World Journal of Microbiology & Biotechnology (2006) 22: 577–585 DOI 10.1007/s11274-005-9074-4
Ó Springer 2006
Isolation and characterization of a metal-resistant Pseudomonas aeruginosa strain Chelliah Edward Raja, Kolandaswamy Anbazhagan and Govindan Sadasivam Selvam* Department of Biochemistry, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, 625 021, Madurai, India *Author for correspondence: Tel.: +91-452-2459213, E-mail:
[email protected] Received 8 June 2005; accepted 29 September 2005
Keywords: 16S rDNA, antibiotic resistance, biosorption, heavy metal resistance microorganisms, oil mill industry effluent, Pseudomonas aeruginosa
Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin. Abbreviations: LB – Luria–Bertani; AFM – Atomic force microscopy; ppm – Parts per million; AAS – Atomic absorption spectrophotometer; rpm – Revolution per minute
Introduction Pollution due to heavy metal toxicity is an everincreasing problem in the developing nations. Heavy metals are major pollutants in marine, ground, industrial and even treated wastewater (Valdman et al. 2001). Toxic heavy metals are found in effluents and discharged wastewater of industries like electroplating, steel, alloy, motor vehicles, aircraft, paint, chemical, textile, pigment etc. (Sani et al. 2001; Saithong & Prasertsan 2002). Release of Cd, Cr, Pb and Ni into the environment has increased in the recent years at an alarming rate. Cadmium discharged into the environment in large amounts as an industrial waste has led to its current rank as a major anthropogenic pollutant (Cunningham & Lundie 1993). It enters the human food chain through plants, smoking materials and diet (Dabeka & Mckenzie 1992; Kalcher et al. 1993). Cadmium is a potent carcinogen, embryogen, teratogen and mutagen and it may cause hyperglycemia, reduced immuno-potency and anemia, due to interference with iron metabolism (Sanders 1986). Cadmium and Pb, though non-essential and non-beneficial, are considered highly toxic to plants, animals and microbes (Ajmal et al. 1998). Chromium(VI) species are
also extremely toxic and exhibit mutagenic and carcinogenic effects on biological systems (DeLeo & Ehrlich 1994; McLean & Beveridge 2001). Microbial reduction of Cr(VI)) to Cr(III) is a potentially useful process for remediation of Cr(VI)-affected environments (Michel et al. 2001; Ganguli & Tripathi 2002; Camargo et al. 2003). Nickel, another toxic metal, reduces growth and development in plants and is teratogenic (Chen & Lin 1998). Microbes showing resistance to Ni have generally been isolated and characterized from anthropogenically polluted sites (Schmidt & Schlegel 1994; Singh & Kumar 1998; Grass et al. 2001; Pal et al. 2004). Lead, a major pollutant that is found in soil, water and air is a hazardous waste and is highly toxic to humans, animals, plants and microbes (Low et al. 2000). Lead resistance has been widely reported and well studied in both gramnegative bacteria and gram-positive bacteria isolated from Pb-contaminated soils. Pseudomonas marginalis shows extracellular Pb exclusion while Bacillus megatarium has been reported to demonstrate intracellular cytoplasmic Pb accumulation (Roane 1999). Conventional methods as precipitation, oxidation/reduction, ion exchange, membrane filtration and evaporation, though capable of
578 eliminating these toxic metals from the environment, are extremely expensive and inefficient for metal removal from dilute solutions ranging from 1 to 100 mg of dissolved metal per liter (Volesky 1990). Microorganisms and microbial products have been reported to efficiently remove soluble and particulate forms of metals, especially from dilute solutions, though bioaccumulation and therefore microbe-based technologies provide an alternative to the conventional techniques of metal removal/recovery (Ozdemir et al. 2004). Microorganisms change/reduce the toxicity of metallic contaminants through pH changes, bioaccumulation or biosorption (Al-shahwani et al. 1984; Vesper et al. 1996). Microbes are capable of accumulating toxic metal ions by two well defined processes viz: (i) biosorption: an energy-independent binding of metal ions to cell walls and (ii) bioaccumulation: energy-dependent process of metal uptake into the cells. Both live and inactivated microbial mass of bacteria, fungi and algae are utilized for removing toxic metal ions (Volesky 1994; Karna et al. 1996; Li et al. 2004). Recent research into clean-up of heavy metals from wastewaters and sediments has led to development of bio-based materials with increased affinity, binding capacity and selectivity for target metals (Pazirandch et al. 1998; Ozdemir et al. 2004). The present study evaluates the isolation, characterization and biosorption of Cd, Cr, Ni and Pb by P. aeruginosa BC15 from oil mill effluent treated wastewater samples.
