescence microscopic studies were carried out to examine the effect of different concentrations of Cd on P. aeruginosa JP-11 biofilm architecture. With an increase in Cd concentration, biofilm-EPS formation varied significantly (Fig. 3). Biofilms formed at 250 ppm Cd showed aggregation of biofilm-EPS. Maximum biofilm-EPS production was observed at 650 ppm. The concentration of Cd at 50, 250, 450 and 650 ppm elucidated the uniform distribution of biofilm-EPS, whereas at 850 ppm, the biofilm density was found to be less. The effect of cadmium on biofilm-EPS of P. aeruginosa JP-11 was studied by analyzing and quantifying the integrated density followed by its analysis of surface parameters using ImageJ software. Values of integrated density and CTCF were also determined for the isolate at different concentrations of cadmium. The mean integrated density of the isolate without cadmium from ten measurements was 4,452,573.4 ± 1,183,430. On increasing cadmium concentration, the density dropped down a bit but it attained a significant maximum value at 650 ppm of 4,385,183.95± 456,921.3 (P ≤0.05). The C T C F o f b i of i l m - E P S p r o du c ed b y t he i s o l a t e P. aeruginosa JP-11 is represented in Fig. 4. The CTCF of the biofilm-EPS in the presence of 50, 250, 450, 650 and 850 ppm of Cd were 1,359,837.368±1,641,261.807; 1,682,263.384 ± 1,218,428.425; 2,459,089.03 ± 1,964,254.095; 1,688,757.666 ± 3,087,990.596 and 890,127.552±988,266.6862, respectively.

ATR-FTIR The EPS extracted from the isolate P. aeruginosa JP-11 and its interaction with cadmium at different concentrations of 50, 250, 450, 650 and 850 ppm showed spectral overlap at 3,855.33 and 3,652.124/cm corresponding to O–H and N–H stretching vibrations (Fig. 5). A peak at 2,365.09/cm was observed in the presence of 50, 250, 450 and 650 ppm Cd which depicted the presence of sulphydryl group (–SH), whereas there was a peak shift to 2,314.837/cm in the presence of 850 ppm Cd suggesting that this functional group plays a major role in the binding with cadmium. In the absence of Cd, a peak was observed at 1,637.19/cm, whereas on treatment with Cd in different concentrations, the peak shifted to 1,655.56/cm indicating the C–O stretching mode. Peaks were obtained at 1,454.764, 1,510.907 and 1,562.143/cm in the presence of Cd, which depicted a combination of CH2 stretch, N–H bending and C–N stretching. In the absence of Cd, vibrations were observed at 1,090.700/cm associated with phosphate and polysaccharide moieties of the EPS. SEM and EDS The cellular morphology of JP-11 grown in cadmium was observed under scanning electron microscope at×10,000 magnification to be long rod shaped (Fig. S3, supplementary material). EDS analysis showed the accumulation of cadmium (11.64 %) in their cell wall in the presence of cadmium (Fig. 6a, b). Genomic DNA isolation and PCR amplification of czcABC gene The genomic DNA amplified with the Cd-resistant gene primers showed the presence of a band depicting the presence of cadmium resistance gene (czcABC) in the genome of the isolate P. aeruginosa JP-11 (Fig. S4, supplementary material). Expression profile of czcABC gene Changes at the transcript levels of genes under cadmium stress were analyzed by qRT-PCR. As shown in Fig. 7a, under

Table 2 The percentage removal of cadmium by biomass and EPS of P. aeruginosa JP-11 Components

Initial Cd supplement (ppm)

Final Cd (Supernatant) (ppm)

Total Cd removed (pellet) (ppm)

Cd removed (%) Mean±SD

Biomass

1,000 1,000 1,000 1,000 1,000 1,000

400.44 415.98 420.76 711.89 705.81 695.98

599.56 584.02 579.24 288.11 294.19 304.02

58.76±10.62

EPS

29.54±8.02

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Fig. 3 Fluorescence microscopic images showing P. aeruginosa JP-11 biofilm-EPS (48-h old) grown in MBD media with 0.2 % glucose in different concentrations of cadmium. a Control, b 50, c 250, d 450, e 650

Fig. 4 Comparison of integrated density and corrected total cell fluoresence of P. aeruginosa JP11 grown in the presence of different concentrations of cadmium (50, 250, 450, 650, 850 ppm) at P≤0.05 in MBD media supplemented with 0.2 % dextrose

and f 850 ppm. Biofilm architectures were stained with acridine orange, red colour indicates the presence of biofilms (synthesizing EPS)

Environ Sci Pollut Res Fig. 5 FTIR spectra of untreated (control) and Cd (50, 250, 450, 650 and 850 ppm) treated biofilm-EPS of P. aeruginosa JP11 within the range of wave no. 4,000–1,000/cm interpreting the important functional groups required for the interaction

various concentrations of cadmium at 100, 500 and 1,000 ppm, czcABC was highly up-regulated to 3.52-, 17and 24-fold, respectively. With respect to optimum pH (6) and change just below (5) and just above (7) it, czcABC showed a down-regulation of 2.17E−04 and 2.40E−03, respectively (Fig. 7b). Similarly, with respect to optimum salinity (20 g/l) and change in salinity to 5, 10 and 15 g/l, the gene showed a down-regulation of 4.43E−04, 0.128, 0.248, respectively (Fig 7c).

Discussion Many indigenous species of microorganisms have been isolated from heavy metal-contaminated sites having high tolerance to heavy metals especially cadmium (Desouza et al. 2006; Xie et al. 2010). Microorganisms get affected in an environment contaminated with cadmium due to its extreme toxicity, but some of them undergo selection pressure and develop resistance. Previous reports by Wang et al. (1997) showed that they have isolated a fluorescent pseudomonad (strain CW-96-1) from a deep-sea vent that could resist 5 mM (916.16 ppm) cadmium. However, this is a report on P. aeruginosa isolated from marine environment possessing high resistance towards cadmium. In this study, the isolate P. aeruginosa JP-11 showed the ability to grow in SWN broth medium supplemented with 1,000 ppm of CdCl2. The isolate P. aeruginosa JP-11 could tolerate heavy metals like mercury, lead, chromium, nickel, zinc and arsenic. Similar resistance pattern by bacteria to multiple heavy metals was also reported from Bay of Bengal by Gunaseelan and Ruban (2011) and Dash et al. (2014). It also demonstrated high resistance to antibiotics and a variety of other toxic metals as previously

described cadmium-resistant terrestrial and marine bacterial isolates, i.e. Bacillus sp., Ralstonia sp., Sphingomonas sp., Moraxella sp., Pseudomonas sp., Flavobacterium sp., Vibrio sp., Xanthomonas sp., Alcaligenes sp., Micrococcus sp., Aeromonas sp., Acinetobacter sp. and Pseudoalteromonas sp. (De souza et al. 2006; Xie et al. 2010; Zhou et al. 2013). Like other Pseudomonas sp. isolated from sludges and industrial effluents (Sinha and Mukherjee 2008), marine sediments of Antarctica (DeSouza et al. 2006), cadmium-polluted area of North East China (Zhang et al. 2008) and from uranium mine of Jaduguda, India (Choudhary and Sar 2009), the isolate P. aeruginosa JP-11 also showed resistance to cadmium and other heavy metals and various antibiotics. A similar study on biosorption of cadmium by the multiple metal-resistant marine bacterium Alteromonas macleodii ASC1 isolated from Hurghada harbour, Red Sea, was also reported (Moselhy et al. 2013). Different carbon and nitrogen sources were used in the presence of cadmium to examine the optimum growth conditions of P. aeruginosa JP-11, in which simple sugars as carbon sources and yeast extract used as the nitrogen source showed better growth. This was in accordance with the report of Chen et al. (2007), who showed that yeast extract is a superior growth stimulant for bacteria. It was reported that the biofilm formation capability of various isolates have enhanced bioremediation potential (Vu et al. 2009; Tribelli et al. 2012; Mangwani et al. 2012). Hence, the biofilm-EPS adsorbing cadmium metal detected by the isolate P. aeruginosa JP-11 adds to its bioremediation potential. Comparative analysis of cadmium removal by the biomass and the EPS of the isolate showed that the biomass could reduce a greater percentage of cadmium (58.76±10.62 %) with respect to the EPS extracted. This report was in accordance with the study of Ueshima et al. (2008) where they used