C.E. Raja et al. Determination of optimal growth conditions The optimal growth conditions with reference to pH and temperature were determined. The strain was grown in LB medium, in the presence and absence of heavy metals with varying pH values, i.e., 5, 6, 7, 8, 9 and incubated at 25, 30, 37 and 40 °C. The optical density of the growing cultures in all the above-mentioned conditions were observed at 600 nm using an ultraviolet visible spectrophotometer (Model 4050, LKB Biochrom, Cambridge, England) to determine the optimum growth. Determination of MIC Maximum resistance of the selected isolates against increasing concentrations of Cd, Cr, Ni and Pb on LB agar plates was evaluated until the strains unable to give colonies on the agar plates. The initial concentration used (100 ppm) was added from 100 mg/100 ml stock solution. The stock solutions of CdSO4, K2Cr2O7, NiCl2Æ6H2O and Pb(NO3)2 (Ranbaxy, Mumbai, India) were prepared in double distilled water and sterilized by autoclaving at 121 °C, 15 psi for 15 min. The growing colonies at a given concentration were subsequently transferred to the next higher concentration. Based on the evaluation minimum inhibitory concentration (MIC) was determined after 48 h of incubation at 37 °C. Sensitivity to antibiotics
Materials and methods Sample collection and isolation of microorganisms Heavy metal-resistant bacteria were isolated from treated oil mill effluent wastewater samples. Samples were collected from oil mill industry site in Madurai and Virudhunagar districts in India. For isolation and enumeration of microorganisms, samples were serially diluted in sterile distilled water and plated on Luria– Bertani (LB) agar supplemented with 5 mg/l (ppm) level of heavy metals CdSO4, K2Cr2O7, NiCl2Æ6H2O and Pb(NO3)2 one metal at a time or as heavy metal mixture (HM), plates were incubated at 37 °C for 48 h to screen resistant colonies. Identification and characterization of the bacterial isolates Selected isolates were grown on MacConkey agar media (HiMedia, Mumbai, India). The shape and colors of the colonies were examined under the microscope after Gram staining. Isolates were biochemically analyzed for the activities of oxidase, catalase, V-P test, MR-VP test, starch hydrolysis and gelatin hydrolysis, motility, indole production and citrate utilization. These tests were used to identify the isolate according to Bergey’s Manual of Systematic Bacteriology (Claus & Berkeley 1986).
Antibiotic sensitivity of the heavy metal-resistant isolates was determined by the disc diffusion method. Antibiotic-impregnated discs (6 mm diameter, HiMedia, Mumbai, India) were placed on nutrient agar plates poured with individual isolates and incubated at 37 °C for 24 h. The diameter of the inhibition zones around the discs was measured. The antibiotic concentrations of the disc used were ampicillin (10 lg), tetracycline (30 lg), chloramphenicol (30 lg), erythromycin (10 lg), kanamycin (10 lg) and streptomycin (20 lg) respectively. Effect of metals on bacterial growth To study the effect of metals on growth in LB, King’s A medium and minimal medium varying concentrations of heavy metals (0.1 mM, 0.05 mM) were supplemented in the culture media. Culture was grown aerobically in 15–25 ml medium in 100 ml conical flasks at 37 °C for 24 h. Culture grown in absence of metal was treated as control. Growth was monitored as a function of biomass by measuring the absorbance at 600 nm against blank. Atomic force microscopy analysis P. aeruginosa BC15 cells cultured in LB medium supplemented with Cd, Cr, Ni, Pb or metal mixture (100 lg/ml) were incubated at 37 °C with agitation for 24 h. Cells were harvested by centrifugation at 4000 rev/ min for 15 min and washed twice with double distilled
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Metal-resistant Pseudomonas aeruginosa water. Samples for AFM analysis were mounted on the cover glass and air dried as described (Bolshakova et al. 2001), and mounted directly on the specimen metal disc using a double adhesive tape. Samples were scanned at different areas using AFM (Shimadzu SPM 9500-2J). For high resolution, contact mode micro cantilever was used for the analysis. Digital images were stored in the computer and processed.