Environ Sci Pollut Res Fig. 6 SEM-EDS of P. aeruginosa JP-11 strain a in the presence of cadmium (1,000 ppm), and b in the absence of cadmium

both the biomass as well as EPS for cadmium interaction study and found that the binding capability of bacterial EPS is identical to bacterial biomass. Integrated density helps to determine the level of fluorescence from an object under fluorescence microscope. In our study, clusters of cells were observed to be glued together in biofilms in the presence of high concentration of Cd which indicated enhanced cohesion in the presence of this divalent toxic cation. The integrated density significantly changed in the presence of Cd (P≤0.05) up to 650 ppm, which suggested that different mechanisms are responsible for the biofilm production in the presence of Cd ion, as the EPS exhibits a higher binding ability in different concentrations of cadmium. This result was consistent with the Cd-binding mechanisms of EPS and the cell wall as illustrated by Ueshima et al. (2008). There are many reports which establishes the role of Ca2+ and Mg2+ in biofilm formation and biofilm architectural determination (Sarkisova et al. 2005; Shukla and Rao 2013; Mangwani et al. 2014). However, there are relatively fewer studies on the effect of toxic metals like cadmium on microbial biofilms-EPS formation. With increase in cadmium concentration, the integrated density of biofilm formation was seen to increase, attaining the maximum value at 650 ppm of Cd. This study was in agreement with the report of Zhou et al. (2013) who found that the deep-sea bacterium

Pseudoalteromonas sp. SCSE709-6 grown in increased cadmium concentration resulted in increased cell adsorption and cadmium removal. The EPS molecules of most bacterial species are negatively charged under nearly neutral pH conditions (pH 6.5–7.5) due to the presence of predominantly carboxylic and phosphoryl functional groups (Beveridge 1988), and many experimental studies have examined metal-EPS binding reactions over the past 20 years (Lau et al. 2005; Comte et al. 2006a, b; Guibaud et al. 2006). According to the study of Huang et al. (2013), the bioaccumulation of Cd by Bacillus cereus RC-1 was found to depend largely on extracellular biosorption rather than intracellular accumulation; therefore, dead biomass of cells appears to be more efficient biosorbent for the removal of Cd from aqueous solution. FTIR spectrum exhibited the presence of amino, carboxyl, hydroxyl, thiol and phosphate groups on EPS interaction with cadmium. In its presence, strong bands in the region 3,800– 3,000/cm were found which are characteristic of N–H and O– H stretching vibrations. A peak observed in the region 9001,100/cm in the absence of cadmium, which is assigned to C– N stretching vibrations and phosphate group was shifted to the region 1,400–1,600/cm in the presence of cadmium. This depicted a combination of CH2 stretch, N–H bending and C–N stretching. This IR absorption pattern indicated the presence of proteins in the EPS. In the presence of different

Environ Sci Pollut Res Fig. 7 Relative expression of czcABC gene in P. aeruginosa JP11 a at 100, 500 and 1,000 ppm Cd, b with different pH—pH 5 and pH 7—and c at salinity 5, 10 and 15 g/l

concentrations of cadmium, the band shift is primarily due to the C–O stretching mode. The FTIR bands on Cd2+ adsorption revealed that the peaks attributed to C–O and N–H (amide) shift, suggesting that the nitrogen atom may be the main

adsorption site for Cd2+ attachment on EPS. On supplementation with 850 ppm Cd, another significant change was the splitting of the peak corresponding to –SH, C–H and O–H stretch. These changes suggested that the oxygen atoms in the

Environ Sci Pollut Res

hydroxyl groups and sulphydryl groups present in the EPS were involved in this adsorption process as well. A disappearance of the peak in the region 900-1,100/cm on cadmium adsorption depicted the phosphate and polysaccharide moieties of the EPS (Brandenburg and Seydel 1996; Naumann et al. 1996; Jiang et al. 2004; Parikh and Chorover 2005, 2006). Thus, we can infer that the bond stretching at this point occurred to a lesser degree due to the presence of cadmium. The FTIR results suggest that the overall composition of binding sites within the EPS may be similar to that of the cell wall in general. These results are in agreement with Oh et al. (2009) who showed that the presence of Cd affected the peak shape and intensity between EPS-treated and EPS-untreated cells. The involvement of these functional groups in the biosorption process is in good agreement with those obtained by other researchers (Panda et al. 2006; Vijayaraghavan and Yun 2008) deducing the fact that the main functional groups responsible for biosorption are carboxylic, hydroxyl, amine, thiol and phosphate groups (Zhang et al. 2013). P. aeruginosa JP-11 possesses czcABC genes for cadmium resistance as reported in other Pseudomonas strains to efflux out the cadmium (Zeng et al. 2012). This gene encodes proteins expressing ion transporters by which metal ions can be pumped out of cytoplasm (Nies 1992). The present study provides information about cadmium resistance though efflux mechanism as well as removal of cadmium by binding to its biofilm-EPS. The czcABC gene is known for encoding proteins showing resistance to Co2+, Zn2+ and Cd2+ (Nies 1992). However, the ability of resistance often varies with the test conditions, such as initial biomass concentration, metal, pH and salinity (Selatnia et al. 2004; Svecova et al. 2006). Thus, it is difficult to compare the resistance and removal mechanisms under varying environmental conditions. Growth of the isolate P. aeruginosa JP-11 in different concentrations of cadmium (100, 500 and 1,000 ppm) highly up-regulates czcABC gene expression. This shows that a gradual increase in cadmium concentration activates the cadmium resistance genetic mechanism. Similar kind of reports was documented by Zeng et al. (2012) where an increase in cadmium stress conditions proficiently expressed the efflux proteins. Another report by Wu et al. (2014) demonstrated that Enterococcus faecalis strain LZ-11 isolated from the petrochemical waste water discharge near Yellow River was able to resist and absorb cadmium. The cadA gene of this isolate was up-regulated 2–3-fold on treatment with Cd. Although this isolate P. aeruginosa JP-11 was obtained from marine sediments having an alkaline pH, it showed its optimum pH at 6. In this study, we have found a significant influence of pH on the cadmium resistance gene. With respect to an optimum pH of 6, a change in pH (±1) showed a downregulation. This effectively illustrated that an optimum pH is essential for proper functioning of the genes required for cadmium resistance.