with the sequences of other representative bacterial 16S rDNA regions by using ClustalX software version 1.83 (Thompson et al. 1997; Jeanmougin et al. 1998). Further phylogenetic analysis was performed by using PHYLIP (DNADIST, NEIGHBOUR, and SEQBOOT) (Felsenstein 1993). Based on the phylogenetic tree, similarity index was generated and compared with known sequences. 16S rDNA sequences of BC15 have been deposited in GenBank.
Estimation of heavy metals The biosorption of heavy metals was carried out using bacteria grown in 250 ml conical flasks containing 50 ml of LB medium supplemented with heavy metals at the concentration of 100 mg/l (Cd, Cr, Ni and Pb) and incubated at 37 °C for 48 h. At selected intervals of time, samples were harvested by centrifugation at 5000 rev/ min, supernatant was collected and stored at –20 °C for heavy metal analysis. The heavy metals present in the solution were determined by a Varian Atomic Absorption Spectrophotometer (Model SpectrAA-220). The amount of metals in samples was estimated by using known concentrations of metals in the medium as control. 16S rDNA sequence determination Genomic DNA from strain BC15 was isolated by the butanol extraction method (Mak & Ho 1991). Bacterial 16S rDNA was amplified from the extracted genomic DNA by using the universal bacterial 16S rDNA primers, forward primer (5’-AGA GTT TGA TCC TGG CTC AG-3’) and reverse primer (5’-GGT TACC TTG TTA CGA CTT-3’). PCR was performed with a 50-ll reaction mixture containing 1 ll (10 ng) of DNA extract as the template, each primer at a concentration of 0.5, 1.5 mM MgCl2, and each deoxynucleoside triphosphate at a concentration of 50 mM, as well as 1 U of Taq polymerase and buffer used as recommended by the manufacturer (Fermentas, Hanover, Germany). After the initial denaturation for 5 min at 94 °C, there were 30 cycles consisting of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min and then a final extension step consisting of 5 min at 72 °C. PCR was carried out in a Gene AMP PCR system 2700 (Applied Biosystems, California, USA). PCR products were analysed by electrophoresis in 1.5% (w/v) agarose gel in 1x TAE buffer with ethidium bromide (0.5 lg/ml) before being subjected to further analysis. Nucleotide sequencing, alignment, and phylogeny A DNA fragment was eluted by DE 80 membrane from the gel. The elute was again reamplified with universal primers. PCR products were sequenced by 37301 DNA Synthesizer (Applied Biosystems, California, USA). Sequences that matched with previously published bacterial 16S rDNA sequences in the NCBI databases using ADVANCED BLAST (Altchul et al. 1997). Based on the scoring index the most similar sequences were aligned
Results Isolation of heavy metal-resistant strain Three hundred colonies were screened from initial (5 ppm) level of heavy metal supplemented LB medium. After secondary screening, 25 bacterial strains were picked up from treated oil mill industry effluent wastewater samples. One of the potential strains showing a high degree of metal resistance and antibiotic resistance was selected for further studies. The strain BC15 formed pinkish colonies on MacConkey agar, was a Gramnegative motile bacterium, rod shaped, 1–3 mm long and formed yellowish brown colonies. The strain showed positive for catalase, oxidase, gelatin hydrolysis, and utilize citrate, optimum growth at 37 °C and pH 7.0. Based on the morphological, physiological, biochemical characteristics (Table 1) and comparative analysis of the sequence with already available database showed that the strain was close to the members of genus Pseudomonas. The highest sequence similarity (94%) and phylogeny based on ClustalX clearly indicates that BC15 is a strain of P. aeruginosa (Figure 1). An 813 nucleotides-long 16S rDNA sequence was submitted in the NCBI databases under the accession number AY971 518. Table 1. The morphological and biochemical characteristics of bacterial isolate (BC15). Characteristics
P. aeruginosa BC15
Colony diameter Colony colour Cell morphology Motility Oxidase Catalase Indole production V-P reaction MR-VP reaction Citrate utilization (Simmons) Starch hydrolysis Gelatin hydrolysis MacConkey agar Temperature (°C) 4 25 37 40 50
1–3 mm Yellowish brown Rod Motile + + ) ) ) + ) + +
Note: ) negative; + positive.