Salinity also plays an important role in influencing bioremediation potential of bacteria (Rhykerd et al. 1995). Marine isolates are susceptible to salinity conditions for their growth and survival. Terrestrial bacteria dwelling in low salinity conditions increases their protein solubility which leads to proper functioning of proteins. However, in high saline conditions, the salts produce a dehydrating effect on the protein which tends to aggregate, ultimately decreasing its solubility and limiting its function (Missimer et al. 2007; Ogungbenle et al. 2009). But in the case of marine isolates, the high salt concentrations are essential for their survival. In this study, with a change in optimum salinity from 20 to 5, 10 and 15 g/l, the gene showed a down-regulation. This shows that decrease in the salinity level results in greater down-regulation of the czcABC gene. Marine bacteria are highly efficient in adaptation to contaminated natural environments, hence monitoring and assessment of their microbial resistance can be a powerful tool in bioremediation. In this perspective, the isolated biofilmforming cadmium-resistant marine bacterium P. aeruginosa JP-11 has been found to be a suitable candidate species for resistance to cadmium by effluxing it out of the cell and its entrapment in the biofilm-EPS produced by the bacterium. With further advancement in technology, genetic engineering approaches may be utilized, and this aspect of metal resistance could be harnessed for removal of pollutants from contaminated sites. Acknowledgment Authors would like to acknowledge the authorities of NIT, Rourkela for providing facilities. J.C. gratefully acknowledges the receipt of fellowship from Ministry of Human Resource Development, Government of India for doctoral research. S.D. thanks the Department of Biotechnology, Ministry of Science and Technology, Government of India for research grants on marine bacterial biofilm-based enhanced bioremediation.

References Aiking H, Stijnman A, Garderen CV, Heerikhuizen HV, Riet JVT (1984) Inorganic phosphate accumulation and cadmium detoxification in Klebsiella aerogenes NCTC 418 growing in continuous culture. Appl Environ Microbiol 47:374–377 Allesen-Holm M, Barken KB, Yang L, Klausen M, Webb JS, Kjelleberg S, Tolker-Nielsen T (2006) A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 59(4):1114–1128 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410 Bauer A, Kirby W, Sherris J, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Path 45: 493–496 Beveridge TJ (1988) Wall ultra structure: how little we know. In: Actor P, Daneo-Moore L, Higgins ML, Salton MRJ and Shockman GD (eds) Antibiotic inhibition of the bacterial cell: surface assembly and function, Amer Soc Microbiol, Washington, DC pp 3-20

Environ Sci Pollut Res Bortman M, Peter B, Cunningham MA (2003) “Cadmium” Environmental encyclopedia. Vol. 1, 3rd edition Brandenburg K, Seydel U (1996) Fourier transform infrared spectroscopy of cell surface polysaccharides. In: Mantsch HH, Chapman D (eds) Infrared spectroscopy of biomolecules. Wiley-Liss, New York, pp 203–238 Brisou JF (1995) Biofilms: methods for enzymatic release of microorganisms. CRC press, Boca Raton, p 204 Chakravarty R, Banerjee PC (2012) Mechanism of cadmium binding on the cell wall of an acidophilic bacterium. Bioresour Technol 108: 176–183 Chen M, Xia L, Xue P (2007) Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate. Int Biodeter Biodegr 59(2):85–89 Choudhary S, Sar P (2009) Characterization of a metal resistant Pseudomonas sp. isolated from uranium mine for its potential in heavy metal (Ni2+, Co2+, Cu2+, and Cd2+) sequestration. Bioresour Technol 100(9):2482–2492 Comte S, Guibaud G, Baudu M (2006a) Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods. Enzyme Microbial Technol 38:237–245 Comte S, Guibaud G, Baudu M (2006b) Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and complexation properties of Pb and Cd with EPS: Part II. Consequences of EPS extraction methods on Pb2+ and Cd2+ complexation. Enzyme Microbial Technol 38:246–252 Congiu L, Chicca M, Pilastro A, Turchetto M, Tallandini L (2000) Effects of chronic dietary cadmium on hepatic glutathione levels and glutathione peroxidase activity in starlings (Sturnus vulgaris). Arch Environ Contam Toxicol 38(3):357–361 Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, LappinScott HM (1995) Microbial biofilms. Annu Rev Microbiol 49(1):711–745 Cunningham DP, Lundie LL Jr (1993) Precipitation of cadmium by Clostridium thermoaceticum. Appl Environ Microbiol 59(1):7–14 Daane LL, Harjono I, Zylstra GJ, Häggblom MM (2001) Isolation and characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with the rhizosphere of salt marsh plants. Appl Environ Microbiol 67(6):2683–2691 Dash HR, Mangwani N, Chakraborty J, Kumari S, Das S (2013) Marine bacteria: potential candidates for enhanced bioremediation. Appl Microbiol Biotechnol 97(2):561–571 Dash HR, Mangwani N, Das S (2014) Characterization and potential application in mercury bioremediation of highly mercury-resistant marine bacterium Bacillus thuringiensis PW-05. Environ Sci Pollut R 21:2642–2653 Davey ME, O’Toole GA (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol R 64(4):847–867 De Souza MJ, Nair S, Bharathi PL, Chandramohan D (2006) Metal and antibiotic-resistance in psychrotrophic bacteria from Antarctic Marine waters. Ecotoxicology 15(4):379–384 De J, Ramaiah N, Vardanyan L (2008) Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury. Mar Biotechnol 10(4):471–477 Deb S, Ahmed SF, Basu M (2013) Metal accumulation in cell wall: a possible mechanism of cadmium resistance by Pseudomonas stutzeri. Bull Environ Contam Toxicol 90(3):323–328 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 Flemming H, Wingender J (2001) Relevance of microbial extracellular polymeric substances (EPSs)—Part I: structural and ecological aspects. Water Sci Technol 43(6):1–8 Guibaud G, van Hullebusch E, Bordas F (2006) Lead and cadmium biosorption by extracellular polymeric substances (EPS) extracted

from activated sludges: pH-sorption edge tests and mathematical equilibrium modelling. Chemosphere 64:1955–1962 Guibaud G, Bhatia D, d’Abzac P, Bourven I, Bordas F, van Hullebusch ED, Lens PN (2012) Cd (II) and Pb (II) sorption by extracellular polymeric substances (EPS) extracted from anaerobic granular biofilms: evidence of a pH sorption-edge. J Taiwan Inst Chem Engin 43(3):444–449 Gunaseelan C, Ruban P (2011) Heavy metal resistance bacterium isolated from Krishna-Godavari basin, Bay of Bengal. Int J Environ Sci 1(7): 1856–1864 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98 Huang F, Dang Z, Guo CL, Lu GN, Gu RR, Liu HJ, Zhang H (2013) Biosorption of Cd(II) by live and dead cells of Bacillus cereus RC-1 isolated from cadmium-contaminated soil. Colloids Surf B 107:11–18 Jain K, Parida S, Mangwani N, Dash HR, Das S (2013) Isolation and characterization of biofilm-forming bacteria and associated extracellular polymeric substances from oral cavity. Ann Microbiol 63(4): 1553–1562 Jiang W, Saxena A, Song B, Ward B, Beveridge TJ, Myneni SCB (2004) Elucidation of functional groups on Gram-positive and Gramnegative bacterial surfaces using infrared spectroscopy. Langmuir 20:11433–11442 CLSI (Clinical and Laboratory Standards Institute) (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, seventh ed., Approved Standard M7-A7, CLSI, Wayne, PA, USA Lau TC, Wu XA, Chua H, Qian PY, Wong PK (2005) Effect of exopolysaccharides on the adsorption of metal ions by Pseudomonas sp. CU-1. Water Sci Technol 52:63–68 Longe JL (2005) “Cadmium poisoning” The gale encyclopedia of alternative medicine. Gale Cengage, Vol. 1, 2nd edition Mack D, Nedelmann M, Krokotsch A, Schwarkopf A, Heesemann J, Laufs R (1994) Characterization of transposon mutants of biofilms producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: genetic identification of a hexosamine-containing polysaccharide intracellular adhesion. Infect Immun 62:3244–3253 Mangwani N, Dash HR, Chauhan A, Das S (2012) Bacterial quorum sensing: functional features and potential applications in biotechnology. J Mol Microbiol Biotechnol 22(4):215–227 Mangwani N, Shukla SK, Rao TS, Das S (2014) Calcium-mediated modulation of Pseudomonas mendocina NR802 biofilm influences the phenanthrene degradation. Colloids Surf B 114:301–309 Missimer JH, Steinmetz MO, Baron R, Winkler FK, Kammerer RA, Daura X, Van Gunsteren WF (2007) Configurational entropy elucidates the role of salt-bridge networks in protein thermostability. Protein Sci 16(7):1349–1359 Mitra RS, Bernstein IA (1978) Single-strand breakage in DNA of Escherichia coli exposed to Cd2+. J Bacteriol 133:75–80 Monachese M, Burton JP, Reid G (2012) Bioremediation and tolerance of humans to heavy metals through microbial processes: a potential role for probiotics? Appl Environ Microbiol 78(18):6397–6404 Moselhy KM, Shaaban MT, Ibrahim HAH, Abdel-Mongy AS (2013) Biosorption of cadmium by the multiple-metal resistant marine bacterium Alteromonas macleodii ASC1 isolated from Hurghada harbour, Red Sea. Arch Sci 66:2 Naumann D, Schultz CP, Helm D (1996) What can infrared spectroscopy tell us about the structure and composition of intact bacterial cells? In: Mantsch HH, Chapman D (eds) Infrared Spectroscopy of Biomolecules. Wiley-Liss, New York, pp 279–310 Nies DH (1992) Resistance to cadmium, cobalt, zinc, and nickel in microbes. Plasmid 27(1):17–28 Nies DH (1995) The cobalt, zinc, and cadmium efflux system Czc ABC from Alcaligenes eutrophus functions as a cation-proton-antiporter in Escherichia coli. J Bacteriol 177:2707–2712