) + + + )
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Figure 1. Phylogenetic analysis of 16S rDNA gene sequences of P. aeruginosa and related taxa. The scale bar corresponds to 5% nucleotide sequence difference.
Growth studies of P. aeruginosa BC15 was carried out on different growth media such as LB, King’s A medium, and minimal medium. The concentrations of the heavy metals were 0.1 mM in LB; King’s A medium and 0.05 mM in minimal medium. The biomass measurements of the cultures incubated for 30 h were in good agreement according to bacterial resistance for each heavy metal. The order of resistance to metals by the strain BC15 was found to be Pb>Ni>Cd>Cr on the agar plates. However, the MICs of the heavy metal in solid media were higher than those in liquid media due to the conditions of diffusion, complexation and availability of metals were different form those observed in solid media. When the cells were grown in LB medium, Cd showed the high-peak values when compared to other metals whereas in the minimal medium Ni and Pb showed highest peak values. In general the growth rate of the strain BC15 in the presence of heavy metal was consistently slower than the control (Figure 2a–c). Resistance to heavy metals and antibiotics The strain BC15 showed very high degree of resistance to the selected heavy metals, MIC values varied from
100 to 800 ppm (Table 2). BC15 showed resistance against 800 ppm of Pb, 700 ppm of Ni, 500 ppm of Cd and 400 ppm of Cr, respectively. Among the heavy metals Ni and Pb were less toxic, whereas Cd and Cr were highly toxic to strain BC15 with the order of resistance is Pb>Ni>Cd>Cr. The bacterial strain BC15 was tested for the ability to grow in various antibiotic-supplemented media, and the strain showed resistance to ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin (Table 3). Microscopic observations of bacterial cells (AFM) P. aeruginosa BC15 cells were imaged with AFM operated in contact mode in liquid conditions. Cells analyzed under AFM showed significant difference in their cell wall morphology and cell dimensions differ considerably Table 2. Minimal inhibitory concentration (mg/l) of heavy metal tolerance of P. aeruginosa BC15. Cadmium
Chromium
Nickel
Lead
100–500
100–400
100–700
100–800
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Metal-resistant Pseudomonas aeruginosa Table 3. Antibiotic resistance of P. aeruginosa BC15.
Discussion
Antibiotics
Concentration (lg)
Diameter of inhibition zone (mm)
Ampicillin Tetracycline Chloramphenicol Streptomycin Kanamycin Erythromycin
10 30 30 20 30 10
NZ (R) 13 (R) NZ (R) 14 (R) 13 (R) NZ (R)
In this paper we report high degree of tolerance to selected heavy metals by an isolate P. aeruginosa BC15. The limit of tolerance to the highest concentration of selected heavy metals in the media was evaluated based on the ability of BC15 cells to grow on the subsequent higher concentration. The degree of growth in response to metal ions varied with the metal and the concentration of the metal ion supplementation in the medium. Further it was observed that inability to grow at higher concentration was associated with decrease in growth and formation of localized depressions in the cell surface of BC15 as shown in the AFM micrograph. This may be due to the presence of polarizable groups on bacterial surfaces that are capable of interacting with cations of the heavy metals and are responsible for the reversible metal-binding capacity of the microorganisms. Friis et al. (1986) have previously reported that reduction in growth is mainly because of the interaction between the cell surface and of metal cations along with phosphate, carboxyl, hydroxyl and amino-groups. Growth rates of the strain BC15 in the presence of heavy metals (Cd, Cr, Ni and Pb) were consistently slower than that of the control (similar observation have been reported earlier, Suresh Kumar et al. 1998, 2001; Pal et al. 2004). Verma et al. (2001) reported that metal tolerance holds an association with