Environ Sci Pollut Res Nies DH (1999) Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51(6):730–750 Nies DH, Silver S (1989) Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus. J Bacteriol 171(2):896–900 Novelli ELB, Marques SFG, Almeida JA, Diniz YS, Faine LA, Ribas BO (2000) Toxic mechanism of cadmium exposure on cardiac tissue. Tox Subst Mech 19(4):207–217 Nucifora G, Chu L, Misra TK, Silver S (1989) Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux ATPase. Proc Natl Acad Sci U S A 86(10):3544– 3548 O’Toole G, Kaplan HB, Kolter R (2000) Biofilm formation as microbial development. Annu Rev Microbiol 54:49–79 Ogungbenle HN, Oshodi AA, Oladimeji MO (2009) The proximate and effect of salt applications on some functional properties of quinoa (Chenopodium quinoa) flour. Pakistan J Nutr 8(1):49–52 Oh S, Kwak MY, Shin WS (2009) Competitive sorption of lead and cadmium onto sediments. Chem Eng J 152:376–388 Panda GC, Das SK, Chatterjee S, Maity PB, Bandopadhyay TS, Guha AK (2006) Adsorption of cadmium on husk of Lathyrus sativus: physico-chemical study. Colloids Surf B 50:49–54 Parikh SJ, Chorover J (2005) FTIR spectroscopic study of biogenic Mnoxide formation by Pseudomonas putida GB-1. Geomicrobiol J 22: 207–218 Parikh SJ, Chorover J (2006) ATR-FTIR spectroscopy reveals bond formation during bacterial adhesion to iron oxide. Langmuir 22: 8492–8500 Renner LD, Weibel DB (2011) Physicochemical regulation of biofilms formation. MRS Bull 36(5):347–355 Rhykerd RL, Weaver RW, McInnes KJ (1995) Influence of salinity on bioremediation of oil in soil. Environ Pollut 90(1):127–130 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evo 4(4):406–425 Saluja B, Gupta A, Goel R (2011) Microbial management of cadmium and arsenic metal contaminants in soil. In: Khan, MS, Zaidi A, Goel R, Musarrat J (eds) Biomanagement of Metal-Contaminated Soils. Springer-Verlag, pp 257–275 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Sarkisova S, Patrauchan MA, Berglund D, Nivens DE, Franklin MJ (2005) Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. J Bacteriol 187(13):4327–4337 Schmidt T, Schlegel HG (1994) Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A. J Bacteriol 176(22):7045–7054 Selatnia A, Bakhti MZ, Madani A, Kertous L, Mansouri Y (2004) Biosorption of Cd2+ from aqueous solution by a NaOH-treated bacterial dead Streptomyces rimosus biomass. Hydrometallurgy 75:11–24 Shukla SK, Rao TS (2013) Effect of calcium on Staphylococcus aureus biofilms architecture: a confocal laser scanning microscopic study. Colloids Surf B: Biointerfaces 103:448–454

Sinha S, Mukherjee SK (2008) Cadmium–induced siderophore production by a high Cd-resistant bacterial strain relieved Cd toxicity in plants through root colonization. Curr Microbiol 56(1):55–60 Sutoo DE, Akiyama K, Imamiya S (1990) A mechanism of cadmium poisoning: the cross effect of calcium and cadmium in the calmodulin-dependent system. Arch Toxicol 64(2):161–164 Svecova L, Spanelova M, Kubal M, Guibal E (2006) Cadmium, lead and mercury biosorption on waste fungal biomass issued from fermentation industry. I. Equilibrium studies. Sep Purif Technol 52:142– 153 Teitzel GM, Parsek MR (2003) Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl Environ Microbiol 69(4):2313–2320 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876–4882 Tribelli PM, Di Martino C, López NI, Iustman LJR (2012) Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis. Biodegradation 23(5):645–651 Ueshima M, Ginn BR, Haack EA, Szymanowski JE, Fein JB (2008) Cd adsorption onto Pseudomonas putida in the presence and absence of extracellular polymeric substances. Geochimi Cosmochim Acta 72(24):5885–5895 USEPA (2000) Technology transfer network-cadmium compounds. http://www.epa.gov/airtoxics/hlthef/cadmium.html Vijayaraghavan K, Yun YS (2008) Bacterial biosorbents and biosorption. Biotechnol Adv 26:266–291 Vu B, Chen M, Crawford RJ, Ivanova EP (2009) Bacterial extracellular polysaccharides involved in biofilm formation. Molecules 14(7): 2535–2554 Wang CL, Michels PC, Dawson SC, Kitisakkul S, Baross JA, Keasling JD, Clark DS (1997) Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture. Appl Environ Microbiol 63(10):4075–4078 Wu G, Sun M, Liu P, Zhang X, Yu Z, Zheng Z, Chen Y, Li X (2014) Enterococcus faecalis strain LZ-11 isolated from Lanzhou reach of the Yellow River is able to resist and absorb cadmium. J Appl Microbiol 29 doi:10.1111/jam.12460 Xie X, Fu J, Wang H, Liu J (2010) Heavy metal resistance by two bacteria strains isolated from a copper mine tailing in China. Afr J Biotechnol 9(26):4056–4066 Zeng X, Tang J, Liu X, Jiang P (2012) Response of P. aeruginosa E1 gene expression to cadmium stress. Curr Microbiol 65(6):799–804 Zhang Y, Zhang H, Li X, Su Z, Zhang C (2008) The cadA gene in cadmium-resistant bacteria from cadmium-polluted soil in the Zhangshi area of Northeast China. Curr Microbiol 56(3):236–239 Zhang HO, Zhou WZ, Ma YH, Zhao HX, Zhang YZ (2013) FTIR spectrum and detoxification of extracellular polymeric substances secreted by microorganism. Guang Pu Xue Yu Guang Pu Fen Xi 33(11):3041–3043 Zhou W, Zhang H, Ma Y, Zhou J, Zhang Y (2013) Bio-removal of cadmium by growing deep-sea bacterium Pseudoalteromonas sp. SCSE709-6. Extremophiles 17(5):723–731