antibiotic resistance. A similar kind of multiple antibiotic resistance property was observed in the isolate BC15, which clearly indicates that the high degree of antibiotic resistance might be associated with higher levels of tolerance to various heavy metals (Dhakephalkar et al. 1994; Rosen 1996; Hassen et al. 1998). Under conditions of imposed stress, metal and antibiotic resistance in microorganisms possibly helps them to adopt spontaneously than by mutation and natural selection (Bhattacherjee et al. 1988; Silver & Misra 1988). P. aeruginosa BC15 was capable of removing significant amount of Ni, Pb, Cd and Cr during growth within 48 h even though it has been reported that the metal biosorption capacity of microbial cells varies with the growth phase (Maceskie & Dean 1984; Tamai et al. 1993; Suresh Kumar et al. 1998). Significant variation in the growth pattern was observed for each of the heavy metals used in the study individually and as a mixture of heavy metal ions. In previous studies Cr(VI)-resistant microorganisms have been found capable of growing in 10–1500 mg of Cr(VI)/l (McLean & Beveridge 2001; Basu et al. 1997). BC15 was able to remove 65% Pb and 30% of Cr (VI) from mixed metal solutions (100 mg/l) within 48 h when compared to Staphylococcus saprophyticus which could remove 100% Pb, 25% Cr (VI) and 24% Cu respectively (Ilhan et al. 2004). Previously reported Enterobacter cloacae and Klebsiella species have been resistant against Cd, Cr and Pb. Enterobacter cloacae resisted Cd (220 mg/l) Cr (800 mg/l) and Pb (1400 mg/l); Klebsiella strain (CMBL-Cd-2 & CMBL Cd-3)
Note: NZ – no zone; R – resistant.
from solution with and without metals. In some regions, cell wall changes were observed (Figure 3a–f). Biosorption of metals Metal uptake capacity was investigated using heavy metals at different time intervals of growth upto 48 h. Decrease of metal concentration in solutions was observed with the increase in growth due to efficient uptake of metals with a maximum time at 48 h. The highest biosorption capacity was observed with Ni (93%) and the lowest corresponds to Cr (30%), 65% for Pb and 50% for Cd during uptake studies. The uptake capacity was measured in AAS analysis (Figure 4).
Figure 2. Growth pattern of BC15 on different media: (a) LB medium; (b) King’s A medium; (c) minimal medium supplemented with Cd, Cr, Ni, Pb at various concentration and in combination.
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Figure 3. Atomic force micrograph images of P. aeruginosa BC15 showing (marked) collapse of the cell structure. Culture grown aerobically in LB medium 9: (a) control; (b) HM; (c) Cr; (d) Ni; (e) Pb; (f) Cd, grown in LB supplemented with 100 lg/ml concentration of metals individually. Scale: normal view Bar = 5 lm and Magnification view Bar = 2 lm.
showed resistance to Cd at the concentration of 110 mg/l and 100 mg/l, Cr 600 mg/l and 500 mg/l, Pb 1200 mg/l and 900 mg/l supplemented in the medium
respectively (Riazul et al. 1999). A significantly higher resistance was observed in the strain BC15 which showed resistance against at the concentration of Cd
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Figure 3. Continued.
Figure 4. Metal biosorption of P. aeruginosa BC15 in LB broth containing Cd, Cr, Ni, Pb (each at 100 mg/l concentration) against growth in h.
(500 mg/l), Cr (400 mg/l) and Pb (800 mg/l) of the medium. Thompson & Walting (1987) and Nelson et al. (1995) reported up to 25% Pb removal using pure cultures of Pseudomonas, Bacillus and Aeromonas through nonspecific processes. But BC15 removed 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h through biosorption process. Efficient heavy metal-removing capabilities and the ability to grow over a wide range of metal concentrations under aerobic conditions along with antibiotic resistance are clear indications of the advantages that may offer to employ this organism for metal remediation in simple reactors or in situ.
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